Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2022-02

An efficient hemodynamics with minimal thrombosis risk post-surgery is essential for short- and long-term success of a cardiac surgery. Achieving this is a challenge in cardiac surgery that involves the design of a complex flow pathway. Aortic arch reconstruction, aneurysm repair, Fontan surgeries are a few examples. The Fontan surgical procedure is the most effective palliative treatment for patients with single ventricle defects (SVD). SVD refers to a collection of congenital heart diseases where one of the lower ventricular chambers of the heart remains underdeveloped. Fontan procedure involves re-routing of deoxygenated blood from upper and lower body to flow directly to lungs allowing the single functioning ventricle to pump blood for systemic circulation. Though lifesaving, the Fontan physiology creates a non-natural pathway for venous return of the blood to the lungs thus producing a non-physiological blood flow. A successful Fontan procedure should involve 1) well-balanced overall and hepatic venous flow return to lungs to prevent pulmonary arteriovenous malformations (PAVMs) that can lead to poor gas exchange, 2) minimal energy loss, and 3) minimal thrombosis (blood clot) risk. Complications such as PAVMs and thrombosis post-surgery can result in a Fontan failure. To improve Fontan surgical planning, its efficacy and predictability, we propose to develop an automated image-based computational fluid dynamics (CFD) workflow capable of optimizing and predicting all the above determinants for a successful Fontan physiology. CFD models have been developed in the past to assess energy loss and hepatic venous flow distribution, but an automated computational tool for rapidly optimizing the patients' Fontan physiology in terms of factors affecting success does not exist. To fill this gap, we will integrate our existing patient-specific Fontan surgical planning protocol to predict energy loss and hepatic venous flow distribution with 1) a shape optimization algorithm and 2) our validated model of blood coagulation to provide a computational tool to virtually improve the planned Fontan physiology for optimal hepatic and overall venous return to lungs, minimal energy loss and thrombotic potential and quantitatively predict thrombosis risk. We will completely automate our workflow with custom scripts to minimize errors and user intervention. Our biochemical model of blood coagulation has all the components representing platelet and fibrin deposition and is 2-way coupled with blood flow. The continuum-based approach of this model allows it to be used in large geometries. After rigorous validation of our surgical optimization workflow using MRI-based patient specific in-vitro models, we will perform virtual surgeries using our tool and retrospective patient data to establish clinical applicability. Our tool could potentially be 1) included in the current surgical planning workflow to perform virtual surgeries using patient pre-op data to improve and predict surgical outcomes, and 2) used to evaluate risk of clotting post- Fontan so that patients can be selectively monitored. Our long-term objective is to provide a prospective surgical planning tool for Fontan and then extend it other surgeries where such optimization can improve surgical efficacy.

NIH Research Projects · FY 2025 · 2022-02

PROJECT SUMMARY/ ABSTRACT Our premise is that the fetal stage of human brain development is the most dynamic, the most vulnerable and the most important for lifelong behavioral and cognitive function. As many neurological disorders have their genesis in fetal life, there is a need to accurately quantify normal and abnormal fetal brain development from both the perspective of fetal brain structure and body motion. Better imaging tools would enable us to explore how fetal neurological disorders as well as environmental exposures, such as opioids, maternal obesity, or COVID-19, impact early brain structure and body movements. Magnetic resonance imaging (MRI) T2-weighted, single-shot fast-spin-echo (e.g. HASTE) images provide a unique window into this critical phase of structural brain development, with the potential to detect subtle abnormalities. However, fetal brain MRI is challenging due to fetal motion, which leads to image artifacts, double oblique acquisitions and incomplete brain coverage. As a result, trained MR technologists must “chase the fetus” to amass the necessary images to diagnose the presence or absence of lesions, resulting in long scan times and higher RF energy deposition. Thus, fetal brain MRI is inefficient, limited to specialized centers, and diagnosis is still difficult because fetal motion results in each image being an independent slice that cannot be referenced to another slice, making confirmation of suspicious findings difficult. At the same time, fetal motion is an important measure of functional neurological integrity, informing postnatal outcomes. However, current clinical MR and ultrasound assessments of fetal motion do not fully capture the complex 3D motions of all body parts simultaneously. Better assessment of fetal neurological health requires novel tools to automatically and efficiently obtain coherent, high quality HASTE fetal brain volumes and to characterize 3D fetal whole-body motion. To address these unmet needs, we will leverage convolutional neural network (CNN) models and propose the following aims: (1) Develop a self-driving engine for efficient acquisition of high-quality HASTE fetal brain volumes and (2) Enable automated fetal whole-body motion tracking and characterization. We will deploy the proposed tools in a prospective study that compares fetuses with Chiari II malformation (spina bifida), a disorder known to have brain abnormalities and often associated with decreased leg movement, to typical fetuses with the following aim: (3) Assess performance of the self-driving HASTE engine and whole-body motion characterization in Chiari II vs typical fetuses. For Aims 1 and 2, we will include data from collaborating sites and strategies for CNN generalization to increase robustness and potential to deploy our tools to other scanners. The ability to automatically obtain high-quality coherent fetal brain volumes and characterize fetal motion will improve stratification for fetal treatments and characterization of response to fetal interventions. Success will also enable sites without fetal imaging experts to locally assess and triage fetuses with suspected abnormalities to specialized treatment centers, as well as facilitate large population-based studies to understand the impact of environmental influences on early brain development and fetal behavior.

NIH Research Projects · FY 2026 · 2022-01

Abstract Apical extracellular matrix (aECM) coats the outward-facing surface of every organ, forming a barrier between the organism and its environment. Although it has been viewed historically as a static layer, aECM has been recently revealed to be dynamic across development and highly varied between cell types. It plays important roles in shaping organ morphogenesis and modulating cell activity. Identifying the regulatory mechanisms that control aECM composition and structure is therefore critical to understanding its role in development. Further, because aECM is highly accessible, there is enormous potential to manipulate it for targeted delivery of therapeutics or in tissue engineering. The major obstacles to studying aECM remodeling are that changes in aECM are difficult to visualize and need to be studied in vivo during highly dynamic processes that involve complex cell rearrangements. This project overcomes these obstacles by using an innovative model of developmentally programmed aECM remodeling. Preliminary data lead to the hypothesis that aECM structure is a discrete modular feature of cell identity, analogous to neurotransmitter types in neurons, rather than a continuum of stiffness/density. The C. elegans cuticle is an aECM that forms barrier between the animal and its environment. In order to directly access the external environment, the ciliated endings of some sensory neurons protrude through nanoscale pores in the cuticle, while those of other sensory neurons are embedded in specialized sheets of cuticle. Both types of cuticle structure are produced by glial cells that wrap the sensory neuron endings. One of these glial cells offers a remarkable example of developmentally programmed aECM remodeling: in juveniles of both sexes and in adult hermaphrodites it produces a cuticle sheet, but in adult males it produces a cuticle pore. This represents a discrete aECM remodeling event that occurs at a defined developmental stage without any major cell rearrangements. Preliminary data show that this aECM remodeling event is accompanied by a switch in gene expression in the glial cell. This includes expression of GRL-18, a novel class of aECM component that forms nanoscale rings in the cuticle. These observations leads to the hypothesis that a developmentally regulated switch in gene expression induces remodeling of aECM to form a nanoscale pore. To test this hypothesis, this project will (Aim 1) use mutant analysis and transcriptional profiling to define the gene expression switch that accompanies aECM remodeling; (Aim 2) determine how a novel developmentally regulated protein (GRL-18) contributes to aECM structure; and (Aim 3) test if changes in expression of GRL-18 and co-regulated genes are sufficient to remodel aECM into a nanoscale pore.

