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Daiwa Anglo-Japanese Foundation

Supports UK-Japan collaboration in education, science, arts, and culture.

NHLBI - National Heart Lung and Blood Institute

PROJECT ABSTRACT Congenital heart disease (CHD) occurs in approximately 40,000 infants in the United States each year. The National Heart, Lung, and Blood Institute (NLHBI) launched its Bench to Bassinet Program (B2B) in 2009 to overcome the major barriers in translational research, identify the causes of human CHD, and improve outcomes for individuals with CHD. Through the Congenital Heart Disease GEnetic NEtwork Study (CHD GENES), the B2B program has enrolled over 14,000 participants with CHD and 18,000 family members, conducting genomic sequencing to identify an estimated 25% of previously unexplained CHD cases. Despite these advances, critical gaps persist in our understanding of how genetic variants influence genotype- phenotype correlations and long-term outcomes in CHD. Although not initially designed as a longitudinal cohort, the NHLBI recognized the unique potential of the CHD GENES and directed the coordinating center (CC) to organize deep phenotyping and re-enrollment of a subset of participants for an in-person clinical assessment and to make the data available to the scientific community as the B2B Congenital Heart disease Advancing New understanding in GEnomics (CHANGE) Cohort. The new iteration of the CC is a unique, integrated combination of world-leading cardiovascular and clinical/translational research expertise, advanced infrastructure, outstanding operational support, and state-of-the-art technology. The specific aims are to: 1) Establish and maintain the B2B CHANGE Cohort, 2) Create a unique resource for CHD research by integrating new data sources with the existing clinical and genomic information maintained in the B2B DataHub (HeartsMart) and shared with NHLBI’s BioData Catalyst system, and 3) Ensure the CHD community has the necessary access, tools, and support to translate B2B data into improved health and quality of life for those affected by CHD. B2B CHANGE will be established using a multifaceted and patient-informed cohort outreach and engagement approach incorporating nationally recognized expert leadership and consultation and adaptation to local contexts as appropriate. Innovative clinical assessments and technical advancements to HeartsMart will expand and enrich existing phenotyping approaches, extend the duration of follow-up, and allow for new biological sample acquisition for future mechanistic and translational studies. Through resources including HeartsMart and BioData Catalyst, and extensive outreach, education, and engagement, the CC will ensure the CHD community has access to this vital resource to support rigorous, independently funded, investigator-initiated ancillary studies. The B2B CC has provided excellence in administrative support and coordination for the B2B program for the previous two funding cycles and will continue to be a successful partner with site investigators, the NHLBI, and the CHD community, leading the coordination of knowledge and data for this important cardiovascular research effort.

NIDA - National Institute on Drug Abuse

Project Summary/Abstract Optimizing safe perioperative opioid use is a critical priority. Despite being intended for short-term use, a significant number of patients who are first exposed to opioids (opioid-naïve) following surgery continue using them longer than the typical recovery period (i.e., 3months). This prolonged exposure is termed new persistent opioid use (NPOU). To date, the existing literature has heavily focused on identifying pre-existing risk factors. However, to better inform practice and facilitate actionable interventions, there is a critical need to discern which patients are at higher risk and characterize trajectories of post-operative opioid use that develop into unhealthy opioid use (i.e., misuse, abuse, and addiction). Thus, the proposed work has two specific aims: (1) to develop an optimal prediction model to identify patients at risk for NPOU, and (2) to determine the extent to which NPOU reflects unhealthy opioid use and identify patient characteristics associated with such use. To achieve these aims, we will analyze electronic health records from a large academic health system that serves diverse sociodemographic populations using state-of-the-art analytic methods, including machine learning and natural language processing. Complementing the proposed training plan, the overall objective is to promote the safe perioperative use of opioids among opioid-naïve patients by generating insights to prevent the transition from acute to chronic opioid use, and to discern chronic opioid use that reflects unhealthy use. This proposed research and training will be strongly supported by structured training activities, an interdisciplinary mentorship team consisting of experts in medicine, nursing, health informatics, and computer science, as well as robust research resources from the University of Pennsylvania. Supporting NIDA’s strategic priority of preventing prolonged drug use, addiction, and related adverse consequences through data science, the proposed research training proposal will provide the applicant an essential foundation for a sustained research career focused on identifying mechanisms underlying the continuum from initial opioid use to addiction, informing the development of novel, effective, and timely interventions to mitigate opioid-related harms, specifically among the large and growing population of opioid-naïve patients undergoing arthroplasty.

United Indians of All Tribes Foundation

Daybreak Star houses Indigenous Art the Sacred Circle Gallery and also hosts large community events such as Seafair Indian Days Powwow and Indigenous Peoples' Day. Daybreak Star's restrooms are not ADA-compliant. This project will develop a design to turn a closet into a universal, accessible, single restroom on the upper level of the building and modifications for the lower level restrooms to comply with current accessibility codes. This project will be completed by February 2019.

West Seattle Community Orchestras

We will provide free concerts for the West Seattle community in December 2024. The first concert will be held on December 9, 2024 at 6pm at Chief Sealth High School located at 2600 SW Thistle St, Seattle, WA 98126. This program promotes the arts in our community through supporting children's musical education as well as local artists conducting the ensembles.

