Boston Children'S Hospital

NIH Research Projects · FY 2024 · 2022-08

ClinGen CHD ECP Project Summary Abstract Congenital heart defects (CHD), defined as structural malformations of the heart and great vessels that are present at birth, are the commonest birth defects, affecting 2-3% of newborns when bicuspid aortic valve is included. While understood to be potentially a heritable trait as early as the mid-1800s, definitive epidemiological evidence supporting the notion that CHD was primarily genetic in its origins has only emerged in the last 35 years. To date, 253 genes contributing to CHD have been established but a far smaller number of genes have been designated as CHD-causing using formal ClinGen gene curation. Furthermore, and there are clinically relevant discrepancies for individual variants (i.e., pathogenic/likely pathogenic vs. benign/likely benign) in a substantial number of these genes. Sequencing-based clinical genetic testing using CHD gene panels facilitates diagnoses for which there are actionable co-morbidities that would often go otherwise undetected, particularly in young infants in whom syndromic features may not yet be evident but available commercial CHD genetic testing panels vary substantially in their gene content. The proposed CHD ECP will bring together experts in CHD genes from around the world which, in a well-organized, ClinGen-compliant manner, will curate gene-CHD pairs and classify their variants. The MPI's and many of the Expert Curation Panel members have extensive prior collaboration on a long-standing NIH-supported consortium (Pediatric Cardiac Genomic Consortium) that has and continues to elucidate the genetic architecture of the trait of interest (CHD) thus bringing significant gene and variant curation experience. An emphasis will be placed on developing experimental evidence criteria for a given CHD trait, drawing from a notably broad range of science (biochemical, cell-based, and animal models of heart development). This effort is well timed as the numbers of potential genes available for clinical genetic testing is rising rapidly and the clinical utility of such testing is well established. Further, candidate CHD genes with compelling human genetic evidence for pathogenicity but scant functional data can be shared with the cardiac developmental research community for consideration. The CHD ECP will fill an important gap in clinical care.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY Abdominal pain is a common symptom of digestive disease that is poorly addressed by existing therapies. Probiotics are widely used to treat abdominal pain even though most studies that have examined their effects have had disappointing results. A more effective strategy might be to stimulate specific pathways of microbes already present in the gut that benefit the host. The overarching goal of this proposal is to determine if a microbial pathway that reactivates steroids in the gut lumen normally regulates the activity of sensory neurons that mediate visceral sensation, and whether this pathway can be manipulated to influence abdominal pain. Abdominal pain is mediated by visceral afferents, primary sensory neurons located outside the gut that communicate information from the gut to the central nervous system. Prior studies suggest that commensal microbiota normally limit visceral afferent sensitivity. Depletion of commensal microbes causes exaggerated responses to colorectal distention, evidence that visceral afferents become hypersensitive to non-noxious stimuli in the absence of microbes. The full extent of microbial effects on visceral pain and the signals that mediate them, however, are largely unclear. Androgens, steroid hormones that circulate at higher levels in males than females, are compelling candidates. Androgens are anti-nociceptive in somatic pain and emerging evidence suggests they have similar effects in visceral pain. In irritable bowel syndrome (IBS), a disorder defined by chronic abdominal pain, we found that low androgen levels were associated with both diagnosis and symptom severity in males and females. Furthermore, androgen homeostasis has clear links to gut microbiota. Like other steroids, androgens are inactivated by glucuronidation in the liver and excreted into bile. In the gut lumen, these inactive forms become substrates for microbial β-glucuronidase enzymes (GUS) that remove the glucuronide moieties, regenerating a large pool of active androgens. Thus, androgen reactivation could be a key mechanism by which commensal microbes limit visceral hypersensitivity, linking previous observations. The central hypothesis of this proposal is that androgens reactivated by microbial GUS signal directly to host visceral afferent neurons to limit peripheral sensitization and pain. First, we will establish the independent effects of commensal microbes, microbial GUS activity, and androgen signaling to visceral afferent neurons on abdominal pain. Then, we will test for mechanistic links between each component. Incorporating genetic and gnotobiotic mouse models as well novel inhibitors of microbial GUS enzymes developed by the co-I, a leader in GUS chemistry, this innovative project moves the PI's research program into new directions of visceral pain and host-microbe interactions. The impact of this work will be to advance the understanding of visceral sensation and generate key evidence for new rational therapeutic targets in abdominal pain.

NIH Research Projects · FY 2024 · 2022-08

Project Summary This grant application is focused on capillary malformation (CM), a sporadic, non-hereditary vascular anomaly affecting 1/300 newborns. CMs are present at birth and may affect any area of skin. They grow darker and thicker over time. The lesions contain excessive, enlarged capillary-like vessels and cause soft-tissue and skeletal overgrowth. Patients suffer severe psychosocial morbidity from the appearance of the lesions and associated overgrowth can cause bleeding and functional disability. Sturge-Weber syndrome (SWS) affects 1 in 20,000 to 50,000 individuals and is characterized by a facial CM with extension to either the brain and/or eyes. Patients with SWS may develop neurological impairment, seizures, glaucoma, and blindness. CM is caused by a somatic activating mutation in GNAQ (p.R183Q) that is enriched in the endothelial cell (EC). GNAQ encodes Gαq, the α- subunit of the heterotrimeric Gq protein that activates phospholipase Cβ. The overactivation of Gαq leads to a strong increase in ANGPT2 expression. Drugs do not exist for CM and management consists of pulse-dye laser to lighten its color and surgical removal. Seizures in SWS are controlled by anti-epileptic drugs. Pharmacotherapy is desperately needed to prevent CM progression and recurrence following traditional treatments. Completion of these studies will be major steps towards this goal. Aim 1 will create a cell-based assay for EC dysfunction in CM/SWS using GFP knocked into the ANGPT2 locus as an easily detectable readout. We will use this cell system for high-throughput screening of FDA- approved drugs and bioactive compounds. This could lead to the identification of druggable pathways. Our understanding of how CM forms and grows, as well as our ability to test potential drug treatments, is hampered by the absence of a mouse model. Aim 2 will focus on creating CMs in mice. We have generated a mouse line in which we can activate expression of Gαq-R183Q in ECs using Cdh5CreER. To turn on Gαq-R183Q expression in a manner that produces CMs resembling the human condition we will use topical tamoxifen. We will test different doses of tamoxifen and time points (new-born to adult). We also will induce CM formation by injection of Adenovirus-Cdh5Cre into the limbs of prenatal and postnatal animals, as well as into the brain of ROSA-GT-GNAQ-R183Q animals to obtain a SWS phenotype. Creation of an animal model will enable future studies to test drug candidates from Aim 1 for their ability to stop the formation and growth of CMs. The most efficacious drugs will be translated to humans and undergo clinical trials.