NIH Research Projects · FY 2026 · 2022-01

Strabismus can be both visually and socially debilitating and its underlying pathophysiological mechanisms remain poorly understood. Current treatments often do not restore full visual function and do not address the underlying pathology. Strabismus has a clear hereditary component, but precise genetic mechanisms have not been defined. We recently identified three rare, recurrent genetic duplications that increase risk of esotropia. Each of these duplications includes a long non-coding RNA (lncRNA), which are often involved in chromatin remodeling and regulation of gene expression. Duplications can also affect gene expression by insertion of regulatory elements in new locations or disruption of the 3D chromatin structure. We therefore hypothesize that regulation of gene expression is an important mechanism underlying strabismus. This is bolstered by the findings that known environmental risk factors for strabismus, including prematurity, maternal smoking, and low birth weight, affect epigenetic regulation through changes in methylation. This proposal aims to (1) define the consequences of these duplications on gene expression, chromatin structure, and neuronal morphology and function, (2) evaluate esotropic and exotropic patients for single nucleotide variants (SNVs) or small insertions or deletions (indels) in the genes and regulatory regions included in the duplications or affected by the duplications, and (3) identify additional genetic causes of strabismus through whole genome sequencing of large strabismus families. The precise breakpoints and insertion points of the duplications will be determined by long-read whole genome sequencing, then each duplication will be introduced into induced pluripotent stem cells (iPSCs) through CRISMERE (a variant of CRISPR/cas9). Gene expression, enhancer activity, and chromatin conformation will be compared between stem cells, neuroprogenitors, and differentiated neurons with and without each duplication. The effects of each duplication on neuronal morphology and function will be assessed. Fluidigm multiplexing and next-generation sequencing will allow cost-effective screening of our large strabismus cohort for SNVs and indels in the coding and regulatory regions of the genes included in the duplications, as well as genes whose expression is altered by the duplications. Variants identified in multiple individuals and predicted to be damaging bioinformatically will be evaluated with in vitro functional studies. Additional families with multiple members with strabismus will be enrolled, and coding, non-coding, and structural variants will be identified through whole genome sequencing. Variants will be prioritized based on linkage, bioinformatic predictions, and population frequency. In addition, the epigenetic and 3D interactome maps from neuroprogenitors and neurons will be used to prioritize variants. Functional studies will be done on high priority identified variants. This work, by identifying genes and signaling pathways that contribute to development of strabismus, will provide insights into strabismus pathogenesis, which will allow development of new strabismus treatments or preventative interventions based on the underlying pathophysiology.