NIA - National Institute on Aging

Project Summary/Abstract Pneumonia is the leading infectious cause of morbidity and mortality in older adults. The incidence of pneumonia increases exponentially with age, resulting in over 3 million US emergency department (ED) encounters and 1 million hospitalizations per year in individuals ≥65 years of age. Although aspiration pneumonia has traditionally been considered a distinct clinical entity, there is an emerging consensus that pneumonia should be considered on a continuum as aspiration of organisms from the oropharynx is a shared pathogenic mechanism for nearly all pneumonias. Oral hypofunction and dysphagia (swallowing dysfunction) are geriatric syndromes and established factors in oral dysbiosis and pneumonia risk. However, the prevalence and interaction between them has not been well characterized as it relates to the pathogenesis or microbial etiology of pneumonia in older adults. This knowledge gap represents a patient safety threat as diagnostic misclassification of pneumonias can result in poorly targeted antibiotic therapy and failure to refer for swallowing and/or oral rehabilitation. There is an urgent need to comprehensively characterize profiles of oral hypofunction and dysphagia in older adults with pneumonia of various bacterial etiologies and evaluate their role in pneumonia pathogenesis. Previous studies examining oral and swallowing profiles in adults with pneumonia are limited due to a lack of objective diagnostic evaluations or consideration of the upper airway microbiome. To address these knowledge gaps, we propose an observational study of older adults presenting to the ED with pneumonia that involves comprehensive assessments of both oral and swallowing function in combination with cutting edge metagenomic analyses and application of saliva to a microphysiological lung model of aspiration. Our overarching objective is to significantly advance the understanding of oral hypofunction and dysphagia in pneumonia pathophysiology in older adults as a foundational step towards reducing diagnostic error, improving targeted antibiotic therapy, optimizing referral to oral and swallowing rehabilitation, and reducing the significant morbidity and mortality observed in this population. Our multidisciplinary team of experts will achieve this objective via the following specific aims: 1a. Determine the prevalence and profiles of oral hypofunction and dysphagia among a cohort of older adults with pneumonia; 1b. Compare prevalence and severity of oral hypofunction and dysphagia between older adults with and without pneumonia and between patients with pneumonia due to normal respiratory flora vs. respiratory pathogens; 2. Compare microbiome profiles in older adults with pneumonia based on the presence of oral hypofunction and dysphagia; 3. Identify targetable mechanisms of saliva-induced lung bronchial epithelial injury in a lung microphysiological system. The proposed work is highly innovative as it will be the first to comprehensively assess oral and hypofunction to elucidate relationships with pneumonia development; include state of the art microbiome characterization of upper and lower respiratory sites; and utilize a microphysiological lung model to examine the impact of salivary characteristics on host response mechanisms.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Project Summary/Abstract Epigenetic modifiers govern cell fate transition during animal development and their mutations drive multiple human congenital disorders; however, the molecular mechanisms underlying the roles of epigenetic modifiers in these normal and pathological processes remain poorly understood. It is widely believed that epigenetic modifiers function through the epigenetic marks they catalyze. Nevertheless, the discoveries of catalytic- independent role of epigenetic modifiers challenge this view, raising the question about the biological function of epigenetic marks. Mono-methylation of histone H3 at lysine 4 (H3K4me1) is a reliable mark of enhancers that shape cell identity, and its reconfiguration accompanies the differentiation of pluripotent stem cells, suggesting that the regulation of H3K4me1 plays an instructive role in cell fate transition. To examine this hypothesis, we investigated the catalytic function of LSD1 and LSD2, two paralogous histone demethylases targeting H3K4me1, in regulating gene expression during cell fate transition. Using state-of-the-art approaches such as precise genome engineering, epigenetic and transcriptomic profiling, and stem cell differentiation, we demonstrate functional synergism between the demethylase activity of LSD1 and LSD2 in regulating cellular differentiation. Based on these compelling preliminary data, here we propose to dissect the molecular mechanisms underlying how the demethylase activity of LSD1/2 regulates cell fate transition. The results generated from our proposed studies will not only reveal novel molecular mechanisms underlying the roles of H3K4me1 in gene regulation and cell fate transition, but also provide insights into understanding the pathogenesis of diseases driven by LSD1/2 loss-of-function. This research aligns with the NIH mission to advance our understanding of fundamental biological processes and contribute to knowledge relevant to developmental disorders and regenerative medicine.

NIGMS - National Institute of General Medical Sciences

Project Summary / Abstract Cells optimize their functional capacity in response to extracellular signals to meet metabolic demands and maintain cellular and organism-level homeostasis. Endoplasmic reticulum (ER) is a central hub for protein folding, trafficking, lipid synthesis and secretion. ER also regulates the function of other organelles by exchanging ions and other hydrophobic molecules through inter- organelle contact sites. The structure of the ER is highly dynamic with specialized subdomains such as ER sheets, tubules, 3-way junctions and exit sites. ER exhibits complex architectural configuration related to cell’s functional capacity. However, whether and how different ER subdomains play a role in optimizing the functional capacity of the cell in response to metabolic demand is unclear. Moreover, the upstream signals that regulate ER shape dynamics are not known. In this proposal, we aim to use state-of-the-art metabolic flux approaches to investigate how the structural regulation of ER controls lipid and glucose fluxes in cells. Additionally, by using enhanced Focused Ion Beam Scanning Electron microscopy and super-resolution fluorescent microscopy, we will interrogate the ER’s architectural response to nutrient sensing and signaling pathways. Lastly, we will perform CRISPR-Cas9 screens, coupled with high-content imaging and deep learning-based analysis to discover novel regulators of ER shape and function. This work will provide novel insight into how organelle shape and dynamics regulate cellular function and health. It will introduce a new perspective to the field by incorporating subcellular architectural remodeling as a new layer of metabolic regulation.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY HIV infection is the second leading cause of viral infection-related mortality in the United States. Furthermore, the inability of the immune system to clear the virus renders this chronic infection a major driver of inflammation- associated co-morbidities, including cardiovascular disease and neurological disorders. This emphasizes the need to understand mechanisms that contribute to immune-mediated control of infection and how these could be exploited to develop cure strategies. Natural Killer (NK) cells help control infection by killing infected cells; their function is tightly regulated by a balance of activating and inhibitory receptors present on both the NK cell and the target cell. NK cell killer-cell immunoglobulin-like receptors (KIRs) interact with different members of MHC-I proteins and regulate the activation or inhibition of NK cytotoxic activity. Population studies have identified combinations of KIR and MHC-I alleles associated with slower HIV disease progression. Among them, KIR3DS1 (whose sequence is relatively conserved compared to other KIRs) was the first to be associated with delayed disease progression. KIR3DS1 interacts with the nonclassical MHC-I molecule, HLA-F, which is also relatively conserved compared to classical MHC-I molecules that bind to KIR2 and KIR3 proteins. This interaction triggers NK cytotoxicity towards HIV-infected cells. Our recently published work suggests that this KIR3DS1/HLA-F interaction can be augmented by IL-15 and retinoids. While these are clinically approved for cancer therapy and are safe in ART-suppressed people with HIV, the mechanisms that contribute to this augmentation, with the potential for further manipulation are unknown. The overall goal of this proposal is to decipher the mechanisms that regulate the HLA-F/KIR3DS1 interaction in the context of infection, cytokines, and retinoids. Our central hypothesis is that the pathways that regulate the KIR3DS1/HLA-F interaction can be exploited to enhance NK cell-mediated control of infection in vitro and in vivo. In Aim 1, we will evaluate the contribution of human genetic polymorphisms, viral subtype, and HIV accessory genes on HLA-F expression. In addition, we will assess the effects of various cytokines and new retinoids on HLA-F expression. In Aim 2, we will evaluate the interplay of KIR3DS1 and HLA-F in controlling HIV infection in vitro. This will include assessing the effects of the cytokines and retinoids described in Aim 1 on NK cell phenotype and function. In addition, a custom CITE-Seq panel and single cell metabolomic profiling will be used for a comprehensive analysis of KIR3DS1-expressing vs non- expressing NK cells to determine whether pathway differences between these populations could be exploited to expand or enhance the function of KIR3DS1+ NK cells. In Aim 3, using samples from two clinical trials, we will assess the in vivo effects of the IL-15 superagonist, N-803 (ACTG A5386), and the retinoid, isotretinoin (ACTG A5323), on CD4+ T cell HLA-F expression, NK cell phenotype/metabolomic, and whether these associate with reservoir dynamics. Together, our studies will yield insights into the pathways that modulate the KIR3DS1/HLA- F interaction that could help develop cure strategies with FDA-approved biologics and small molecule inhibitors.