NIH Research Projects · FY 2026 · 2022-07

Our goal is to understand the mechanisms of cell type- and stimulus-specific regulation of the human TNF gene and the TNF/LT locus genes (LTA and LTB) in T cells and monocytes/macrophages and to identify genomic regions that could potentially be targeted in TNF-driven disease states. Using unbiased next generation sequencing (NGS) approaches and CRISPR editing of human cells and mice, we will identify and elucidate function of transcriptional regulatory elements that modulate TNF, LTA, and LTB gene expression in T cells and monocytes/macrophages during activation and differentiation conditions and infectious challenges. Our preliminary studies using the NGS approaches of stranded RNA-, ATAC-, and HINT-seq reveal multiple novel highly conserved non-coding elements that transcribe eRNA in a cell type- specific manner in naïve T cells and in human monocytes/macrophages. They also show the cell type- specific hHS-8 enhancer that controls IFN-γ priming in monocytes/macrophages and enhances TNF and LTA in activated T cells we previously described. Our first goal will be to define the transcriptional territories and potential intrachromosomal interactions between the novel elements and hHS-8 with the TNF, LTA, and LTB genes. We will use ChIP-seq to determine the recruitment of the architectural protein CTCF, which mediates chromatin conformation, and the enrichment of H3K27Ac and H3K24Me, which are associated with enhancers. To select high potential regulatory areas this data will also be evaluated by a phylogenetic analysis of the TNF/LT locus in non-human primates to define highly conserved regions that predict regulatory function. These studies will guide our 3-dimensional analysis of locus architecture with Hi-C and CRISPR deletion of potential regulatory elements in cell lines and primary cells to establish their function. These studies then will provide a powerful framework and data set from which to interrogate these sites and new regulatory elements we will uncover in our analyses of (i) different states of human T cell and macrophage differentiation stimulated with TCR ligands or LPS and/or IFN-γ, respectively; (ii) the TNF/LT locus in primary T cells and BMDM from C57BL/6 and Balb/c mouse strains to evaluate concordance between the regulation of the murine and human TNF/LT loci as a baseline for performing studies in CRISPR-edited mice and testing the role of elements in acute (sepsis) and chronic (arthritis) TNF-mediated disease models. We will also characterize the role of distal elements that regulate TNF and the IL-6 gene expression, which shares regulatory similarities with TNF during infection with M. tuberculosis (MTb) or RNA viruses (Sendai and SARS-CoV-2), to elucidate broader gene expression programs. We anticipate that these studies will lead to a new understanding of how the TNF/LT genes are coordinately regulated, provide fundamental insights into gene regulation and the role of distal elements, and provide potential genomic targets to regulate TNF in a cell type-and inducer-specific manner in disease states.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY Somatic post-zygotic mutations are increasingly recognized as a cause of neurologic disorders ranging from epilepsy to autism to neurodegeneration. Somatic mutations accumulate with each cell division during fetal life, a developmental process of not yet fully defined scale and mechanism, hampering interpretation of disease states. The first part of this study implements a clinically applicable somatic-aware algorithm to identify early somatic mutations that lead to epilepsy and brain malformations. The latter aspect applies cutting-edge single- cell DNA technology to human fetal brain in order to define the rates and mechanisms driving accumulation of somatic mutations in neurons during normal development. The insights from this study have the potential to impact the detection and diagnosis of somatic disorders in clinical practice, and defines the scope of normal brain developmental mosaicism in neurons to serve as a framework for future studies of neurological disease. The candidate’s career goal is to become an independent physician-scientist contributing to the understanding of genetic and functional implications of post-zygotic mutations in childhood neurological disorders. The candidate trained clinically in child neurogenetics with deep research experience, including in cellular and molecular biology and statistical analysis, acquired in the cancer field. During the mentored training period, the candidate will prioritize activities to transition skills from a cancer background to human genetics and neuroscience: specifically working with primary human postmortem tissue, single cell analytical methods, and evaluating genotype-phenotype relationships in somatic disorders, and additionally, preparing for a transition to independence. The candidate will be mentored by Dr. Christopher Walsh, a renowned neurobiologist who has mentored dozens of successful independent investigators and will be supported by an advisory team with expertise in epilepsy, neuroscience, and bioinformatics. The proposed research and training plan will take place in the laboratory of Dr. Walsh at Boston Children’s Hospital (BCH), which is affiliated with Harvard Medical School and Howard Hughes Medical Institute, embedded within a world-class life sciences community of the Boston- Cambridge area. The candidate will benefit from both the outstanding resources and intellectual community of this tremendous network in addition to the close-knit communities within the BCH Department of Neurology and Division of Genetics.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY: Congenital heart disease is the most common congenital anomaly and affects approximately 1% of infants. Hypoplastic left heart syndrome (HLHS), a severe form of congenital heart disease in which the left ventricle is underdeveloped, has a 10-year mortality of 40%. Only 6% of HLHS patients have a genetic cause identified on exome sequencing, limiting the ability of patients to receive a diagnosis and potentially benefit from targeted treatments. There are two theoretical mechanisms for HLHS: a cardiomyocyte origin, where there a defect in cardiac muscle cells causes underdevelopment of the ventricle, or an endothelial origin, where value abnormalities attenuate flow through the left ventricle. Two known HLHS genes, RBFOX2 and NOTCH1, are primarily expressed in cardiomyocytes and cardiac endothelial cells, respectively and provide an opportunity to study these mechanisms. Discovery of additional pathogenic HLHS variants could increase the proportion of diagnosed patients and improve our molecular understanding of cardiac development. Currently, most pathogenic variants in exome sequencing are loss-of-function variants that reduce gene expression. To test my hypothesis that missense and noncoding variants also contribute to HLHS by altering gene expression or activity, I propose to use machine learning on HLHS patient genome sequencing, three-dimensional protein structure, and enhancer assay data to identify new genetic contributors to HLHS. By completing these aims, I will advance my training in functional assays and machine learning to be best prepared for a career as an independent physician scientist. My scientific goal is to identify new variants and loci that contribute to HLHS. First, in Aim 1 I will use machine learning to predict the pathogenicity of missense variants in RBFOX2 from HLHS patients. Accuracy of these predictions will be determined by genome editing of induced pluripotent stem cells to introduce the RBFOX2 missense variants, followed by assessment of RBFOX2 expression and function during cardiomyocyte differentiation. In Aim 2, NOTCH1 missense variants will be similarly assessed for pathogenicity during cardiac endothelial cell differentiation. Finally, in Aim 3 I will use massively parallel reporter assays to identify active cis-regulatory regions near RBFOX2- and NOTCH1-pathway genes, and then determine if rare variants in HLHS patients within these regions cause gene dysregulation. I will use linear models and machine learning to determine which cardiac genomic annotations that best predict enhancer activity, and use those annotations to identify additional candidate HLHS loci. Together this proposal will employ machine learning on biological data in a way that uses my background in developmental biology and develops new skills in computational and functional genomics. These results will contribute towards the long-term objective of understanding the molecular basis of heart development and human disease to improve diagnosis, better define risks, and inspire novel treatments for patients.