NIH Research Projects · FY 2026 · 2022-01

Project Summary In pediatric epilepsy patients with drug-resistant seizures, surgical resection is the most effective treatment option. The goal of resective surgery is to maximize removal of epileptic foci to attain seizure-freedom while minimizing damage to surrounding brain regions to avoid permanent post-surgical functional loss. Diffusion MRI enables rapid and non-invasive pre-surgical mapping of language, motor skills and other critical functional brain regions with high spatial resolution. However, excessive head motion presents a major limitation for acquiring high-quality diffusion MRI in pediatric patients with focal brain lesions, who usually have difficulty remaining still for long scan durations. Unfortunately, current retrospective and prospective approaches cannot adequately compensate for the complex effects of motion in diffusion MRI. As echo planar imaging (EPI) is highly susceptible to local magnetic field variations, motion-induced geometric distortions can lead to potentially significant mislocalization of important brain regions, even with accurate head motion tracking. The overarching goal of the research proposed under this application to the NIH is to dramatically improve the quality of diffusion MRI for pre-surgical mapping in pediatric epilepsy patients. We are proposing a solution based on a dual-echo EPI sequence, which was shown to produce high quality slice level distortion maps that can be used to correct motion related artifacts. We will generate a pipeline that produces motion and distortion free images on the scanner with the utilization of an online reacquisition and distortion correction strategy. We hypothesize that this improved diffusion MRI acquisition strategy will produce technically useful tractography in pediatric epilepsy patients evaluated for a resection surgery at a higher rate than previously thought possible. To achieve these ambitious goals, we will undertake the following specific aims: Specific Aim 1: Develop, optimize and evaluate a dual echo sequence for slice level geometric distortions correction; Specific Aim 2: Develop and evaluate a novel prospective motion correction technology that estimates and corrects geometric distortions at each position; Specific Aim 3: Develop and evaluate tools for on- scanner motion and distortion correction, reacquisition and diffusion parameter estimation; Specific Aim 4: Apply and evaluate motion and distortion compensation technologies in DW-MRI of pediatric candidates for epilepsy surgery: If successful, our project will facilitate widespread clinical adaptation of diffusion MRI for pre-surgical mapping in epilepsy, and enable high resolution diffusion MRI for research studies in incompliant patient populations.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY Attention is comprised of several component processes, including sustained attention, selective attention, and attentional flexibility. The first phase, the initial focusing of attention, precludes engagement of other components making it an important building block of many of our more complex behaviors. Additionally, attention impairments often present as a comorbidity in conditions including but not limited to schizophrenia, autism, depression, and epilepsy, making identifying therapeutic targets a significant public health concern. Convergent data point to the medial prefrontal cortex (mPFC) as a hub for supporting attentional shifting along with other key structures, while its role in the initial engagement of attention is less clear. Somehow, mPFC pyramidal neurons integrate information from multiple sources, represent task rules, cue, and response-related information in dynamically active ensembles, and select appropriate behavioral responses. Previous physiological data suggests this astounding computational feat is made possible due to the powerful regulation of pyramidal neuron activity by cortical inhibitory interneurons, but many studies lacked the ability to monitor distinct cell-classes with specificity. Using fiber photometry of GCaMP-mediated Ca2+ signals in mPFC parvalbumin-expressing interneurons (PVINs), we have collected preliminary data demonstrating that PVINs play a novel role in cue-perception during a visual attentional engagement task (AET). Specifically, we have observed that cue-evoked population increases in PVIN activity are necessary, sufficient, and can be predictive for successful attentional engagement. We hypothesize that this may represent a universal mechanism of attention that is consistently disrupted across diseases with attentional impairments. PVINs however, do not operate in isolation, and mPFC pyramidal neurons are also regulated by long-range inputs from the mediodorsal thalamus (MD) among others, and local circuit interactions with other interneuron subtypes, such as somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) expressing interneurons, all of which have also been linked to cognition and psychiatric disease dysfunction. Separate studies have identified distinct disinhibitory circuits involving VIP and SST interneurons, and SST and PV interneurons that can regulate mPFC-dependent behaviors. However, the precise circuit motifs which regulate attentional processes are largely uncharacterized. Specifically, how individual interneurons in specific classes represent information during attentional assays capturing distinct components of attention remains to be determined. Through the K99 phase, I will receive critical training in in vivo Ca2+ imaging and design and implementation of attentional tasks to test my overall hypothesis that PVINs provide broad inhibition to suppress distracting information during attentional engagement, while the R00 phase will examine how separate disinhibitory circuit motifs allow pyramidal neuron ensembles to signal cue and response information to appropriately guide separate attention functions.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT Congenital heart disease is the most common birth defect, affecting 0.5-2% of all live births. As medical and surgical advances have dramatically increased survival, the burgeoning population of children and adults with congenital heart disease has exposed a high prevalence of neurodevelopmental disabilities in survivors. By adolescence, more than 2 out of every 3 children with critical congenital heart disease experience deficits requiring developmental/special education services. As these children reach adulthood, their disabilities may limit educational opportunities, employment, and quality of life. Abnormal fetal brain development may contribute to neurodevelopmental disability in patients with congenital heart disease. Neonates with congenital heart disease have abnormal brain structure before surgery. Neurobiological processes that lay the foundation for long-term structural brain organization begin in utero. Components of fetal brain critical for this process, in particular neural progenitor cells, premyelinating oligodendrocytes, and subplate neurons, are sensitive to hypoxia-ischemia, rendering this system vulnerable to prenatal circulatory disturbances. The impact of abnormal fetal brain development on long-term brain structure and function in congenital heart disease is unknown. To date, there are no congenital heart disease cohorts that have been studied in both the fetal period and later in childhood when these deficits are typically detected. This proposal will leverage an existing fetal MRI cohort, including children both with and without congenital heart disease, to acquire long-term neuroimaging and neurodevelopmental data at 7 years of age, thereby determining the fetal contribution to long-term outcome. Specifically, the proposed study will investigate 1) the association between fetal brain structure and school-age structural brain connectivity; 2) the relationship between fetal brain structure and school-age neurodevelopmental functioning; and 3) the potential for a clinical risk stratification tool harnessing measures available in utero to predict school-age neurodevelopmental outcome. The overarching hypothesis is that abnormal fetal brain structure is associated with long-term differences in structural brain connectivity and neurodevelopmental functioning in congenital heart disease. This project will support the development of clinical risk stratification and advance the development of interventions designed to protect the brain in children with congenital heart disease.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Children with autism who receive early intervention services have better outcomes than those who do not. It is therefore imperative to lower the age of diagnosis. There is strong evidence that there are reliable behavioral signs/symptoms of the disorder that emerge in the second year of life. However, there is mounting evidence from our laboratory that there are patterns in the EEG that emerge as early as 3 months that are reliably associated with autism outcomes at 2-3 years. In this proposal we seek to extend our previous work in two important ways. First, we will deploy our high-dimensional EEG data collection in a large pediatric primary care clinic, thus demonstrating the potential scalability of EEG as a biomarker of autism risk. Second, we will focus our efforts on a population of infants who have historically been underserved and understudied: primarily Black and Hispanic infants growing up in low-income homes. We will enroll 720 infants over 3 years (240/year), and based on previous work, anticipate a retention rate of 85%. We will collect resting EEG data at 4, 9 and 12 months in conjunction with their well-baby visits at the clinic. At 24 months diagnostic outcomes will be evaluated using the ADOS, developmental measures, and expert clinical judgement. In addition to the EEG assessment in the first year of life, a general developmental screener will be included (Ages and Stages Questionnaire-3) and indices associated with a number of non-genetic variables associated with increased autism risk (e.g., infant sex, parental age, prenatal maternal health, etc.) will be obtained from a demographic questionnaire and medical records. The specific aims of the project are: Aim 1: Using a prospective study design in a racially, ethnically and socioeconomically diverse primary care population, we will identify EEG features measured <1 year of life that are associated with ASD at 2-years of age. Aim 2: To develop predictive models with EEG biomarkers and other risk factors that reliably predict later diagnosis of ASD. Aim 3: To determine the specificity of predictive features for ASD versus other neurodevelopmental outcomes such as language or cognitive delays. Our ultimate goal is to create a scalable, practical, neurobiologically-based tool that can be readily integrated into a pediatric primary care setting, and in so doing, greatly improve our ability to identify autism in the first year. We believe our approach will allow us to demonstrate scalability of EEG in the primary care setting, develop usable models for children at greatest risk of delayed diagnosis, and improve our understanding of the underlying neural mechanisms of idiopathic autism.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Heart failure (HF) has a high morbidity and mortality. Its incidence is increasing worldwide. One hallmark of HF is endothelial cell (EC) dysfunction which initially manifests as impaired endothelium-dependent vasodilation of the epicardial coronary arteries and the microvasculature. Another important hallmark of HF is the presence of interstitial fibrosis, which increases myocardial stiffness and cardiac work, elevates diastolic pressures and increases pulmonary interstitial fluid to impair oxygenation. Genetic lineage tracing showed that most HF fibroblasts originate from tissue-resident fibroblasts, which expand and differentiate into myofibroblasts. However, the molecular mechanisms regulating fibroblast activation and myofibroblast transdifferentiation remain poorly understood. Intercellular communication, especially EC-fibroblast crosstalk, plays a substantial modulatory role in the normal and failing heart. More specifically, factors secreted by cardiac microvascular EC modulate cardiac performance and cardiac fibrosis. Thus, targeting endothelial dysfunction has the potential to be a promising therapeutic avenue for HF. Recently, we and other groups discovered that genes important for the control of cell identity exhibit a unique epigenetic signature, e.g., broad enrichment of the activating histone modification H3K4me3 and super- enhancer marks. These discoveries prompted our pilot work to develop the first computational model for the discovery of new EC master regulators. This novel model employs an analysis of both the epigenetic landscape as well as the gene expression network. It successfully recaptured known EC identity genes with high sensitivity and accuracy. The model further revealed a number of top ranked genes with no reported role in EC, making them promising candidates as novel EC identity genes. One of the most top-ranked genes is transcription factor 4 (TCF4), which displays typical features of cell identity gene in EC. Interestingly, we have preliminary data showing that TCF4 function is a master regulator that maintains EC identity. Further, TCF4 is downregulated in cardiac ECs of HF patients compared to non-failing controls. The silencing of TCF4 in EC leads to an increase of EC-secreted proteins TGFβ1, which stimulate fibroblast activation and myofibroblasts transdifferentiation, and thus promote cardiac fibrosis. In this proposal, we will investigate the role of TCF4 in EC identity maintenance. We will further investigate the role of TCF4 in the crosstalk between ECs and fibroblasts, and reveal TCF4 as a therapy target to prevent cardiac fibrosis in HF. Successful completion of this proposal will be the first to define TCF4 as a novel EC master regulator that maintains EC phenotype and function. We will uncover an overlooked determinant of HF -- loss of EC identity. TCF4 dysregulation disturbs EC-fibroblast crosstalk within the heart, aggravating cardiac fibrosis in HF. Therapeutic modulation of EC-specific TCF4 delivery may be a novel and promising approach for treating HF.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY/ABSTRACT Cardiovascular diseases are often associated with impaired responses from the endothelium, which results from endothelial cell dysfunction. Endothelial cell dysfunction causes endothelial activation and sub-endothelial retention of modified low-density lipoprotein (LDL) particles, leading to the recruitment of immune and inflammatory cells to the intima, which initiate atheromatous plaque build-up. Of major importance, transitioning from a stable to vulnerable atheroma fuels myocardial infarction and stroke, posing enormous health challenges with the highest morbidity and mortality in the United States. New research is urgently needed to uncover critical pathophysiological mechanisms and identify molecules that limit endothelial dysfunction. This has led us to determine a novel and indispensable role for an endocytic adaptor protein called Disabled homolog 2 (Dab2), which participates in clathrin-mediated endocytosis in addition to moonlighting as a tumor suppressor. Curiously, little to no prior work has been done to identify its role in endothelial cells. Our pilot assessment has revealed that Dab2 levels are strikingly decreased in the atherosclerotic endothelium of mouse and human fatty streaks—suggesting a protective role in atherogenesis. As the atheroprotective effects of Dab2 are poorly understood in the context of endothelial cells, we created endothelial-specific inducible Dab2 knockout mice (EC-iDab2KO) and bred them to an ApoE-null background (EC-Dab2iKO/ApoE-/-). Western diet-fed EC-iDab2KO/ApoE-/- mice exhibit heightened arterial inflammation and more severe plaque formation; yet, the molecular mechanisms and signaling pathways that direct Dab2 to combat arterial inflammation are completely unknown. Our initial investigation indicates that Dab2 expression is upregulated in response to atheroprotective flow, and Dab2 deficiency in human aortic endothelial cells suppresses endothelial nitric oxide synthase (eNOS) activation. To ensure the clinical relevance of our work, we are employing an innovative nanotechnology to deliver Dab2 mRNA to the atherogenic endothelium using an engineered nanoparticle to restore Dab2 function. This technology increases Dab2 expression in the atheroma, which restrains plaque progression in ApoE-/- mice. The goal of this proposal is to define the signaling mechanisms underpinning the essential role of Dab2 in protecting the atherogenic endothelium. To this end, we seek to determine molecular mechanisms by which Dab2 curbs arterial inflammation and activates eNOS in endothelial cells. Our possession of innovative targeting reagents and novel animal models will greatly facilitate our paradigm-shifting endeavor. If fruitful, the exciting work proposed in our application will provide a foundation for the development of new treatments to benefit patients at-risk for heart attacks and strokes.