NIGMS - National Institute of General Medical Sciences

Project summary/abstract. Proteins orchestrate cellular processes as dynamic ensembles of interconverting conformations, characterized by the underlying free energy landscape (FELs). Understanding these FELs is paramount for deciphering biological mechanisms, elucidating disease pathogenesis, and engineering novel therapeutics. However, resolving complete FELs, predicting how they respond to perturbations like mutations or ligand binding, and designing them de novo present formidable challenges, limiting our ability to rationally control protein function. This application seeks to bridge this critical gap by developing an integrated computational and experimental platform for the comprehensive decoding, modulation, and de novo design of protein FELs. I am a postdoctoral researcher in Dr. Anum Glasgow’s laboratory at Columbia University, with a strong background in computational biophysics, protein engineering, and advanced hydrogen-deuterium exchange mass spectrometry (HX/MS) analysis. My development of PIGEON-FEATHER, a state-of-the-art Bayesian framework for deriving site-resolved energetics from HX/MS data, exemplifies my commitment to advancing methods for studying protein ensembles. Building on this foundation, my K99 research will establish a transformative framework for resolving, manipulating, and designing protein FELs, providing fundamental insights and practical tools for protein science, drug discovery, and synthetic biology. Aim 1 will develop PF- MetaD, a novel enhanced sampling approach that incorporates HX/MS-derived protection factors (PFs) into meta dynamics simulations. This will be enabled by two deep learning tools I propose to develop—PFNet and PFBoost—for accurate, residue-level PF determination. Together, these will allow the reconstruction of complete protein FELs. Aim 2 will apply these landscape insights to a critical biomedical challenge by designing state- selective protein binders to modulate the FEL of BRAF kinase, aiming to rationally control its activity in cancer- associated mutants by reshaping its conformational ensemble. Aim 3 will push the boundaries of protein engineering by pursuing the de novo design of a universal, ligand-responsive allosteric protein switch based on the PAS domain scaffold, programming its FEL for custom molecular recognition and regulation. Under the primary mentorship of Dr. Anum Glasgow and Dr. Barry Honig, and with the support of collaborators and the rich research environment at Columbia University and affiliated New York City institutions, I will train in single- molecule FRET, high-throughput screening methodologies, advanced machine learning for integrating multimodal biophysical data, scientific leadership, and grant writing. These skills will enable my long-term goal: an independent multidisciplinary lab at a leading R1 institution, focusing on FEL-guided design of functional and therapeutic proteins. This K99/R00 award is critical for my transition to an independent investigator, transforming our ability to rationally program biomolecular behavior.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Influenza viruses cause seasonal and epidemic outbreaks that pose a recurring burden on global public health systems. Despite annual vaccination efforts, severe influenza virus infections occur each year and disproportionally impact children, the elderly, and those with pre-existing conditions. Influenza-induced lung damage and persistent inflammation are highly variable across infected individuals, with limited understanding of the pathogenic signals that contribute to these processes. This proposal hopes to shed light on the pathogenic signals that promote the development of damage-associated niches in the lung leading to more severe disease outcomes. Prior members of the Thomas laboratory discovered a subset of damage- responsive fibroblasts (DRfibs) that reside in damage-associated lung niches and uniquely contribute to influenza-induced lung damage. DRfibs produce high levels of ADAMTS4, an enzyme that degrades versican, an extracellular matrix component produced in the lung during development and infection. Interestingly, when mice lack ADAMTS4, they are protected from influenza-induced mortality compared to wildtype littermate controls. It was found that a dense versican barrier prevented CD8 T cell: DRfib crosstalk, leading to fewer IFNg-producing CD8 T cells, less lung damage, and improved hypoxemia in ADAMTS4 KO mice. This proposal seeks to exploit ADAMTS4 KO mice as a model of damage-associated niche disruption to elucidate how preventing T cell: DRfib communication affects T cell phenotype, clonality, and specificity using spatial transcriptomic, scRNAseq, and TCR sequencing approaches. Low blood oxygen saturation (hypoxemia) is included as a predictor of poor outcomes in 9 out of 12 influenza and pneumonia severity scores, highlighting a strong correlation between impaired oxygenation and influenza severity. Tissue-level hypoxia in the lungs is also a characteristic of influenza illness. Previous research has shown that a cell’s microenvironment can significantly impact its phenotype; however, the impact of hypoxia on immune and stromal cell subsets in the lungs during and following a respiratory virus infection has not yet been explored. This F32 proposal will employ a unique mouse model to reveal how hypoxia in the lung microenvironment during severe respiratory viral infection influences the phenotypes of T cells and fibroblasts. Successful completion of the proposed will generate a unique atlas of hypoxic cell phenotypes that could have broad implications for the field as many severe respiratory viruses induce hypoxemia and lung damage. Dr. Paul G. Thomas, a well-established influenza immunologist and member of the Center of Excellence for Influenza Research and Response, and St. Jude Children’s Research Hospital will be integral to achieving the goals outlined in this proposal by providing technical training, practice in scientific communication, mentorship experience, on-site access to state of the art resources, and networking opportunities with influenza experts. Completion of the research and training goals outlined in this F32 proposal will unveil novel mechanisms of influenza pathogenesis while supporting the development of the applicant’s independent research career.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The mammalian heart undergoes profound transcriptional and phenotypical remodeling during postnatal development, a process known as cardiac maturation. However, the molecular mechanisms driving this transition is not fully understood, posing a major challenge in cardiac regenerative medicine, where induced cardiomyocytes from pluripotent stem cell differentiation or non-myocyte reprogramming exhibit an overall immature phenotype that severely limits their application in cell therapy and in vitro disease modeling. In this K99/R00 application, I propose to integrate cutting-edge single cell multiomics with state-of-the-art computational methods to unravel the cell-type-specific gene regulatory networks governing cardiac maturation, and develop a novel dual-reporter system to model and enhance cardiac maturation in vitro and in vivo. During the K99 phase, I will characterize the epigenomic changes of the mouse heart during postnatal development at a single cell resolution using various single cell multiomic technologies (Aim 1), and construct cell-type-resolved gene regulatory networks underlying cardiac maturation using bioinformatic approaches coupled with deep learning (Aim 2). I will also establish cell culture and mouse models with CRISPR-mediated knock-in of dual-fluorescent reporters to track and assess cardiomyocyte maturation (Aim 3a). During the R00 phase, I will experimentally characterize key regulatory elements and novel transcriptional regulators using functional genomic approaches (Aim 3b). I will also leverage these findings to enhance the maturation of in vitro-derived cardiomyocytes for improved therapeutic potential (Aim 3c). The expected outcomes of my proposed research will deepen our understanding of postnatal cardiac development and uncover new therapeutic strategies to improve cardiac function after injury. My career goal is to lead an independent research group that develops and employs innovative technologies to study the regulatory mechanisms underlying cardiac development, regeneration, and disease. In my K99 phase, I will acquire crucial knowledge and skills in advanced single cell genomics and computational biology to complement my previous expertise in developmental biology and cardiac research. My career development will be supported by an exceptional mentoring and advisory committee from UCSD/Salk/HHMI, along with world-class resources, training opportunities, and institutional support at UC San Diego. These elements will provide a strong foundation for my successful transition to an independent tenure- track faculty position.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Direct CD8+ T cell recognition of allo-HLA plays a major role in acute cellular rejection (ACR) of transplanted tissues. However, much is unknown about the T cells responsible for direct ACR. For decades, alloreactive T cells have been viewed as generally nonspecific, responding either to a plethora of allopeptides in the context of allo-HLA, or unique determinants on allo-HLA independent of peptide. Recent data though has suggested that many, if not most, alloreactive T cells are allospecific, responding to individual peptide/HLA complexes. Despite these advances, and despite their critical role in transplant rejection, systematic studies of intragraft T cells, their specificities, and their fundamental biology are lacking. We now have an unprecedented opportunity to study the T cells driving direct ACR. Our initial work using scRNA sequencing on biopsies and urine from 10 patients undergoing kidney transplant rejection found a surprisingly limited number (~20/patient) of clonally expanded CD8+ T cells (CD8EXP) with unique CDR3 α/β sequences. Remarkably, some of these CD8EXP persist for months in rejecting allografts, despite histologically successful anti-rejection therapy. In some cases, these “rogue” cells can re-expand and contribute to additional rejection episodes. However, the specificities, transcriptomic programs, functionality, and environmental niches that favor persistence of these and other CD8EXP remain unclear. We are now poised to make substantial breakthroughs in these critical areas. Supported by a wealth of exciting preliminary data demonstrating both rigor and feasibility, the goals of this project are to capitalize on our discoveries to advance our understanding of the specificity and biology of CD8+ T cells driving ACR. In our 1st Aim, we will determine the specificities of intragraft CD8EXP from several patients undergoing kidney allograft rejection, allowing us to decode the underpinnings of allospecificity, including its structural and biochemical features, the contributions of TCR affinity and specificity to persistence of CD8EXP cells, and the potential for shared or immunodominant targets in patients with the same HLA mis-matches. In preliminary data for Aim 1, in what we believe is a first for the field, using yeast display coupled with state-of-the-art structural informatics, we identified a bona fide allopeptide that evokes a prominent response from one of these allospecific TCRs. In our 2nd Aim, we will define the spatially resolved environments and cell-cell communications that define the gene expression and functional states of persisting, intragraft, allospecific CD8EXP cells. Overall, we will greatly expand our knowledge of the T cells driving ACR. We will identify the ligands of T cells driving rejection, assess the potential for immunodominance, and deconstruct the biochemistry of TCR allospecificity. We will dissect the phenotypes and genetic programming of the allospecific T cells that are retained in various allograft niches. Beyond the advances in basic immunology and mechanistic biology of allorecognition, this work will significantly improve our understanding of the biology of ACR, allowing for more targeted prediction, monitoring, and control of rejection.

