Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Challenges. The airway epithelium consists of various cell types – understanding cellular and functional heterogeneity will have a significant impact on diagnosing and treating diseases. However, few analytical tools are available to investigate spatiotemporal phenotypes of these cells on a global population scale. Conventional high-throughput microscopy (HTM), although powerful for dissecting heterogeneous biological processes, is significantly limited in multiscale imaging and analytics. Most HTM systems are constructed by combining high-magnification microscopes with scanning stages; this configuration would entail high complexity in the system design and operation, high cost, and slow image acquisition rates. Follow-on data analyses, based on traditional ensemble averaging approaches, often lead to the loss of detailed mechanistic information. Innovations. We will advance a “smart” imaging platform, M3 (Multiscale Machine-learning Microscopy) for large-scale, live-cell analyses. M3 will integrate cutting-edge breakthroughs: Fourier ptychographic microscopy (FPM) and deep learning (DL). FPM is based on a spatially coded-illumination technique, collecting low-resolution image sequences while changing the position of a point-light source. These images are then numerically combined to restore the whole Fourier space, allowing FPM to achieve both wide field-of-view and high spatial resolution simultaneously. DL is potent in discovering intricate, hidden structures in high-dimensional data sets with limited human supervision. We will integrate DL with time-series modeling to learn disease-related cellular traits. Goals. We will implement the M3 platform and adopt it to analyze cellular phenotypes during airway epithelium development. Aim 1. We will construct the M3 imaging system based on the FPM technology. This system will feature i) a new numerical algorithm to reconstruct 3D volumetric images and ii) multi-color imaging capacity for molecular detection. Aim 2. We will advance a DL framework for M3 image analyses. This framework will be designed to recognize different cell types and learn their spatiotemporal features to unravel multiscale cellular heterogeneity. Aim 3. We will apply M3 to phenotype cells in the airway epithelium. We will use an in-vitro model that uses induced pluripotent stem cells (iPSCs) to derive lung epithelium. M3 will monitor cellular differentiation during epithelium development and examine the correlation between cellular phenotypes and functionals. Impact. The M3 will bring unprecedented analytical power to characterize diverse cells within the airway epithelium, allowing us to discover many hidden phenotypes in cellular and tissue levels. Such knowledge would have implications for early disease detection as well as designing effective therapeutics.