NIH Research Projects · FY 2025 · 2021-12

Atherosclerosis pathogenesis is multifactorial, involving hyperlipidemia and inflammation, as well as hyperglycemia. Individuals with type 1 diabetes show a four-fold increase in cardiovascular disease risk that has persisted despite the spectacular advances in drugs for risk factor management and insulin therapy4. Thus, new strategies and approaches are necessary. Because insulin is administered to diabetic patients subcutaneously, rather than into the portal vein, which is physiological, the liver remains relatively under-insulinized. We considered the possibility that this could contribute to the pro-atherogenic milieu, even beyond hyperglycemia. The long-term goal of this project is to develop drugs that mimic key atheroprotective effects of insulin on the liver. In our preliminary data, we identify Cyp7b1 as an exquisitely sensitive target of insulin in the liver: Cyp7b1 was increased by acute insulin stimulation; reduced by insulin deficiency; reduced by hepatic knockout of the insulin receptor; and one of only four genes significantly altered in all three conditions. CYP7B1 plays a central role in cholesterol, oxysterol and bile acid metabolism6-8. Based on our strong preliminary data, we hypothesize that insulin induces CYP7B1 to maintain lipid homeostasis and suppress inflammation, and that this regulation is lost in type 1 diabetes, leading to atherosclerosis. To test this hypothesis, we will (1) determine the extent to which restoration of Cyp7b1 in a mouse model of type 1 diabetes can prevent atherosclerosis; and (2) define the signaling pathways by which insulin regulates Cyp7b1. We expect to find that CYP7B1 reduces atherosclerosis in diabetic mice via two mechanisms: (1) reducing oxysterols and vascular inflammation; and (2) reducing dietary cholesterol absorption (via modulation of the bile acid profile) and plasma cholesterol. Validation of our hypothesis could lead to the development of drugs that mimic insulin action on CYP7B1. Such drugs, which would restore homeostasis in type 1 diabetes, could be more effective than present lipid-lowering therapies as they would lower both plasma cholesterol and inflammation.

NIH Research Projects · FY 2025 · 2021-09

Neurodevelopmental processes are shaped by dynamic interactions between genes and environments. Maladaptive experiences early in life can alter developmental trajectories, leading to harmful and enduring developmental sequelae. Pre- and postnatal hazards include maternal substance exposure, toxicant exposures in pregnancy and early life, maternal health conditions, parental psychopathology, maltreatment, and excessive stress. To elucidate how various environmental hazards impact child development, it is imperative that a normative template of developmental trajectories over the first 10 years of life be established based on a sufficiently large and demographically heterogeneous sample of the US population. To accomplish this, the Healthy Brain and Child Development (HBCD) Consortium has been formed to deploy a harmonized, optimized, and innovative set of neuroimaging (MRI, EEG) measures complemented by an extensive battery of behavioral, physiological, and psychological tools, and biospecimens to understand neurodevelopmental trajectories in a sample of 7,200 mothers and infants enrolled at 27 sites across the United States (US). The HBCD Study will carry out a common research protocol under direction of the HBCD Consortium Administrative Core (HCAC) and will assemble and distribute a comprehensive and well-curated research dataset to the scientific community at large under the direction of the HBCD Data Coordinating Center (HDCC). The overarching goal of the HBCD Study is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize typical neurodevelopmental trajectories in US children and that will assess how biological and environmental exposures affect those trajectories. A special emphasis will be placed on understanding the impact of pre- and postnatal exposure to opioids, marijuana, alcohol, tobacco and/or other substances. To address these broad objectives, the sample of women enrolled will include: 1) a varied cohort that is representative of the US population; 2) pregnant woman with use of targeted substances (opioids, marijuana, alcohol, tobacco); and 3) demographically and behaviorally similar women without substance use in pregnancy to enable valid causal inferences. In addition, the HBCD Study will identify key developmental windows during which both harmful and protective environments have the most influence on later neurodevelopmental outcomes. The large, multi-modal, longitudinal, and generalizable dataset that will be produced for the first time by this study will provide novel insights into child development using state-of-the-art methods. The HBCD Study will inform public policy to improve the health and development of children across the nation. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis. The NIH HEAL Initiative bolsters research across NIH to improve treatment for opioid misuse and addiction.