NIMH - National Institute of Mental Health

Delusions – unfounded and often bizarre beliefs – can be a highly distressing feature of psychotic illnesses. They frequently do not respond to pharmacological or psychosocial treatments, and advancing treatment development requires a clearer understanding of their specific underlying mechanisms. Prior research has implicated abnormalities in social cognition, particularly in paranoid, persecutory delusions. Other work suggests that more general mechanisms of belief formation may underlie delusions. Prediction errors may play a central role in both social and non-social accounts of delusions; however, the content, computations, and implementation of these aberrant error signals have yet to be established. The goal of the current proposal is to evaluate the roles of social and non-social prediction errors in delusions among individuals with schizophrenia, compared to control participants without delusions. This proposal includes three specific aims that span various levels of analysis: Specific Aim 1: Examine social and non-social Kamin blocking as behavioral metrics of prediction error processing in patients and controls and explore their relationship with delusion severity. Specific Aim 2: Apply state-of-the-art computational modeling to social and non-social Kamin blocking behaviors to quantify prediction errors, learning rates, and weighting parameters relevant to delusions. This aim will investigate whether these factors differ by task frame and if such differences are associated with symptoms. Specific Aim 3: Use functional neuroimaging and neuromelanin scanning to determine how social and non-social Kamin blocking is implemented in the brain and explore their relationships to dopamine and noradrenaline sytem integrity and delusions. Together, these aims will provide a rigorous account that spans the bio-psycho-social processes implicated in delusions. This integrated approach will provide the foundation for developing rational and targeted treatment strategies.

NLM - National Library of Medicine

PROJECT SUMMARY Fractures are a common manifestation of physical abuse, with children <2 years at highest risk. The identification of healing fractures is crucial in the evaluation of physical abuse in a young child as these can suggest ongoing violence within the home and have serious implications for child protection. However, estimating time-since-injury of healing fractures based on imaging is often difficult and imprecise. Although deep learning (DL) models could vastly improve accurate dating of healing fractures in children presenting with suspicious injuries, a critical gap remains for accessible large digital pediatric imaging datasets and needed artificial intelligence (AI) infrastructure. Notably, this gap has recently been designated a critical pediatric health priority by the American College of Radiology. This project closes this gap by establishing the framework for deidentified image sharing and storage between three PEDSnet sites (Nationwide Children’s Hospital, Cincinnati Children’s Hospital Medical Center, Riley Hospital for Children) via a Secure File Transfer Protocol and providing the AI infrastructure needed for better image interpretation and diagnosis. We will train and validate DL models with state-of-the-art transformers such as DINOv3 and benchmark to the well-established convolutional neural network architecture ResNet-50 using skeletal imaging of accidental fractures of long bones in children <4 years to directly and accurately age healing fractures. In parallel, we will use meta- learning with a combination of labeled accidental fractures and unlabeled abuse fractures, followed by few-shot learning to regress the age of abuse fractures. Deliverables include establishing the framework for image sharing within pediatric health systems and the development of DL algorithms for aging of healing fractures that could be implemented widely as a virtual consultant for radiologists faced with the task of interpreting imaging completed in children presenting with high-risk injuries. This is the first study to propose the development of DL algorithms for aging healing fractures by 1) training on multicenter imaging data and 2) using real-world data of patients evaluated for abuse. This proposal is a key first step towards development of a national resource to stimulate and support high-quality, collaborative imaging research within pediatrics, dramatically improving patient outcomes within both pediatric and community settings. By providing a mechanism for cross-site image sharing, this project enables future scalable multi-institutional model development and validation for improved interpretation of imaging completed in child abuse evaluations.