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/ Abstract While substantial progress has been made to delineate the somatic mutations that drive myeloproliferative neoplasms (MPNs), epidemiologic studies demonstrate a significant heritable risk for disease acquisition. We have recently conducted the largest genome-wide association study of MPNs to define inherited risk variants that promote the acquisition of this disease. Our initial analyses suggest a role for modulation of HSC self- renewal and function by these inherited risk variants. However, we lack a complete mechanistic understanding of how the selective pressures that arise from these inherited variants can promote MPN acquisition. Here, in this R01, we specifically want to understand how inherited risk variants can provide selective pressures to enable MPN initiation. In the first aim of this grant, we seek to use rigorous mouse models to define how a variant- containing HSC-selective enhancer of Gfi1b can promote MPN acquisition. We will study how this enhancer plays a role in native hematopoiesis to promote MPN initiation, as well as how this enhancer can cooperate with somatic mutations in this process. In the second aim of this grant, we seek to understand how loss-of-function of CHEK2 can promote MPN acquisition and clonal hematopoiesis by coupling genome editing of primary human hematopoietic stem/ progenitor cells and whole genome sequencing to assess how such germline genetic mutations can promote oncogenesis. Finally, in the third aim, we seek to more holistically define how variant- harboring regulatory elements and target genes may play a role in HSC self-renewal and function through the use of a multiplexed guide-swap Cas9 genome editing approach in primary human hematopoietic stem and progenitor cells. Collectively, the studies we propose in this grant will provide new insights into how inherited risk variants can provide selection pressures that enable MPNs to arise.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Genome-wide screens using CRISPR-Cas9 technology have revolutionized studies of host-pathogen interactions, leading to identification of many key host cellular factors required for the actions of human pathogens and bacterial toxins. However, this powerful approach has yet to be utilized in insect cells to uncover host factors required for insect-borne pathogens, which are responsible for a long list of infectious diseases such as malaria, Dengue, West Nile, Zika, and Lyme diseases. This is largely due to a technical barrier: the inability to efficiently deliver genome- wide guide RNA library into insect cells. We recently overcame this barrier and developed a genome-wide CRISPR-Cas9 screening method in cultured Drosophila cells. Using this method, and by leveraging the expertise of Dr. Norbert Perrimon’s lab in insect models and of Dr. Min Dong’s lab in bacterial toxins, we carried out extensive preliminary studies, leading to the identification of a potential insect receptor for a member of the major bacterial toxin family known as Tc toxins, demonstrating the power and feasibility of our unbiased genome-wide screen approach. Building on these successes, in Aim 1 we will focus on further development and validation of genome-wide screens with major Tc toxin family members to establish a mechanistic understanding of toxin-receptor interactions and their role in pathogenesis in vitro and in vivo. We further propose to expand our approach to establish the first genome-wide CRISPR-Cas9 screening method and tools in mosquito cells in Aim2, and then utilize this approach to identify key host factors for two novel bacterial toxins that showed selective toxicity on mosquito but not Drosophila cells. The success of our proposal will uncover receptors and key host cellular factors for important bacterial toxins and establish generalizable methods and essential tools for investigating pathogens and toxins at genome-wide scale in insect cells relevant to transmitting human infectious diseases.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY This application for a K23 Mentored Patient-Oriented Research Career Development Award aims to support the career development of Dr. Hiu-fai Fong, a child abuse pediatrician at Boston Children’s Hospital and Harvard Medical School, in becoming an independent investigator whose work improves mental health care and outcomes for families affected by child abuse. Dr. Fong proposes to develop and pilot test a novel engagement intervention for Black and Latino caregivers of sexually abused children to address the significant prevalence of unmet mental health need for these children. Engagement interventions, in which child-serving professionals (e.g., social workers) work with caregivers to increase children’s receipt of mental health services, represent a promising approach to address unmet mental health need but have not been well studied with Black and Latino caregivers of sexually abused children. This K23 project seeks to develop a novel engagement intervention for this new population of caregivers that integrates components from two evidence-based interventions (McKay’s engagement intervention and the DECIDE intervention). The research will use principles of community based participatory research and a systematic process of cultural adaptation to develop the engagement intervention over three Aims. Aim 1 will use in-depth interviews with Black and Latino caregivers of sexually abused children to identify sociocultural perceptions about child sexual abuse and mental health care seeking. Aim 2 will use focus groups to elicit feedback from social workers that serve Black and Latino caregivers of sexually abused children and input from a community advisory board to culturally and contextually adapt intervention components. Aim 3 will pilot test the new engagement intervention for feasibility and acceptability with Black and Latino caregivers of sexually abused children. This research will lead to the development of a new engagement intervention ready for large-scale testing as a strategy to reduce racial and ethnic disparities in mental health care after sexual abuse. Dr. Fong has assembled a highly experienced mentorship team, including Dr. Margarita Alegria (intervention development for multicultural populations), Dr. Megan Bair-Merritt (trauma-focused research), and Dr. Michael Lindsey (treatment engagement for minority families) to guide her through a rigorous training plan to gain expertise in: 1) behavioral intervention development; 2) sociocultural and ethical issues in trauma-focused research; and 3) clinical trial methodology for behavioral interventions. Dr. Fong will leverage the unique resources available at Boston Children’s Hospital (a leading pediatric healthcare and research institution, and a specialty referral center for child sexual abuse) and the broader Harvard community (Harvard Medical School, Harvard T.H. Chan School of Public Health, Harvard Catalyst) to carry out her research and career development plan. The K23 award will prepare Dr. Fong to tackle the substantial challenges of disparities in mental health care for abused children and their families as a future independent researcher.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT The Society for Inherited Metabolic Disorders (SIMD) requests support to provide scholarships for trainees to attend its annual meetings in years 2023 to 2027. The 2022 meeting will be held in Orlando, FL April 10-13, 2022. The 2022 meeting will be supported by no-cost extension of the previous grant. The 2023 meeting will be held in Salt Lake City, UT March 18-21, 2023 in conjunction with the American College of Medical Genetics (ACMG) following their meeting. The 2024 meeting is TBD. The 2025 meeting will be held in conjunction with the International Congress on Inborn Errors of Metabolism in Kyoto, Japan. The 2026 meeting is TBD. The 2027 meeting is TBD. Inborn errors of metabolism (IEM) are an important cause of intellectual disability, cerebral palsy, neuromuscular disease, cardiac disorders, hepatic and renal dysfunction, arthritis, diabetes, growth failure and blindness. As the wide clinical and molecular spectrum of these disorders is being elucidated, in part fueled by the development of tandem mass spectroscopy and the rapid expansion of newborn screening programs, the number of affected individuals is now known to be much larger than originally recognized. In addition, therapies are available for some conditions, but few clinical trials have been performed to evaluate their efficacy. Thus, much remains to be done to better understand these severe disorders and develop effective treatments for them. For the U.S. to remain pre-eminent in this important area of research, it is essential to attract young investigators into the field. One effective mechanism to achieve this goal is to provide them with the opportunity to participate in the SIMD meeting, where they can explore the field and develop scientific ties to other established investigators. The SIMD meeting is held annually and participation, especially by young investigators, has been steadily increasing each year. The availability of NIH travel awards has been a major reason for this increase. Trainees seeking funding are required to submit an abstract describing original research to be presented at the meeting. We anticipate submission of 40 abstracts for presentation at each meeting from trainees/young investigators with twice that number for the international meeting (2025). Applications for travel awards will be competitively reviewed 4 months prior to each meeting, with the goal of making up 10 annual awards of $1,000 each for the national meeting and up to 5 awards of $2,000 for the International meeting. Additional funds will be solicited from private sources. All applicants will be actively recruited.