NIH Research Projects · FY 2024 · 2021-09

Image-based approaches to single-cell transcriptomics represent one of the most exciting emerging biomedical research tools. These technologies leverage massively multiplexed single-molecule RNA imaging to provide a direct measure of not just the expression profile of every cell within intact samples but also the location of every RNA molecule within those cells. As such, these techniques combine the ability of single-cell RNA sequencing to generate whole-transcriptome expression measurements and discover and catalog cell types, states, and lineage with the ability of high-resolution, fluorescence microscopy to interrogate the molecular organization of cells, define their morphology, and reveal their interactions and organization. Thus, in situ transcriptome-scale molecular imaging promises advances in a vast array of topics, from the role of intracellular RNA organization in synaptic remodeling, to the spatial organization of commensal microbial communities and its effect on host gene expression, to the modulatory role of the microenvironment in tumorigenesis, to name only a few examples. One image-based single-cell transcriptomics technique—MERFISH (multiplexed error robust fluorescence in situ hybridization)—has emerged as a leading technology given its high resolution, high capture efficiency, single-molecule sensitivity, and unparalleled throughput combined with its proven ability to map the intracellular organization of large fractions of the transcriptome and discover, functionally annotate, and map cell types within intact tissues. However, MERFISH remains a nascent technology, and to fully unlock the transformative potential of both MERFISH and spatially resolved single-cell transcriptomics in general, this technology must be matured. First, MERFISH must be made whole-transcriptome. Multiplexing is not the barrier, rather several RNA categories—highly expressed RNAs, short RNAs, and highly homologous RNAs—remain challenging for this technique. Through a combination of new experimental and computational advances, we will extend MERFISH to these categories, creating whole-transcriptome MERFISH and allowing hypothesis-free discovery. Second, the biological demands for single-cell throughput are staggering, as even small tissues often contain tens of millions of cells. By combining new sample preparation techniques, an emerging approach to ultra-high- throughput microscopy, and advanced image storage and analysis tools, we will increase the throughput of MERFISH by orders of magnitude, allowing characterization of large tissue areas and tens of millions of cells. Finally, the transformative potential for whole-transcriptome imaging could be very broad, yet MERFISH has been validated in only a few tissues. Thus, we will provide a robust suite of sample preparation protocols and quality metrics to make routine the application of MERFISH to all tissues and organisms. Here we will unlock the potential of this emerging technique by delivering rapid, robust, and routine whole- transcriptome MERFISH. As gene expression is key to cellular identity and behavior in all domains of life, this general tool could empower a truly remarkable range of basic and translational biomedical research.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Genomic medicine has rapidly advanced in the past decade enabling earlier diagnosis and personalized treatment. However, only a few highly specialized centers in the US have the resources to take advantage of these advances in patient care. This has created a gap in access whereby patients cared for in typical community settings do not receive the same medical care. Another barrier to the wider utilization of genomic medicine is the poor dissemination of knowledge among clinicians, especially in community settings. A wide gap exists in the implementation of genomic medicine from diagnosis to personalized therapies, a field experiencing huge advances but still subject to wide differences in access. Our proposal aims to develop and test the implementation of a strategy to break down these barriers to genomic medicine, aligned with RFA-HG-20-036. Our target population is sick newborns. We propose a novel center, VIrtual GenOme CenteR (VIGOR), building upon our past and ongoing research as investigators for the NIH-funded Babyseq study (U19HD077671), Undiagnosed Disease Network (U01HG007690), and Center for Mendelian Genomics (UM1HG008900). VIGOR will be a center that can remotely support clinicians and families working in community NICUs. In AIM 1, we will establish the VIGOR center, and enroll and follow 250 eligible newborns and their families for 6 months within a network of community NICUs from across the United States. In AIM 2, we will facilitate exome sequencing and create and return timely, comprehensive interpretive reports to families and physicians that: (1) relay diagnostic findings, (2) recommend clinical actions, (3) offer reanalysis of data for those with negative or inconclusive findings; and (4) provide additional research opportunities. In AIM 3, we will comprehensively assess implementation outcomes. Among neonatologists and within NICUs, we will examine 1) Appropriateness; 2) Feasibility; 3) Penetration; and 4) Satisfaction of VIGOR use; among families, we will examine 1) Satisfaction; 2) Adverse mental health (stress and depression) and 3) Newborn clinical outcomes. This study will provide rigorous evaluation of implementing a virtual genome center into community clinical settings without highly specialized resources, thereby offering generalizable insights as to how best to implement genomic medicine at scale and for other age groups. Our intervention has great potential to broaden access to genomic medicine and will enhance capacity for providers and health systems to utilize highly specialized genomic techniques in all communities.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT In the fetal brain, cortical plate (CP) thickness is thought to be related to the number and size of cells within a column, packing density, intracortical myelin, and synapses, and subplate (SP) thickness associated with the number of thalamic and cortical afferents and the amount of cortico-cortical connections. Estimation of cortical thickness postnatally with MRI has contributed greatly to our understanding of human brain development and cognitive function and disease onset and progression in various brain disorders. However, our knowledge and research of human in utero CP and SP thickness remains limited due to the lack of available techniques that automatically measure regional CP and SP thickness from fetal brain MRI. Compared to child or adult brains, fetal brains are much smaller in size and have different image contrast. Fetal brain MRI shows lower effective resolution and suffers from head motion which causes artifacts. Thus, it is challenging to extract accurate CP and SP regions and define geometrically appropriate thickness between the CP and SP surfaces. This study will develop a fully automatic pipeline to extract regional CP and SP thickness using multi-site fetal MRI datasets. We will develop the method for CP and SP segmentation with the identification of sulcal cerebrospinal fluid regions using deep convolutional neural networks. Based on the accurate segmentation, a deformable model method that is optimized and specialized for fetal brains will be developed to extract the CP and SP surfaces. CP and SP thickness will be measured based on vertex-wise correspondence between all CP and SP surfaces. We will perform reliability and sensitivity tests using different imaging subsets within the same subject and artificial data created by moving the CP and SP boundary. We will then define the growth rate of CP and SP thickness in all cortical regions in typically developing (TD) fetuses from 18 to 37 gestational weeks (GW). We hypothesize that the growth rate of CP and SP thickness, the maximum SP thickness, and/or the maximum growth GW of CP thickness will be variable across different cortical areas in TD fetuses. The growth of CP and SP thickness in fetuses with cerebral abnormalities (polymicrogyria and agenesis of corpus callosum) will be statistically compared to the growth of TD fetuses. Malformations of cortical development and cortico-cortical connections may result in altered growth of CP and SP thickness in fetuses with polymicrogyria and agenesis of corpus callosum. This study will lay the foundation for a novel biomarker that can lead to greater insight into the mechanisms of normal and altered in utero brain development. Our methods developed from the proposed study will be publicly distributed using a web-based neuroimage computation platform, which will enable more clinical applications of fetal CP and SP thickness analysis.