NIAID - National Institute of Allergy and Infectious Diseases

During suppressive antiretroviral therapy (ART), HIV-1 persists in long-lived resting memory CD4+ T cells of children and young adults with perinatal HIV-1 as both intact and defective proviral genomes. The intact, replication-competent proviruses contribute to the latent reservoir and are a lifelong barrier to cure. In perinatal HIV-1, the reservoir is established early and shaped by unique immunologic factors. A growing body of evidence suggests that while defective proviruses cannot contribute to rebound in the absence of ART, these proviruses are transcriptionally and translationally active, potentially leading to adverse immune effects. However, the frequency, composition, and potential immunologic effects of defective proviruses across pediatric age groups remain poorly understood. In this proposal, we aim to characterize the defective proviral reservoir in children and young adults living with perinatal HIV-1 by determining the abundance and sequences of proviruses that are maintained for years despite ART and assessing their ability to produce viral mRNA and proteins. This project leverages well-characterized, bio-banked peripheral blood mononuclear cell (PBMC) and plasma specimens from pediatric HIV-1 cohorts to systematically characterize the landscape of defective proviruses in perinatal infection from infancy through adolescence. Our hypothesis is that in longstanding treated perinatal HIV-1, defective proviruses are transcriptionally and translationally active and drive persistent residual HIV-1 viremia during ART, promoting immune activation and exhaustion despite replication incompetence. Defective proviruses may also serve to produce decoy viral proteins that elicit autologous neutralizing antibodies, thereby reducing the efficacy of autologous neutralization of the latent reservoir. We propose three specific aims. In Aim 1, we will quantify and characterize intact and defective proviruses across pediatric age groups using near full-length single genome sequencing. In Aim 2, we will assess the transcriptional activity of defective proviruses following ex vivo stimulation in co-culture for HIV-1 mRNA analyses and their correlation with immunologic and clinical measures, including markers of immune activation and exhaustion. We will then compare it to sequences from low level plasma viremia to determine whether defectives are the source. In Aim 3, we will perform the quantitative viral outgrowth assay (QVOA) with the ultrasensitive p24 Simoa assay to identify if high- and low-level p24 producing wells are harboring intact or defective proviruses. We will then determine whether env-pseudotyped virus derived from intact or defective proviral sequences can be neutralized with autologous plasma IgG. By integrating molecular virology and immunology profiling in a pediatric context, this study will generate novel insights into the role of defective proviruses in HIV-1 persistence in children. Our findings will inform the design of age-specific cure strategies and contribute to the broader goal of ART-free remission in children with perinatal HIV-1 towards a life free of co-morbidities.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Although effective therapeutics that target the dysregulated kinase activity of BCR::ABL1 have been developed for patients with chronic myeloid leukemia (CML), acquired resistance remains an important clinical issue. Additionally, problematic side effects plague a considerable proportion of patients who are expected to require lifelong therapy. The first five approved tyrosine kinase inhibitors (TKIs) for CML target the ATP binding pocket of BCR::ABL1 (“orthosteric” TKIs). Asciminib is the first active “allosteric” TKI for CML and was recently approved as a frontline therapy based on high response rates and excellent tolerability. Asciminib is rapidly being adopted as a preferred treatment in all lines of therapy. We have demonstrated that several mutations that confer resistance to orthosteric TKIs unexpectedly confer in vitro and/or clinical resistance to asciminib. We have further demonstrated that a clinical variant of BCR::ABL1 lacking ABL1 exon 2 is uniquely and highly resistant to asciminib. Notably, these isoforms retain asciminib binding affinity, thereby invoking a novel molecular mechanism of resistance. Our central hypothesis is that asciminib will be vulnerable to multiple resistance- conferring mutations that disrupt its allosteric effect on kinase conformation, in addition to a limited number of mutations that impair its ability to bind BCR::ABL1. Our rationale is that pioneering work on orthosteric TKI resistance mechanisms in CML have informed kinase conformational dynamics, optimal CML patient management, development of next-generation TKIs and successful prediction of TKI resistance mechanisms in several other malignancies. We propose to (i) employ orthogonal approaches to identify and validate single point mutants in BCR::ABL1 that can confer resistance to asciminib, and compound (≥2 on one DNA strand) mutants that arise following subsequent orthosteric TKI therapy, (ii) assess their sensitivities to a novel active investigational allosteric inhibitor, combinations of TKIs, and a novel bitopic TKI, (iii) determine mechanisms of resistance through computational and structural studies, (iv) define residues necessary for adoption of the closed ABL1 kinase conformation, and (v) assess the ability of asciminib-resistant mutants to pathologically activate ABL1 kinase activity. The proposed research is significant due to its potential to rapidly impact clinical investigation and optimize patient management, inform understanding of kinase regulation and other malignancies. The proposed research is innovative because it applies state-of-the-art methodologies to comprehensively define and characterize a novel mechanism of resistance to a first-in-class highly clinically active allosteric TKI and thereby establish a new paradigm. Additionally, it will assess the promise of emerging agents, TKI combinations, and an innovative bitopic TKI with best-in-class features for treating asciminb-resistant single and compound mutants.