NIH Research Projects · FY 2026 · 2022-05

Program Abstract The goal of this training program is to provide the skill-set necessary for translational surgeon- scientists focused on pediatric diseases. Only 3% of T32 programs in the United States are focused on training surgeon-scientists, and only 1 surgical research training program in pediatrics currently exists in Urology. Pediatric surgical diseases are unique because the vast majority result from congenital anomalies. Consequently, pediatric surgical disciplines require specialized training to be best equipped for a translational research career focused on children. This program will provide a 2- year postdoctoral fellowship in translational basic research for individuals in a surgery residency who are planning on a career as a pediatric surgeon. A total of 3 fellows in the program at one time will receive broad training in the fields of vascular diseases and vascular biology. Vascular anomalies affect 5% of the population and involve every anatomical location and organ system. Consequently, all pediatric surgical specialists manage these disorders. Because vascular biology is applicable to almost all pediatric diseases, graduates will be able to apply the skills learned to a broad range of pediatric conditions. The training program consists of 8 pediatric surgical departments and programs. The 13 program faculty are experts in pediatric surgical diseases, vascular anomalies, and/or vascular biology and include surgeon-scientists and basic scientists. The postdoctoral fellows will be based in one of the faculty’s laboratories and will have independent mentored research projects (70% effort), didactic training in translational research methodology (15% effort), participation in conferences and clinics (10% effort), and guidance by a Mentorship Committee to facilitate their transition to independent research careers in pediatric surgery (5% effort). The postdoctoral fellows will be in an outstanding environment to succeed, supported by an internationally-recognized Pediatric Medical and Research Institution, Vascular Anomalies Center, Vascular Biology Program, Lymphedema Program, and Departments of Cardiac Surgery, General Surgery, Neurosurgery, Orthopedic Surgery, and Plastic Surgery. The program faculty have a long history of collaboration and a proven track-record of successful research funding and mentoring. This unique training program will produce the next generation of pediatric surgeon-scientists with the skill-set necessary to improve the lives of children through innovative translational research.

NIH Research Projects · FY 2026 · 2022-05

Project Summary/Abstract Converging lines of evidence support the hypothesis that deviations from typical brain structure development take place prior to psychosis onset, while ‘big data’ neuroimaging studies of adults with psychosis find subtle, widespread gray matter disruptions in the brain. In this proposal, we will synergize knowledge about normative structural neurodevelopment and findings of structural brain aberrations in adults with psychosis to develop cost-effective brain-based markers of psychosis risk in youth. To improve identification of those at greatest risk, we leverage results from large-scale structural neuroimaging studies of psychosis to create a ‘Psychosis Neuroimaging Score’, a cumulative summary score that reflects one’s psychosis liability. We first aim to transport the Psychosis Neuroimaging Score to youth by incorporating crucial aspects of structural brain development. In Aim 1, we will characterize the normative developmental trajectory of the Psychosis Neuroimaging Score by harmonizing many archival datasets of normative development (N>5,000, 2-30 years old). We will then evaluate how greater age-associated deviation from the aggregate Psychosis Neuroimaging Score differentiates youth with psychosis spectrum symptoms from typically developing youth in the Philadelphia Neurodevelopmental Cohort (N=1209, 10-22 years old). In Aim 2, we plan to examine how greater age-associated deviation from the aggregate Psychosis Neuroimaging Score predicts distinct developmental trajectories associated with psychotic-like experiences in youth from the Adolescent Brain and Cognitive Development Study (N=11,875). We will also assess the extent to which known psychosis risk factors (e.g., family history of psychosis, obstetric complications, trauma) contribute to characterization of these trajectories. Finally, in Aim 3, we propose to use measurement-in-error modeling to establish a functional relationship between Psychosis Neuroimaging scores generated from 3T MRI scans and those generated using low-field MRI scans in a community sample of youth. Results from this study will allow us to create more affordable, clinically accessible biological indicators of severe psychopathology, ultimately improving identification of young people at greatest risk and allowing earlier, more effective interventions.