NIH Research Projects · FY 2025 · 2021-09

Abstract/Project Summary Microbes interact with the intestinal epithelium in ways that modulate susceptibility to infection, malnutrition, and predisposition to chronic metabolic diseases such as obesity and diabetes. However, the host signaling pathways utilized by microbes to promote health and disease are poorly understood. The powerful genetic tools provided by the model arthropod Drosophila melanogaster have enabled many discoveries that form the basis of our modern understanding of innate immunity. Here we propose to exploit the Drosophila melanogaster model to define the host signaling pathways that detect intestinal microbes and orchestrate the innate immune response of the intestinal epithelium. Drosophila intestinal stem cells, enterocytes and enteroendocrine cells (EECs) carry out functions similar to those of the mammalian intestine. EECs, which constitute 5-10% of cells in the intestinal epithelium, secrete enteroendocrine peptides (EEPs) that modulate host metabolic functions such as insulin signaling, satiety, and intestinal contractions. We have identified a subset of EECs that responds uniquely to the microbial fermentation product acetate by activating innate immune signaling through the TNF-like Immunodeficiency (IMD) pathway. In these EECs, IMD signaling increases transcription of the genes encoding EEPs. These EEPs, in turn, coordinate the response of the diverse cell types in the intestine to microbes. Here we investigate the mechanism by which microbes activate the intestinal innate immune response and the ultimate impact of this regulatory pathway on susceptibility to infection. In this proposal, we will investigate the role of chromatin remodeling in acetate-mediated IMD signaling, the contribution of peptidoglycan to intestinal IMD signaling, the role of EEPs as cytokines, and finally the cell- specific roles of EEPs in modulating susceptibility to intestinal infection. The overarching objective of this research is to uncover novel paradigms of the intestinal innate immune response to microbes with the goal of informing therapies that modify nutrient utilization in malnutrition, chronic metabolic diseases and susceptibility to intestinal infection.

NIH Research Projects · FY 2025 · 2021-09

Suicide is a top 10 cause of death in the U.S. and the second leading cause of death among adolescents and young adults. Access to knowledgeable healthcare providers, family support, and community support are critical determinants of mental health, yet many young adults lack access to mental health care, and interventions to improve family and community support do not exist at scale. This study aims to reduce suicidal behaviors among young adults through a multifactorial intervention that increases access to trained providers, caregiver support, and community connection. The first intervention component consists of a provider training and support program focused on addressing mental health problems among young adults. The second intervention component is an innovative digital platform for young adults and their caregivers. The digital platform provides expert-generated knowledge via educational modules and uses interactive features to promote communication and strengthen relationships between young adults and caregivers. A Multiphase Optimization Strategy (MOST) framework will be used to optimize the platform for testing in a hybrid effectiveness-implementation trial with an Immediate Arm (upfront access) and Deferred Arm (access at 6 months). The intervention period for each Arm will last 6 months, followed by an observation period of up to 12 months with continued access. Using validated subscales, we will assess changes in the proportion of individuals reporting suicidal ideation in the prior three months (primary outcome) and in psychological distress and anxiety, caregiver support, and health care empowerment (secondary outcomes), as well as dose effects, heterogeneity of treatment effects across groups, and within-group resilience factors. This innovative, multi-level intervention fulfills a significant unmet need for near-term and sustainable solutions to reduce suicidality and improve mental health outcomes among young adults in the U.S.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Vaccines represent a highly effective public health measure to protect individuals from infectious diseases. Many vaccines work by inducing antigen-specific antibodies that neutralize the pathogen or its products and promote their clearance. Vaccines based on protein antigens usually require the addition of adjuvants to enhance potency, breadth and duration of the antigen-specific adaptive immune response. Adjuvants promote vaccine antigen immunogenicity by activating receptors of the innate immune system called pattern-recognition receptors (PRRs) and/or modulating antigen pharmacokinetics. Aluminum salts are the most common adjuvants in FDA- approved vaccines. Recently, vaccines including adjuvants that target specific PRRs, in particular toll-like receptor (TLR)4 and TLR9, have also been approved by the FDA, paving the way for the development of molecularly defined adjuvants. Investigating the potential of additional PRRs as adjuvant targets is of paramount important to expand our vaccine toolbox and probe how different modalities of innate immune cell activation impact the adaptive immune response. Here, we propose to use the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein as a model antigen to test a new adjuvant formulation that contains fungal ligands that target the PRR Dectin-2. Our preliminary results show that mannans (fungal cell wall polysaccharides isolated from Candia albicans) alone or formulated with aluminum hydroxide enhance the immunogenicity of pre-fusion stabilized, Spike trimers in mouse models of immunization. In particular, mannan formulations, compared to aluminum hydroxide only, induce an early increase in anti-Spike antibody levels, potentiate the induction of SARS-CoV-2 neutralizing antibodies, broaden the Spike epitopes that are targeted and favor the switch towards immunoglobulin subclasses associated with higher effector functions and reduced risk of vaccine-associated enhanced respiratory disease (VAERD). Here we hypothesize that mannans formulated with alumOH induce a potent and durable adaptive immune response to SARS-CoV-2 Spike by inducing specific innate immune pathways and activation programs. By combining detailed immunogenicity and mechanistic analyses, our proposal will define a novel adjuvant formulation for SARS-CoV-2 Spike and potentially other viral glycoproteins as well as shed new light on the biology of Dectin-2.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/ Abstract Gastric dysmotility affects 40-80% of critically ill children and is associated with significant morbidity and mor- tality. There are knowledge gaps regarding the mechanisms for gastric dysmotility in critically ill children, which limit the therapeutic options for this cohort. Gastrointestinal (GI) macrophages regulate gastric motility and have been shown to have microbiome-dependent changes on their function. Microbiome-dependent changes on macrophage activation are mediated by trans-epithelial intestinal trafficking of microbial products. Zonulin is a protein that increases trans-epithelial trafficking of microbial products. Models of zonulin expression, includ- ing a Zonulin transgenic mouse (Ztm), have dysbiosis and activation of pro-inflammatory macrophages. In criti- cally ill children who express zonulin and in Ztm mice we have identified gastric dysmotility. Our central hy- pothesis is that zonulin upregulation under conditions of inflammation, increases trans-epithelial trafficking of microbial products from an altered microbiome which activate macrophages associated with gastric dysmotility. In Aim 1 we evaluate whether zonulin-mediated increases in trans-epithelial intestinal trafficking due to system- ic inflammation result in dysbiosis and activation of macrophages associated with gastric dysmotility. We will complete Aim 1 with Ztm mice and the use of a zonulin inhibitor. In Aim 2a, we will identify differences in mi- crobiome composition and markers of trans-epithelial intestinal trafficking in relation to gastric motility in pa- tients with and without the zonulin-producing allele. Direct examination of GI macrophage differences in pa- tients is not feasible. Therefore, in Aim 2b, we employ a translational mouse model of fecal material transplant from patients in Aim 2a to examine whether differences in microbiota from patients with and without the zonu- lin-producing allele and gastric dysmotility result in macrophage activation and gastric dysmotility in the mice. This proposal fills a knowledge gap in our understanding of mechanisms for gastric dysmotility in critical illness. Our long-term goal is to identify novel diagnostic markers and therapeutic targets for gastric dysmotility in criti- cal illness, which can impact clinical outcomes for this cohort. This proposal details a four-year project in which Dr. Martinez will gain experience in clinical-translational research and expertise in GI physiology, mucosal im- munology, host-microbiome interactions and the neuro-enteric system. These educational opportunities, the surrounding ideal research environment and established mentoring team that is working with Dr. Martinez will prepare her to apply for a NIH R01 grant and promote her advancement towards an independent academic career.