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Antiretroviral therapy (ART) effectively suppresses HIV replication, yet treatment interruption results in rapid viral rebound due to persistent viral reservoirs. A major barrier to eliminating these reservoirs is the progressive loss of HIV-specific CD8+ T cell effector function. Although immune checkpoint blockade can partially restore T cell function in cancer, these approaches show limited efficacy in people living with HIV (PLWH), underscoring the need to identify alternative mechanisms contributing to HIV-specific CD8+ T cell dysfunction. Extracellular adenosine (ADO) is a potent immunosuppressive metabolite generated from inflammatory ATP by the ectonucleotidases CD39 and CD73 and signals through the A2a adenosine receptor to inhibit CD8+ T cell function. Under physiological conditions, ADO levels are tightly regulated by adenosine deaminase-1 (ADA-1). In PLWH, increased expression of ADO-generating enzymes and A2aR, together with reduced ADA-1 expression, promotes ADO accumulation and is associated with immune dysfunction. However, the contribution of the ADO/ADA-1 axis to antigen-specific CD8+ T cell impairment in PLWH remains poorly defined. This project will define the role of ADO signaling in HIV-specific CD8+ T cell dysfunction (Aim 1) and evaluate whether targeted ADA-1 supplementation can improve antiviral function (Aim 2). We hypothesize that ADO/ADA- 1 axis impacts antigen-specific CD8+T cell function in PLWH and that restoring ADA-1 activity will improve HIV- specific function. Aim 1 will quantify ADO-driven suppression of antigen-specific CD8+ T cell function in PLWH. Aim 1.1 will determine whether HIV-specific CD8+ T cells are more susceptible to ADO-mediated suppression than CMV-specific CD8+ T cells within the same donor. Preliminary data demonstrate epigenetic and transcriptional repression of ADA-1 and enhanced ADO-pathway signaling in HIV-specific CD8+ T cells, supporting increased vulnerability. Aim 1.2 will determine whether chronic HIV infection broadly increases ADO sensitivity by comparing antigen-specific CD8+ T cell responses from PLWH and HIV-negative donors. Established MDDC–T cell coculture assays will be used to model antigen-specific function ex vivo. Aim 2 will test whether targeted ADA-1 supplementation improves HIV-specific CD8+ T cell function. Building on preliminary feasibility data, ADA-1 mRNA will be delivered selectively to CD8+ T cells using CD8- targeted lipid nanoparticles, alone or in combination with PD-1 blockade, to assess functional improvement. Overall, this exploratory study will define a mechanistically distinct pathway contributing to HIV-associated CD8+ T cell dysfunction and evaluate ADA-1 supplementation as a targeted strategy to restore antiviral immunity, informing future immune-based HIV cure approaches.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT People with HIV-1 (PWH) on antiretroviral therapy (ART) are prone to experiencing chronic inflammation despite effective viral suppression. This sustained inflammation has been linked to an elevated risk of developing a variety of age-associated comorbidities, including chronic kidney disease (CKD). Preliminary data supporting this study demonstrates multiple distinct inflammatory endotypes among aging PWH on ART, several defined by levels of chemokine C-C motif ligand 2 (CCL2), a critical mediator and biomarker of kidney injury and disease. However, the precise cellular and molecular inflammatory immune endotypes in PWH that could lead to disease remain undefined. Moreover, how endotypes defined by circulating inflammatory markers impact organ- compartmentalized inflammation and the functional and molecular states of immune cells are unknown. The overall objective of this project is to define the early cellular and molecular signatures of inflammation associated with the progressive development of CKD in PWH on ART in both blood and urine. This will be achieved by comprehensively defining plasma inflammatory endotypes in a retrospective cohort of aging (50+ years) PWH on ART, sampled as they progressed from early to later stage kidney disease, with comparison to PWH on ART with normal renal function. In addition, systems immunology will be used to characterize the soluble and cellular inflammatory profiles of peripheral blood and urine in a prospective cohort of 200 aging PWH on ART. Urine, a readily accessible non-invasive biofluid, contains proteins and viable cells originating from the kidney that can serve as indicators of renal inflammation and overall kidney function. A combination of advanced machine learning approaches, clinical tests, and human kidneys-on-chips models will be applied to define the relationship between systemic, urinary, and renal cell inflammation and dysfunction in PWH on ART that are associated with onset of CKD. This project will test the hypothesis that specific inflammatory immune endotypes can be identified in PWH on ART that promote the activation and dysregulation of immune and kidney cells, contributing to the development of CKD. The hypothesis will be tested, and the overall objective achieved, with completion of three Specific Aims. Aim 1 will define plasma inflammatory endotypes of aging PWH on ART and identify signatures that predict CKD. Aim 2 will identify how plasma inflammatory endotypes impact the cellular, metabolic, and functional programs in blood and urine of aging PWH on ART. Finally, Aim 3 will determine the mechanisms of activation of renal inflammatory programs using kidneys-on-chips. This research will identify specific endotypes of inflammation associated with development pf CKD and uncover pathways driving chronic inflammation in the blood and urine that promote kidney injury and disease. This knowledge will enable the development of CKD risk prediction tools and of therapeutic interventions like CCL2 signaling inhibitors to reduce inflammation-related kidney disease and other comorbidities in this expanding population.