NIH Research Projects · FY 2026 · 2022-05

Abstract Hypoxic Ischemic Encephalopathy (HIE) is a brain injury occurring in ~5/1000 newborns. In 2005, the NIH Neonatal Research Network (NRN) established therapeutic hypothermia (TH), cooling patients in the first 6 postnatal hours to 33-34°C for 72 hours, as the standard treatment for HIE in high-income countries. However, many patients still experience adverse outcomes (death or cognitive Bayley Scales of Infant Development <85) by 18-22 months. Thus, from 2008 to 2015, the NRN tested if deeper, longer, or later TH further reduced adverse outcomes, with two trials in 21 sites. Unfortunately, results were inconclusive and further progress has been slow, largely because adverse outcomes cannot be reliably assessed until 18-22 months. To expedite therapeutic innovations and assess the impact of novel therapies in a more timely manner, there is an urgent but unmet need to establish a neonatal biomarker of 18-22 month adverse outcomes. To address this gap, the NRN developed such a biomarker using neuroradiological expert scoring of brain injury on clinically acquired neonatal brain magnetic resonance images (MRIs), known as the NRN MRI score. In one dataset with one reader, sensitivity/specificity for adverse outcomes was 81%/78%. However, in another dataset with two readers, the inter-reader agreement was only moderate and specificity for adverse outcomes was only 56-69%. Questions arise for whether this subjective and time-consuming scoring system is reliable or fully characterizes complex HIE injury patterns. Also in many countries, there are no experts available to perform MRI scoring. Finally, important clinical data elements such as birth weight, sex, APGAR scores, socioeconomic status, and aspects of the clinical exam are not fully integrated into the scoring system. Our overall hypothesis is that Artificial Intelligence (AI) algorithms on neonatal brain MRI and clinical data elements can provide higher sensitivity and specificity than the expert NRN MRI scores in predicting adverse HIE outcomes by 18-22 months. Our R61 Aims are as follows: Aim 1, Compile a large HIE dataset (N=430) from two completed NRN multi-site HIE trials; Aim 2, Develop an AI biomarker of outcome using neonatal brain MRI, and compare with NRN scores with Aim 2a focusing on MRI injury patterns and Aim 2b focusing on whole brain MRI signal intensity patterns; and Aim 3, Develop an AI biomarker of outcome combining clinical and MRI data, and compare with NRN scores. Go/No- Go criteria for the R33 is if at least one biomarker (2a, 2b, or 3) outperforms NRN MRI scores in our N=430 cohort (p<0.05; DeLong Test of AUC). The R33 Aim 4 is to further evaluate accuracy and reliability in a new cohort (N=231). Deliverables: Publicly released data and the AI software. Impact: A brain MRI and clinical AI- powered neonatal prognostic biomarker could expedite therapeutic innovations in future HIE trials worldwide.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract Rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) are chronic autoimmune diseases of the joint punctuated by periodic arthritis flares. Clinicians have long recognized that each affected person develops an individual pattern of affected joints, and that this pattern remains stable over time through disease remission and flares. We recently identified the presence of synovial resident memory T cells (TRM) in arthritic joints and showed that they mediate arthritis flares. Correspondingly, depleting these cells ameliorates disease recurrence, indicating that TRM can be targeted as a novel approach in arthritis therapy. The long-term objective of the proposal is to define the mediators of TRM development and maintenance in the synovium and determine if these pathways can be therapeutically targeted to treat arthritis. The specific aims of this proposal utilize 2 complementary approaches in mice and human studies to identify the mediators of synovial TRM development. Aim 1 utilizes a mouse model of inflammatory arthritis developed by the PI to define the lineage and differentiation process of synovial TRM. Aim 2 utilizes a human synovial organoid system to interrogate the impact of the synovial microenvironment, namely synovial stromal cells, on TRM formation and survival. We expect that these studies will identify critical mediators of TRM development, which may represent novel therapeutic targets for inflammatory arthritis. The candidate is an M.D./Ph.D. pediatric rheumatologist at Boston Children’s Hospital. This proposal builds upon her foundational knowledge of immunology to extend her skillset into antibody-coupled single cell sequencing, bioinformatics, organoid models of human synovium, and CRISPR gene targeting. The proposal includes a comprehensive mentoring and didactic plan that will allow her to successfully learn new skills and gain expertise in each of these important areas. The primary mentor, Dr. Peter Nigrovic, is a rheumatologist and expert in the pathophysiology of inflammatory arthritis. The candidate has assembled a K08 advisory committee consisting of Dr. Michael Brenner, Dr. Rachael Clark, and Dr. Soumya Raychaudhuri, who each have specific expertise in various aspects of this proposal such as analysis of single-cell sequencing data, 3D models of human synovium, and expertise in TRM biology in human disease. The candidate is committed to a career in translational research with the goal of becoming an independent lab-based investigator focusing on local mechanisms to autoimmune disorders. The proposed studies, training plan, and exceptional environment at Boston Children’s Hospital, Brigham and Women’s Hospital and Harvard Medical School will enable her to successfully transition to an independent PI and leader in this field.

NIH Research Projects · FY 2025 · 2022-04

ABSTRACT Bacterial keratitis is a serious public health threat associated with significant ocular morbidity and is one of the major causes of blindness worldwide. By one estimate, the annual incidence of bacterial keratitis is approximately 500,000 patients worldwide. Even with modern day treatment, corneal infections can result in poor vision in 50% and surgical intervention in 12% of patients. Several Gram-positive and Gram-negative bacterial pathogens can infect the cornea and cause keratitis. Bacterial pathogens use all resources available to survive in the hostile host environment. Subversion of host extracellular matrix (ECM) components and their receptors as attachment sites is thought to be a common virulence mechanism shared by many bacteria. However, there are few data that clearly support this idea in vivo. We found in preliminary studies that deletion of syndecan-1 (Sdc1), a major cell surface heparan sulfate proteoglycan (HSPG) of epithelial cells, causes a gain of function in a mouse model of scarified corneal infection, where Sdc1-/- corneas are significantly less susceptible to Streptococcus pneumoniae infection. Topical administration of excess Sdc1 ectodomains or heparan sulfate (HS) significantly inhibits S. pneumoniae corneal infection, suggesting that HS chains of Sdc1 promote infection as a cell surface attachment receptor. However, S. pneumoniae does not interact with Sdc1 and Sdc1 is shed upon S. pneumoniae infection, indicating that Sdc1 does not directly support S. pneumoniae adhesion. Instead, Sdc1 promotes S. pneumoniae adhesion by driving the assembly of fibronectin (FN) fibrils in the corneal basement membrane to which S. pneumoniae attaches when infecting injured corneas. Excess Sdc1 ectodomains inhibit S. pneumoniae corneal infection by binding to the heparin-binding domain in FN, and interfering with S. pneumoniae binding to FN. Based on these data, this proposal will examine the overall hypothesis that specific ECM interactions coordinate the assembly of corneal basement membranes, and that certain bacterial pathogens of the ocular surface exploit these normal biological processes to promote their pathogenesis. This hypothesis will be tested in 3 Specific Aims. Aim 1 will define the structural basis of how HS inhibits bacterial corneal infection. Aim 2 will determine the significance and relevance of bacteria-induced Sdc1 shedding in corneal infection, and Aim 3 will elucidate the underlying mechanisms of how Sdc1 regulates FN fibrillogenesis in the corneal basement membrane. These studies are expected to uncover previously unknown functions of the ECM in the cornea and to establish a new integrated virulence pathway in bacterial keratitis.