NIH Research Projects · FY 2025 · 2021-09

Project Abstract Neurogenic bladder from congenital myelodysplasia (Spina Bifida) presents lifelong challenges in bladder management. Even with improved clinical management up to 50% of patients with SB are at increased risk for development of chronic kidney disease associated with urologic complications of neurogenic bladder. Bladder management for patients with neurogenic bladder comprises catheterization to promote emptying, medication to decrease intravesical pressures and inhibit detrusor overactivity, and regular monitoring of bladder function by urodynamics (UDS). If conservative therapy fails, patients may undergo surgical intervention to enhance capacity and reduce intravesical pressure to minimize upper tract damage. Regardless, controversy exists over the best management protocol for the neurogenic bladder. UDS is considered the gold standard for evaluation of lower urinary tract function and the impetus for intervention. However, UDS is invasive, expensive, subject to substantial inter-observer variability and not routinely available beyond tertiary care centers. The availability of a quantitative, non-invasive approach to signal bladder changes that serve as a harbinger of renal deterioration would advance clinical management of this patient population significantly. Notably, no such markers have been validated prospectively as independent markers of functional bladder deterioration. In preliminary studies, we have identified a panel of urine biomarkers enriched in urine from two models of neurogenic bladder (human and rodent), but not detected in kidney urine from human patients with ureteropelvic junction obstruction. This, together with their detection in bladder tissue from rats with neurogenic bladder strongly suggests that this unique panel reflects pathological bladder wall remodeling as opposed to renal damage. Based on these observations we hypothesize that our Non-Invasive Markers of Bladder Deterioration (NIMBLE) represent prognostic markers of deterioration in bladder function in neurogenic bladder patients. We believe that these markers may also serve as early predictors of upper tract damage in this population. We will test the hypothesis with the following aims: Aim 1. Determine the association between bladder-enriched urine biomarkers and functional parameters in a well characterized prospective cohort of children with neurogenic bladder. Aim 2. Investigate bladder-enriched urine biomarkers, their association with function and their response to treatment in a longitudinal cohort of children and rodents with neurogenic bladder. In each aim we will use mass spectrometry-based proteomics to quantify our unique panel in sample cohorts with neurogenic bladder, determine their association with functional UDS and their response to pharmacological intervention. We will also profile the remaining urinary proteome to refine the biomarker panel. At the end of the project we will know the extent to which our NIMBLE panel reflects functional deterioration of the neurogenic bladder and how it could be implemented clinically to improve management of SB patients.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Short bowel syndrome (SBS) is often due to the loss of large amounts of small intestine that compromises digestive absorption. The treatments include a high-calorie diet and feeding through the vein (i.e., parenteral nutrition or PN), among others. Many patients cannot wean from PN due to reduced intestinal length or function. Patients on long-term PN frequently experience serious metabolic complications, sepsis, hepatic biliary disorders including cholestasis, and fibrosis and can progress to liver failure. Full intestinal feeding (enteral nutrition) without PN is the optimal way to prevent the above complications. Enterally administered long chain triglycerides in patients with SBS, especially those with hepatic dysfunction, are not well tolerated due to bile acid malabsorption, which leads to decreased micelle formation and fat digestion. The dietary fat is unable to be emulsified by the bile acids and acted on by lipases before exiting the patient as stool. Switching to other forms of fat such as medium-chain triglycerides (MCTs) that do not require micelles for absorption may be better tolerated in patients with bile acid or pancreatic insufficiency but are not optimal as they increase the osmotic load in the intestine. This may increase the chance of stool dumping; moreover, MCTs do not contain essential fatty acids (FAs). The ability to provide the essential FAs such as those present in enteral formulas in a form that does not require the formation of micelles for absorption, would allow patients with SBS and those who are no longer PN dependent to receive adequate nutrition and continue to maintain the same growth trajectory as when they received the majority of their nutrition parenterally. RELiZORB is an enzyme cartridge connected in-line with enteral feed tubing sets designed to mimic the function of pancreatic lipase. It is hypothesized that by using this external lipase device, enteral nutrition will be better absorbed, and PN dependence reduced as enteral autonomy is increased. This product eliminates the need for intestinal emulsification and eliminates the risk of drugs, including lipases, allowing absorption at the time the diet enters the gut. The device has been shown to digest >90% of fat in most enteral formulas. This is a phase 3, open label single center clinical trial to determine the safety, tolerability, and bioavailability of the RELiZORB enzyme cartridge with enteral nutrition when used daily for 90 days in pediatric subjects with SBS, aged 2 years – 18 years, who are PN dependent. The change in PN calories from baseline, assessed weekly, will be evaluated by area under the curve as a mean percentage increase or decrease and presented with a 95% confidence interval. The number (percent) of treatment-emergent adverse events, grade 2 or above, as well as the incidence of abnormalities in vital signs, changes in stool amount/frequency, ostomy output, the need to decrease enteral feeds, changes in urine color, hematology and biochemistry parameters will be tabulated. Changes in growth z-scores, 72-hour fecal fat and coefficient of fat absorption, plasma FAs, PN volume, enteral/oral nutrition, and ability to wean from PN will also be described.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY With more than 300,000 new cases of Lyme disease each year in the U.S., approximately half of new cases occur in children. Children with Lyme meningitis, a clinical manifestation of Lyme disease, present with headache, fever and fatigue. Previously, an intravenous antibiotic (ceftriaxone) was the recommended first treatment for Lyme meningitis, but it is associated with a high rate of complications related either to the long- term intravenous catheter placed for medication delivery or to complications from the medicine itself. Based on European trials conducted in adults and small observational pediatric studies, some clinicians have begun treating Lyme meningitis in children with an oral antibiotic (doxycycline), avoiding the complications associated with intravenous ceftriaxone and reducing health care costs. Our first goal is to compare oral doxycycline to intravenous ceftriaxone for the treatment of Lyme meningitis, with a focus on both short-term recovery and long-term quality of life. Our second goal is to examine patient, parent and clinician preferences to inform shared decision-making about Lyme meningitis treatments. To accomplish our goals, we propose a comprehensive pediatric Lyme meningitis study, enrolling children at 20 U.S. centers located in regions of the U.S. where Lyme disease is endemic. Treatment decisions will be made by the child’s doctors, per usual practice, and we will obtain informed consent to follow the outcomes over the following six months. We will enroll a total of 210 children with Lyme meningitis to determine whether oral doxycycline is not inferior to intravenous ceftriaxone for the treatment of Lyme meningitis in children. We will interview patients, parents and clinicians to gain a nuanced understanding of the factors that shape treatment decisions. The overall impact of this study will be to inform the best practices for the treatment of children with Lyme meningitis accounting for the preferences of key stake holders.