NHLBI - National Heart Lung and Blood Institute

Project Abstract Pneumonia is a leading cause of infectious deaths worldwide; over 2 million people die of pneumonia each year. The leading bacterial cause of pneumonia is Streptococcus pneumoniae (Spn), an opportunistic pathogen that colonizes the human respiratory tract. While there are >100 known serotypes of Spn worldwide, current immunization strategies protect only against a limited few. Recent advances in our understanding of mucosal immunology have identified lung-resident CD4+ memory T (TRM) cells as critical determinants of broad protection against multiple serotypes of Spn. However, despite their clinical value from the public health perspective, little is known about mechanisms that drive the establishment of these CD4+ TRM cells in the lungs. Furthermore, it is unclear whether Spn may alter CD4+ TRM cell formation in the lungs using its own virulence factors. Understanding these mechanisms is instrumental for development of next generation cross-protective immunization strategies against this pathogen. Relevant to this, our preliminary data suggest that the Spn toxin pneumolysin (Ply) and bacterial sensing by NLRP3 are both key to recruitment and establishment of CD4+ TRM cell in the lungs. However, it remains unclear whether pore-forming activity or complement-activating biology of Ply is required for CD4+ TRM cell formation nor is it known how NLRP3 sensing of Spn may coax CD4+ TRM cell formation. In this proposal we will test the hypothesis that Spn drives CD4+ TRM cell formation via Ply’s pore forming activity and induction of macrophage-epithelial crosstalk via NLRP3. This hypothesis will be tested through two specific aims: Aim 1 will determine whether pore formation activity of Ply drives CD4+ TRM cell formation by boosting T cell recruitment, and Aim 2 will determine whether NLRP3 sensing of Spn is required for macrophage-epithelial crosstalk to drive CD4+ TRM cell formation. These studies will be accomplished by using isogenic Spn mutant strains, genetically engineered mice, intratracheal murine infection models, adoptive transfers, spectral flow cytometry, and single cell- and bulk-RNA sequencing. Findings from these innovative studies will guide development of more effective, broadly protective Spn immunization strategies that will prevent life-threatening Spn-pneumonia and subsequent diseases. This proposal will support the applicant with her scientific, technical, personal, professional, and career development which includes courses and workshops, guidance from a strong mentoring team and dissertation advisory committee, opportunities to develop science communication skills, and opportunities to mentor junior students in the lab and classroom. The University of Michigan offers top academic training, connections with esteemed faculty in pulmonology, microbiology, and immunology, and state-of-the-art resources to achieve the proposed aims. Completion of this proposal will also support the applicant’s rigorous training in experimental design, microbiology and immunology techniques, and data interpretation that will usher her towards becoming a successful, independent scientist.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Asthma is an inflammatory disorder of the respiratory tract triggered in response to inhalation of stimuli like allergens. It is the most common chronic disease in children worldwide, making it a major public health concern. A hallmark feature of asthma is the excessive eosinophil recruitment to the airways (referred to as allergic airway eosinophilia) which inflicts tissue damage, airway wall remodeling, and reduced gas exchange in the affected lungs. The hygiene hypothesis posits that exposure to commensal microbes may educate our immune system and direct it away from development of allergies and asthma. Relevant to this, our preliminary data also suggests that inhalation experience with the frequent human nasopharynx-colonizing bacteria Streptococcus pneumoniae (Spn) confers protection against allergic airway eosinophilia. How such inhaled microbial experience may mechanistically preclude allergic airway eosinophilia, however, remains unclear. In this proposal we will test the hypothesis that Spn experience protects against allergic airway eosinophilia by potentiating a Treg- and a cDC1- dependent immunoregulatory axis; to be tested as part of Aims 1 and 2 respectively. Aim 1 will determine whether inhaled Spn experience is protective due to the production of IL-10 by regulatory T cells (Tregs) formed during bacterial encounter, and Aim 2 will determine whether Spn confers protection through the activity of an enriched cDC1 pool during allergen encounter. These studies will be accomplished using novel genetically engineered mouse lines, human relevant murine models of pneumococcal experience and asthma, spectral flow cytometry, and single cell RNA sequencing. Findings from these innovative studies will delineate pathways that may be exploited as targets for next-generation preventative and treatment strategies against asthma. What is more, this proposal will also support the applicant in scientific, professional, technical, career, and personal development through guidance by an expert mentoring team and dissertation committee, use of state-of-the-art experimental techniques, enrollment in a tailored course curriculum, engagement in workshops and seminars, and engagement in an array of science communication and mentorship opportunities. The University of Michigan boasts elite academic training; connections and collaborations with experts in the fields of immunology, microbiology, pulmonology, and allergy; and cutting-edge facilities to achieve the proposed aims. Completion of this proposal will additionally support the applicant’s thorough and rigorous training in creative experimental design, microbiological and immunological techniques, and data interpretation and presentation that will ensure her future success as an independent researcher and principal investigator.