NIH Research Projects · FY 2026 · 2022-04

Platelets are specialized anucleate cells that play an essential role in hemostasis, angiogenesis, immunity, and inflammation. Thrombocytopenia (platelet counts <150x109/L) is a major clinical problem encountered across a number of conditions including immune (idiopathic) thrombocytopenic purpura, myelodysplastic syndromes, chemotherapy, surgery, and genetic disorders. The demand for platelets—and for an improved understanding of their mechanistic formation—is at an all-time high. This program will use a multi-prong approach to investigate megakaryocytes (MKs) to discover therapeutic strategies and molecular targets that drive proplatelet formation and increase platelet counts. MKs are precursor cells that generate platelets by remodeling their cytoplasm into beaded proplatelet processes, which function as the assembly lines for platelet production. While we know that cytoskeletal mechanics power platelet production, many questions about platelet biogenesis remain unanswered. We know that microtubule-based forces are critical for proplatelet elongation; however, there is a surprising lack of understanding of the mechanisms that trigger platelet production. We hypothesize that centrosome regulation, via super spindle formation and KIFC1 motor involvement, is critical for the initiation of platelet production. We will use a novel high-content microscopy screen to identify the small molecules and signaling pathways that drive platelet production. Using proplatelet image analysis, we will test thousands of drug molecule candidates for their ability to stimulate or inhibit platelet production; target pathway analysis, secondary screens, and dose-response curves will be established to identify compound “hits.” While we know that proplatelet protrusions extend from bone marrow, breach the endothelial barrier, and deposit platelets into the blood, we do not know how. Therefore, we will employ bio-engineering and a unique microfluidic bone marrow on-a-chip to test the idea that actin-driven megakaryocyte podosomes provide a mechanism to penetrate the endothelium. This chip will also be used to study how organelles are transported into assembling platelets under physiological conditions, and to test the hypothesis that super spindle assembly functions as a major transport hub for distributing these organelles. We will determine if vascular thiol isomerases play a role in new platelet granule biology through investigating how they are packaged, transported, and exocytosed from platelets. We expect that findings from this investigation will 1) advance the understanding of the mechanisms that initiate and regulate platelet formation, and 2) identify novel therapeutic targets and approaches to accelerate platelet production in patients with thrombocytopenia. The R35 structure is necessary given the relative immaturity of the MK field and will provide vital time and focus to expanding the current base of knowledge. This proposal will coordinate a group of collaborators, provide the field with novel data and theory, and support junior scientists with consistent mentorship and proven leadership from a laboratory with broad ranging translational experience.

NIH Research Projects · FY 2025 · 2022-04

Abstract. Transforming growth factor β2 (TGF-β2) is critically important for heart and vascular development and repair. TGF-β2 dysregulation is seen in patient TGF-β2 mutations, systemic sclerosis, and Kawasaki disease, which have cardiovascular sequelae such as aortic aneurysms and cardiac fibrosis. TGF-β1, 2 and 3 are synthesized as proproteins that dimerize and associate with milieu molecules that regulate TGF-β tissue localization, such as the transmembrane protein glycoprotein A repetitions predominant (GARP) and latent TGF-β binding proteins (LTBPs) in the extracellular matrix (ECM). Proconvertases cleave between the prodomain and growth factor (GF) domain; however, the prodomain dimer remains non-covalently associated with the GF in a proTGF-β–milieu molecule complex after secretion. ProTGF-β–milieu molecule complexes are inactive because the prodomains encircle the GF and prevent binding to TGF-β receptors. ProTGF-β1 and 3 activation is mediated by binding of integrins αVβ6 and αVβ8 to an RGD-motif in the prodomain and requires proTGF-β association with a milieu molecule. How proTGF-β2, which lacks an RGD-motif, is activated remains a mystery. Aim 1 will define the structure of proTGF-β2 to understand its mechanism of latency. Aim 2 will determine proTGF-β2/milieu molecule complex structures by X-ray crystallography and cryo-EM to define how milieu molecules bind and alter TGF-β2 latency. We will generate antibodies to use as crystallization chaperones in addition to using already developed nanobodies to proTGF-β2. Complementary unfolding studies will test the hypothesis that milieu molecule binding stabilizes proTGF-β2. Aim 3 characterizes TGF-β2 activation. Follow-up studies will identify cell-lines that natively activate TGF-β2 and characterize the physiologically relevant process. The results of this grant will enhance our understanding of TGF-β2 latency and activation in extracellular milieus and lay the foundation for developing therapeutics that target proTGF-β2 and its physiologically relevant complexes with milieu molecules.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY/ABSTRACT Hospitals ineffectively examine the safety of their processes by relying on voluntary incident reporting (VIR) by clinical staff who are overworked and afraid to report. VIR captures only 1-10% of events, excludes patients and families, and underdetects events in vulnerable groups like patients with language barriers. Patients and families are vigilant partners in care who are adept at identifying errors and AEs. Failing to actively include pa- tients and families in safety reporting and instead relying on flawed VIR presents an important missed oppor- tunity to improve safety. To improve hospital safety, there is a critical need to coproduce (create in partnership with families) effective systems to identify uncaptured errors. Without this information, hospitals are impeded in their ability to improve patient safety. In partnership with diverse families, nurses, physicians, and hospital lead- ers, we created a multicomponent communication intervention to engage families of hospitalized children in safety reporting. Known as FACES (“Family Activation and Communication about Errors and Safety”), the in- tervention includes 3 elements: (1) a Spanish and English mobile (email, text, and QR-code) FACES reporting tool prompting families to share concerns and suggestions about safety, (2) family/staff education, and (3) a process for sharing family reports with the unit and hospital so systemic issues can be addressed. After pilot- ing FACES in one inpatient unit, we saw marked improvements in family safety reporting and reductions in dis- parities in reporting by parent education and language. We now propose to conduct an RCT of FACES in 4 geographically, ethnically, and linguistically diverse hospitals. Our specific aims are to: (1) evaluate the effec- tiveness of FACES in improving error detection and other safety outcomes, (2) assess the impact of FACES on disparities in reporting, and (3) understand contextual factors contributing to successful implementation of FACES. If effective, FACES will contribute by: (1) increasing patient/family engagement in reporting, espe- cially from vulnerable groups, (2) identifying otherwise unrecognized events, and (3) enabling hospitals to bet- ter understand safety problems in a 360-degree manner and design more effective, patient-centered solutions. This is significant because hospitals need to identify medical errors reliably in order to improve patient safety. The proposed research is innovative because it extends family safety reporting from the research to the oper- ational real-world context; compares patient/family safety reporting to flawed existing VIR; and is informed by principles of coproduction, communication science, health literacy, and organizational behavior. It also involves a novel strategy to share learnings, ensuring concerns are acted upon to improve patient safety in a manner that matters to patients and families. Finally, it focuses on the intersection between safety and equity. This study will achieve our long-term objective to coproduce with families evidence-based strategies to make hos- pital care safer, higher quality, and more equitable, in line with AHRQ's mission. It intersects multiple priorities highlighted by AHRQ, including children (a priority population), safety, and equity (SEN NOT-HS-21-014).