NIH Research Projects · FY 2025 · 2021-08

SUMMARY People with intellectual or developmental disabilities (IDD)—a sizeable and growing population—now live longer and enjoy better quality of life than in prior decades. However, this group still suffers from excess morbidity, especially during adolescence and young adulthood, the time when they are expected to make health care transitions (HCTs) from child-centered to adult-oriented health care systems. Although it is likely that some adolescents and young adults with IDD experience high-quality HCTs, our understanding of how HCTs proceed longitudinally, in vivo for large populations of adolescents and young adults with IDD is extremely limited. At a population level, we do not know the ages at which HCTs start or end or whether the timing depends on the type of physicians involved. Additionally, scant data exist regarding how health care quality changes for this population during HCTs and the role insurance gaps may play in disrupting that care quality. The proposed study combines three recently developed claims-based tools to create one of the largest, longitudinal, and multi-payer datasets of adolescents and young adults with IDD to date. It uses five years of all-payer claims databases (2014–2018) in three states to identify individuals aged 10–28 with IDD (69,000 persons in Colorado, 85,000 persons in Massachusetts, and 217,000 persons in New York). It then pursues the following four Aims: (1) to characterize HCTs for adolescents and young adults with IDD in terms of the types of physicians involved and the ages across which transfers occur; (2) to assess the quality of care received by people with IDD during the ages at which HCTs occur; (3) to characterize insurance gaps associated with Medicaid age 19 eligibility rules; and (4) to examine the relationship between insurance gaps following Medicaid’s eligibility redetermination at age 19 and clinical care quality using quasi-experimental methods. This study will establish much-needed basic facts about how HCTs proceed for adolescents and young adults with IDD so that doctors, nurses, and allied health professionals can help develop delivery system, insurance, and payment policy interventions that better create health among adolescents and young adults with IDD. It is novel in the database it creates for study, the features of HCTs that it will quantify, its assessment of care quality, and its use of quasi-experimental methods to examine the relationship between insurance gaps around the time of Medicaid’s age 19 eligibility rules and the health of adolescents and young adults with IDD. This research addresses the NICHD’s strategic priority to “improve the transition from adolescent to adult health care…for [those] with disabilities” and the Intellectual and Developmental Disabilities Branch’s priorities to “understand the complexity of comorbid symptoms [of IDD]” and “promote…treatments for IDD that will impact clinical care.”

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Eukaryotic cells employ different mechanisms to sense and respond to their environment and maintain tissue homeostasis. Cells have evolved strategies to co-opt stable reactive oxygen species such as hydrogen peroxide (H2O2) as non-transcriptional signaling molecules in order to rapidly respond and adapt to environmental changes. Numerous examples of the influence of H2O2 signaling have emerged, ranging from abiotic stress in plants to immune responses in humans. H2O2 signaling and subsequent regulation of target proteins is therefore an important but still underappreciated biological control mechanism. H2O2-regulated cell signaling is largely dependent on the presence of redox-sensitive thiol switches in protein cysteine residues, where the reactivity of these switches is highly dependent on the local H2O2 concentration. Our previous studies in epithelial cells have shown that key determinants of cellular H2O2 concentrations include generation of H2O2, by cell membrane surface enzymes such as the NADPH oxidases and permeability across cellular membranes which can be facilitated by Aquaporin (AQP) channels. A number of cellular processes such as innate immune signaling, vesicular trafficking and migration have been shown to be regulated by H2O2, but how cellular membranes allow for specific and privileged signaling by H2O2 remains incompletely understood. We therefore propose studies that aim to establish general rules and emergent concepts related to H2O2 signals at membranes. Our studies will encompass four major areas of inquiry that seek to address i) How does plasma membrane permeability to H2O2 influence redox signaling and regulation? ii.) How do H2O2 signals and subsequent regulation of proteins alter essential vesicular trafficking pathways in the cell? iii) How does H2O2 signaling occur at vesicular membranes? iv.) How does spatial control of cellular H2O2 regulate the directional migration of cells? To address these questions, we will apply and develop high resolution quantitative fluorescence imaging to follow the spatial and temporal dynamics of H2O2 signals at membranes, in combination with proteomic approaches to identify target modified cysteines. Further studies will investigate how oxidative modifications alter target protein structure, function and localization, constructing a mechanistic understanding of how H2O2 signals are relayed from cellular membranes. Future studies will build on this framework to uncover strategies to direct and manipulate H2O2 signals for treatment of human disease. Integrated to these studies will be the development of novel tools and approaches to study H2O2 signals at membranes that can be broadly applied for research in this field.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The family of bacterial toxins, botulinum neurotoxins (BoNTs), produced by spore- forming Gram-positive Clostridium bacteria, are the most potent toxins known. They cause the deadly paralytic disease botulism in humans and animals. These toxins are one of the six most dangerous potential bioterrorism agents (Category A and Tier 1). They target motor neurons with extreme specificity, enter the cytosol of neurons, and block neuronal activity, causing muscle paralysis. A major contributor to the threat of BoNTs is their extremely long half-life within the cytosol of motor neurons – the toxin resides in the cytosol for 4-6 months in humans and causes persistent paralysis for months. To date, no therapeutics can inhibit toxins within the cytosol of neurons. Here we propose to create an effective post-exposure treatment for this top-priority bioterrorism agent and deadly bacterial toxins. This treatment is based on modifying a recently discovered new bacterial toxin BoNT/X and utilizing this engineered protein to deliver a fused neutralizing antibody against BoNTs into the cytosol of motor neurons. Our extensive preliminary studies have provided working prototypes, which completely rescue mice from systemic toxicity of BoNTs in multiple post-exposure models. Here we propose three aims to further validate our hypothesis by (1) refining the chimeric toxin platform; (2) optimizing the nanobody cargo against BoNTs; and (3) establishing pharmacokinetic parameters and validating therapeutic efficacy in both rodent and guinea pig models. Success of these aims will create an effective post-exposure treatment for a top-priority bioterrorism agent, and also develop a novel protein-based delivery platform for targeting motor neurons, thus offering new tools for targeting cytosolic processes and “undruggable” proteins in neurons.