NIDCD - National Institute on Deafness and Other Communication Disorders

Abstract: Noise exposure causes permanent damage to the cochlea. A moderate level of noise causes temporary threshold shifts (TTS), synapse loss, and subsequent repair, whereas a severe level of noise causes permanent threshold shifts (PTS), and loss of hair cells and synapses. Noise also induces inflammation in the cochlea by increasing the macrophage populations and upregulating inflammatory genes among them. Broad ablation of macrophages prevents synapse repair after TTS, suggesting that they mediate this recovery mechanism. However, several knowledge gaps exist and prevent us from identifying precise therapeutic targets to treat noise-induced hearing loss: whether macrophages are heterogenous and whether subsets of macrophages serve different functions in the cochlea during homeostasis and post-damage are not known. To bridge these gaps, we have in preliminary experiments found and characterized 3 subtypes of macrophages with distinct transcriptomes and spatial distributions in the mature mouse cochlea. In this proposal, we have designed two independent aims to interrogate the roles of these macrophage subtypes in mice exposed to noise-induced TTS or PTS. In Aim 1, we will selectively ablate individual macrophage subtypes and determine whether this approach confers protection or increases susceptibility to TTS-induced synapse loss/recovery, spiral ganglion neuron loss, and wave 1 amplitude decrease. Moreover, we will assess the spatiotemporal changes of macrophages, spiral ganglion neurons, and other sensory and non-sensory cochlear cell types using single-cell RNA sequencing on the cochlea after noise exposure. In Aim 2, we will ablate macrophage subtypes and assess their effects on PTS-associated damage at both the histologic, physiologic, and transcriptomic levels. In summary, we will apply state-of-the-art technologies (single-cell RNA-sequencing, novel transgenic mouse models, bioinformatic strategies) to study the roles of macrophage subtypes in noise- induced TTS and PTS. We have assembled a team of experts experienced in macrophage and cochlear biology and physiology. At the end of this 5-year proposal, we will have 1) revealed whether ablating macrophage subtypes affects the cochlea during homeostasis and post-noise, 2) revealed the transcriptome of macrophage subtypes post-noise and identified putative therapeutic targets, 3) revealed the transcriptomes of noise-susceptible sensory and non-sensory cell types post-noise and after ablation of macrophage subtypes.

NEI - National Eye Institute

Project Summary: Neurodevelopmental disorders, including syndromic disorders of retinal and brain development, are a major cause of morbidity in children. A subset of these disorders results from abnormal vascular development, and microcephaly and chorioretinopathy (MCCRP) is a recently identified disease that may belong in this category. It is characterized by small head circumference, brain anomalies, developmental delay, and vision loss due to chorioretinopathy and abnormal retinal vasculature. Autosomal recessive MCCRP results from defects in TUBGCP4 or TUBGCP6, which encode components of the gamma-tubulin ring complex (γ-TuRC), a ubiquitous structure necessary for microtubule nucleation and spindle formation in cells. However, γ-TuRC has not previously been implicated in vascular development and it is unknown why defects in γ-TuRC lead to blindness and microcephaly. We demonstrated that Tubgcp4 and Tubgcp6 expression is highly upregulated in murine vascular endothelial cells (EC) from the retina and brain (relative to EC from other tissues), and that murine EC deficiency of TUBGCP4 results in embryonic lethality, indicating a critical role for TUBGCP4 in EC. Our long-term goal is to identify the role of the γ-TuRC in retinal and brain development. The objective of this application is to define the pathophysiology of TUBGCP4 and TUBGCP6-associated MCCRP. We will test the hypothesis that TUBGCP4 and TUBGCP6 serve critical, EC-specific roles in the retina and brain, and that deficiency of these proteins results in MCCRP due to a primary vascular developmental defect. Since retinal and cerebral vascular development is not complete until several weeks after birth, we will use novel conditional knockout mouse models of Tubgcp4 and Tubgcp6 and a tamoxifen-inducible EC-specific Cre recombinase to eliminate expression of these genes in EC postnatally. Ophthalmic studies will demonstrate the necessity of EC-specific TUBGCP4 and TUBGCP6 in retina and retinal vascular development, including optical coherence tomography (OCT), OCT-angiography, electroretinogram, optokinetic response, and retinal histology (Aim 1). Neurologic studies in mice and/or embryos lacking TUBGCP4 or TUBGCP6 in EC will demonstrate the necessity of these proteins in cerebral and neurovascular development, including MRI brain imaging, neurobehavioral studies, and brain immunohistochemistry (Aim 2). Elucidating the pathophysiology of MCCRP will improve our understanding of the genetic mechanisms controlling retinal and brain vascular development, and may reveal new therapeutic targets for more common blinding retinal vascular diseases. The career development objective of this proposal is to develop the mentorship and expertise needed to become a productive independent clinician-scientist and international leader working at the intersection of inherited retinal diseases (IRD) and disorders of vascular development. OHSU is a center of renowned expertise in IRDs, in vivo retinal vascular imaging, and the neurosciences; it provides state of the art resources and world-class faculty to support Dr. Everett’s scientific and career development goals for this proposal.

Delaware Community Foundation

Delaware Community Foundation ($350M in assets) supports nonprofits in DE through grants focused on education, community development, health, arts. Awards range from $2,000 to $100,000.