NIH Research Projects · FY 2025 · 2022-04

Project Summary/Abstract Acquired severe aplastic anemia (SAA) is a rare bone marrow failure disorder with an annual incidence of 3 per million in North America (>300 cases < age 25 in the US yearly). The disease can be treated and often cured by either immune suppression therapy (IST) or hematopoietic stem cell transplantation (HSCT), with the recommended approach in SAA being early matched sibling donor bone marrow transplantation (BMT). However, only 20% of patients have sibling donors, consequently, the large majority of patients receive IST for initial therapy. From initiation of IST it takes 2-6 months to see hematologic improvement, with responses occurring 70-80% of the time in children. Unfortunately, 20-30% of patients eventually relapse, requiring additional immune suppression, and some become cyclosporin-dependent. The results of matched unrelated donor (URD) BMT for SAA has improved significantly over the past decade, with studies reporting similar outcomes for BMT using URD compared to MSD. Although these data are provocative, URD BMT carries significant risks, and most consensus opinions still conclude that IST should be considered standard of care when a matched sibling donor is not available, until a definitive study shows otherwise. To address this challenge, the North American Pediatric Aplastic Anemia Consortium (NAPAAC), in collaboration with the Pediatric Transplantation and Cellular Therapy Consortium (PTCTC), conducted an NHLBI R34-funded pilot trial to determine feasibility and safety of randomizing between IST and URD BMT. Our recently published results of the first 23 patients showed high rates of acceptance of randomization, receipt of randomized therapy without significant adverse events, and rapid institution of definitive therapy (IST or BMT) (Pulsipher et al., Pediatric Blood and Cancer, 2020). Having demonstrated feasibility, we submit this application to support a paradigm-changing randomized trial in partnership with the Center for International Blood and Marrow Transplant Research (CIBMTR). The study proposes a multi-center phase III trial to compare the percentage of newly diagnosed SAA patients with immune suppression-free survival with adequate counts (ISFS-AC) at 2-years between those randomized to IST vs 9-10/10 HLA matched URD BMT. The study will also address patient-reported outcomes and fertility preservation in each arm and explore critical biological correlates including assessing germline genetic mutations associated with pediatric SAA that may affect response to BMT or IST and the development of clonal hematopoiesis following IST vs BMT in pediatric SAA. The study proposed would represent the largest randomized study in pediatric SAA ever attempted with the goal of providing practice-altering conclusions to the field.

NIH Research Projects · FY 2025 · 2022-04

ABSTRACT The central goal of this R01 proposal is to understand how molecular and cellular interactions of heparan sulfate proteoglycans (HSPGs) modulate the pathogenesis of acetaminophen (APAP)-induced liver injury (AILI). Accidental or intentional misuse of APAP is the leading cause of acute liver failure in the Western world. While mechanisms that trigger AILI are well known, those that facilitate liver recovery are less understood. HSPGs bind and regulate various tissue injury factors through their heparan sulfate (HS) chains, but the significance and mechanisms of HSPGs in tissue injury and repair in vivo remain largely unknown. We examined the role of syndecan-1 (Sdc1), the major cell surface HSPG of hepatocytes, in AILI. Deletion of Sdc1 in mice led to unopposed progression of liver injury in APAP liver disease. However, direct APAP hepatoxicity at early times after APAP overdose was unaffected by Sdc1 deletion, suggesting that Sdc1 regulates later mechanisms that affect the progression and outcome of APAP liver disease. The exuberant AILI phenotypes of Sdc1 null (Sdc1-/-) mice were traced to an exaggerated innate immune response in the liver and a deficiency in pro-survival Akt signaling in hepatocytes and hepatocyte proliferation, which led to amplification of liver damage. Administration of purified Sdc1 or heparan compounds containing 2-O-sulfate motifs rescued Sdc1-/- mice from AILI by inhibiting innate immune responses, and by potentiating hepatocyte proliferation and liver repair. Furthermore, HS showed a significantly prolonged therapeutic efficacy as compared to N-acetylcysteine (NAC), the clinical antidote for APAP overdose. These findings suggest that Sdc1 and HS, either alone or in combination with NAC, could provide a new therapeutic strategy to combat AILI, especially in treating patients admitted after NAC treatment is no longer effective. Based on these preliminary data, we propose that Sdc1 is a critical endogenous factor that halts the perpetuation of liver injury and facilitates liver repair in AILI. This hypothesis will be tested in 3 specific aims. Aim 1 will define how Sdc1 is released from hepatocytes during AILI and establish that discrete structural motifs in Sdc1 HS provide protection against AILI. Aim 2 will elucidate the biological mechanisms of how Sdc1 halts the progression of AILI. Aim 3 will determine how Sdc1 enhances hepatocyte proliferation and facilitates liver repair in AILI. These studies are expected to establish a new integrated pathway in liver injury and repair.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy and is the major form of pancreatic cancer. With an incidence on the rise, it currently ranks as the third leading cause of cancer death in the US. Despite recent advances in the understanding of its biology, genetics and risk factors, PDAC has maintained extremely poor prognosis, with a 5-year survival rate of only 10%. This mainly stems from its late diagnosis, aggressive nature and resistance to therapies. Thus, novel therapeutic approaches are urgently needed. Targeting altered metabolism in PDAC has been an area of extensive investigation for over a decade now. A major hurdle however, for most anti-tumor metabolic strategies, is the high risk of toxic side effects, given the essential roles of metabolic pathways in the maintenance of normal tissue homeostasis. This has indeed been the case for targeting polyamines in cancers. Polyamines are small, highly positively charged molecules involved in multiple fundamental processes of cell growth and survival, including the synthesis of nucleic acids, modifications of chromatin structure, gene transcription and mRNA translation. Polyamine levels are significantly increased in many cancers, including PDAC. Prior anti-tumor strategies focused on pharmacological inhibition of the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC1) with little success, partially due to risk of harming normal tissues at higher drug doses. This project identifies and aims at validating a dependency of pancreatic cancer, both in cultured cells in vitro and in mice in vivo, on an unconventional way for the synthesis of the polyamine precursor ornithine, specifically from glutamine via ornithine aminotransferase (OAT); this is compared to its synthesis in most adult normal tissues from arginine via arginase (ARG) activity. It also aims at identifying potential key players mediating the induction of this metabolic pathway by KRAS, the main oncogenic driver in PDAC, and to characterize the downstream effects of polyamines on transcriptional activation and gene expression in PDAC cells compared to normal pancreatic cells. The high dependency of PDAC, but not normal tissues on de novo ornithine synthesis from glutamine provides an attractive therapeutic window for treating pancreatic cancer patients with minimal toxicity.

NIH Research Projects · FY 2026 · 2022-03

One in 26 people experience epilepsy at some point in their life and temporal lobe epilepsy is the most common adult focal epilepsy. New and innovative therapies are required to treat the many cases of temporal lobe epilepsy that are poorly controlled with conventional medications. Ideally, treatments should be based on a detailed understanding of the mechanisms that give rise to seizures, but these biological mechanisms are poorly understood. The conventional approach is to surgically remove the seizure “focus” in attempts to alleviate seizures, but identifying the focus is challenging. Even in cases when the focus is ostensibly clear, surgery may not prevent seizures. An alternative hypothesis is that even classically focal epilepsies, such as temporal lobe epilepsy, rely on more distributed brain networks. One such network that is well-positioned to support seizures is the Papez circuit, which embeds the hippocampus, a classic seizure focus, in a recurrent excitatory network. Preliminary work demonstrates that the medial mammillary body, the hypothalamic node of this circuit, drives synchronous network events in the hippocampus and highlights a potential role as an external controller for pathologically synchronous states (i.e., epileptiform events). Using transgenic mouse lines and cell-type-specific tools, two divergent pathways from the medial mammillary body will be investigated to determine 1) how seizure activity spreads through these pathways, 2) whether these pathways are necessary and sufficient for seizure activity, and 3) if targeted, non-invasive neuromodulation with ultrasound can control seizure activity. Completing this grant will advance our understanding of the mechanisms that generate seizure activity and take a first step towards translation using a clinically relevant treatment modality. The candidate's long-term goal is to establish an independent research program that sheds light on the organization and function of networks that support physiological processes (e.g., memory) and pathological seizures. To attain this goal, the candidate has outlined a comprehensive, personalized program that identifies plans for career development in research training, neuroscience knowledge, technique development, grantsmanship, scientific management, and others. This training plan outlines a pathway to independence (i.e., from postdoc to establishing their own laboratory) that has a realistic timeline and is supported excellent resources at Stanford University, collaboration, and an established mentor with extensive experience relevant to this goal.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY To date, efforts to define and apply precision endotyping has been limited to studies of adults. However, immune development in early life (IDEAL) is dynamic and varies between individuals suggesting that endotypes corresponding to distinct pathophysiological mechanisms will be age-dependent. We propose therefore a novel approach in which we will study well-defined longitudinal childhood cohorts and use in silico integrative analyses of existing and prospectively collected data coupled with age-specific human in vitro model systems to identify agents that redirect IDEAL away from disease endotypes towards those associated with health. We have selected three clinical endpoints to correlate with systems biology data to identify IDEAL endotypes: a) vaccine responsiveness, as vaccines are the most important biomedical intervention to reduce childhood disease; b) respiratory infection which represents the greatest burden of childhood infectious disease; and c) asthma, an immune-mediated respiratory disease which manifests in childhood and results in substantial health burden. Each of these endpoints demonstrates substantial inter-individual variability enabling powerful systems biology tools to extract meaningful correlations. We will harmonize and study an IDEAL Meta-Cohort (IMC) comprised of longitudinal childhood cohorts enrolled in North America, Africa and Australasia. Our Clinical Core in Rochester, NY, is nationally prominent in the study of childhood immune ontogeny. Project (PR) 1 will employ cutting edge, cross- platform integrative bioinformatics tools to identify endotypes associated with clinical endpoints. PR2, will apply epigenetic analysis tools to the same samples and translate to host immune parameters the in silico-derived signatures. In PR3, key endotype-associated biomarkers and pathways will be dissected in vitro to establish cause and effect and identify agents (e.g., proteins, metabolites, adjuvants, vaccines) that may redirect IDEAL away from unfavorable endotypes and towards favorable ones. We have optimized sample-sparing assays to enable systems biology in infants and our published preliminary data demonstrate feasibility, robust IDEAL, and suggest distinct signatures by clinical status. Our cross- platform validation and correlation with endotypes correlating with clinical phenotypes will identify predictive/actionable biomarkers by i) characterizing IDEAL and microbiome in systemic/mucosal compartments (Overall Aim 1), ii) identifying endotype-specific biomarkers (Overall Aim 2), identifying in vitro interventions that re-direct IDEAL endotypes towards health (Overall Aim 3). Overall, we will enhance and accelerate discovery of new approaches to predict and prevent childhood disease.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Malaria is an important cause of illness and death worldwide, with most of these deaths resulting from Plasmodium falciparum infection. Successful completion of the P. falciparum life cycle and infection of a new human host requires transmission. During the asexual blood stage in human red blood cells, a small population of parasites differentiates into transmission forms known as gametocytes. These gametocytes can complete the sexual stage of the parasite life cycle following ingestion by a mosquito. Gametocyte maturation in human red blood cells occurs over 10-12 days and is associated with major changes in cellular morphology and rigidity. A newly discovered protein PfBLEB (for Baso-Lateral Expansion Boundary) is essential for mature gametocyte formation. In asexual parasites, PfBLEB is part of the basal complex, a ring-like multi-protein complex at the leading edge of the inner membrane complex. While PfBLEB is dispensable for both asexual replication and gametocyte commitment, it is essential for gametocyte development. PfBLEB-knockdown or knockout gametocytes arrest during maturation and are non- transmissible. Furthermore, the PfBLEB-deficient gametocytes have gross morphologic changes with defects in major cytoskeletal features of the maturing transmission-stage parasite, including the inner membrane complex and subpellicular microtubules. In gametocytes with normal PfBLEB expression, PfBLEB defines a new subcellular compartment within the parasite, demarcating the regions of the parasite plasma membrane that are devoid of the underlying inner membrane complex. The PfBLEB-compartment is essential for gametocyte development, but the function and protein constituents of this newly discovered subcellular compartment remain unknown. The goal of the current application is to define and genetically evaluate the protein components of the PfBLEB compartment and to understand the functional defects in PfBLEB-deficient gametocytes. The first aim will utilize proximity labeling and reverse genetics to explore the PfBLEB-containing compartment. The second aim will utilize multiple imaging and microfabrication techniques to gain a functional understanding of what processes are abnormal in PfBLEB-deficient gametocytes. Together, the proposed studies will add a new layer to our molecular understanding of gametocyte development in P. falciparum.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Kabuki Syndrome (KS) is a rare disease caused by heterozygous pathogenic mutations in two known genes: KDM6A (~20% cases) and KMT2D (~80% cases). Both genes are broadly expressed in many tissues and their activity spans temporally from development to postnatal adult life. KS patients present with various degrees of clinical abnormalities, including severe muscular hypotonia and reduced muscle strength. Whether hypotonia develops as a consequence of nerve conduction malfunction or it is due to a cell-autonomous primary defect in skeletal muscle is currently unknown. Further, skeletal muscle tissue from patients affected by KS has not been thoroughly studied. Our main hypothesis is that skeletal muscle tissue is primarily affected by mutations in KMT2D, which results in dysregulated muscle function. We propose to validate our hypothesis via the following specific Aims: 1) Define primary versus secondary muscle function defects using constitutive and conditional mouse models of KS; 2) Determine the gene networks and molecular targets of KMT2D driving muscle hypotonia in constitutive and conditional KS mouse models; 3) Define muscle satellite cell heterogeneity and `immaturity' in conditional and constitutive KS models, as well as in human patients. The work proposed will fill major gaps in our lack of knowledge about etiology of hypotonia in Kabuki syndrome and will pave the way for clinical improvements of patient care.