NIH Research Projects · FY 2026 · 2023-03

Abstract The skin of AD patients is often colonized by S. aureus strains that produce superantigens (SAg), primarily staphylococcal enterotoxin B (SEB). There is a positive association between S. aureus skin colonization and food allergy in AD. The mechanism of this association is unknown. We have made the observation that epicutaneous (EC) application of ovalbumin (OVA) and SAg producer S. aureus, or OVA and SEB, results in the selective exaggeration of anaphylaxis to oral challenge with OVA compared to EC application of OVA alone. Moreover, it results in exaggerated systemic anaphylaxis to oral challenge with BSA-TNP in mice passively sensitized with IgE anti-TNP, indicating that the enhancement of food anaphylaxis was non-antigen-specific and determined by factors beyond differences in IgE Ab levels or affinity. We propose to dissect the mechanisms of SEB enhancement of IgE mediated oral anaphylaxis. Preliminary data show that enhanced susceptibility to oral anaphylaxis in mice EC exposed to OVA+SEB is associated with elevated levels of serum IL-4, dependent on IL-4 and IL-4R expression by intestinal epithelial cells (IECs),and accompanied by increased intestinal permeability (IP). Enhanced susceptibility is inhibited by Divertin, a small molecule that suppresses intestinal absorption of antigen via the paracellular pathway by blocking the recruitment of myosin light chain kinase (MLCK) to the peri-junctional actinomyosin ring, where it disrupts epithelial tight junctions. This suggests a critical role for MLCK in food allergy. In addition, the data show that EC application of SEB causes a massive influx of basophils in skin-draining lymph nodes (dLNs) that was dependent on CD40 keratinocyte (KC)-derived IL-33, and T cells. The recruited basophils enhanced the ability of dendritic cells (DCs) from skin dLNs to drive Th2 polarization. Pretreatment of DCs in vitro with IL-4 also promoted their capacity to drive Th2 polarization. We propose to test the hypothesis that SEB from S. aureus that colonizes AD skin binds to CD40 on KCs and triggers caspase 8 mediated cleavage and release of bioactive IL-33 which induces IL-3 release by T cells leading to recruitment of basophils in dLNs. There, basophil-derived IL-4 promotes the Th2 polarizing ability of DCs that have captured antigen encountered in the skin. These events drive a rise in systemic levels of Th2 derived IL-4. Increased IL-4 signaling in IECs synergizes with mediators released by MCs to promote MLCK dependent barrier loss by causing redistribution of tight junction proteins. The resulting increased antigen absorption triggers a forward amplification cycle of MC activation that exaggerates allergy to foods against which the patient has been sensitized. The studies proposed will define the mechanisms by which S. aureus skin colonization aggravates food allergy and will uncover a central role of MLCK in this disease. They may lead to novel therapies for food allergy that would target S. aureus skin colonization, CD40 in skin, IL-33 and MLCK.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY G-qudruplex (G4) is a noncanonical secondary structure that can form in both DNA and RNA. Human genome contains over 400,000 potential G4 forming sequences (PQS) and they are highly enriched in upstream of oncogene promoters and regulatory genes, strongly suggesting a switch-like function with programed positioning. PQS is also prevalent in e. coli genome, located in important regulatory regions. Indeed, many studies have demonstrated the role of G4 in up or downregulating genomic processes including replication, transcription and translation. Our recent study demonstrated that in transcription, G4 forming sequence located in the non- template strand leads to a robust formation of R-loop (mRNA annealed to template strand), which in turn, induces G4 structure in the non-template strand. Remarkably, such R-loop/G4 structure drives enhanced transcription by a mechanism that involves successive formation and release of R-loop. We show that when positioned in a plasmid i.e under torsional constraint, such G4/R-loop structure can tune the transcription activity up or down depending on the distance from the transcription start site. Furthermore, 5’UTR-G4 bearing mRNA (RG4) leads to over 10-fold enhanced translation in a cell-free system and in e.coli cells. Upon testing several plausible hypotheses, we propose that the RG4 structure promotes translation by blocking ribosomes from sliding off the mRNA. Building on these exciting new findings, we propose to investigate the impact of G4, R-loop and supercoiling in transcription and translation primarily in T7 RNAP system and in e. coli cells. By combining quantitative biochemical tools and newly developed single molecule platforms suited to measure stepwise progression of transcription in a linear or plasmid DNA, we will examine how different sequence, length and position of G4 forming sequence leads to R-loop formation, mRNA output and protein production. Accomplishing the proposed goals will reveal the structure-function relationship of how G4, R-loop and supercoiling regulates transcription and translation activity.

NIH Research Projects · FY 2026 · 2023-03

Inflammation and thrombosis fuel cardiovascular and pulmonary disease: Focus on the interplay of neutrophil inflammasomes with NETs. For many years, we have conducted a successful research program studying the links between thrombosis and inflammation. We plan to continue this line of investigation with the current emphasis on neutrophil extracellular traps (NETs) and the role inflammasomes plays in neutrophils and NETosis. Upon neutrophil activation, peptidylarginine deiminase 4 (PAD4) citrullinates histones and promotes inflammasome assembly needed for effective NETosis and IL-beta production. NETs trap microbes but we have shown a dark side of NETs, i.e., they promote thrombosis, inflammation and age-related heart and lung fibrosis. The central hypotheses of our program are as follows: NETs are involved in the formation of a stable, organized, and vascularized thrombus and breaking up NETs is necessary for thrombolysis. Thrombosis promotes deposition of NETs in both the adjacent vessel wall and in distant organs, leading to post-thrombotic syndrome and an increased systemic pro- coagulant and pro-inflammatory state. Inflammation, activating inflammasome and NET generation as seen in rheumatoid arthritis has systemic consequences such as the development of heart failure with preserved ejection fraction (HFpEF). Inhibiting inflammation by NLRP3 inflammasome or PAD4 inhibitor in mice will reduce the systemic effects and alleviate HFpEF development. We hypothesize further that in many diseases, inhibition of NET formation and reduction of thromboinflammation would be beneficial to the host. We propose to test these hypotheses using mouse and human blood cells, knockout mice and murine disease models we have developed. The work by the “Wagner Lab” is considered innovative and solid; an objective measure is its high citation. Obtaining prolonged funding would free time for more mentoring, innovative thinking, and helpful collegiate activities. In addition, we now study chronic inflammatory and thrombotic diseases and their impact on aging. These experiments take time, and the extended duration of support would assure that we can pursue this exciting research program effectively.

NIH Research Projects · FY 2026 · 2023-03

Project Summary/Abstract Copy number variations (CNVs) of the human 16p11.2 genetic locus, containing 29 coding genes, are associated with a number of neurodevelopmental and psychiatric disorders. The deletion (16pdel) and duplication (16pdup) variants of this region have poorly understood pleiotropic effects. Although autism is more common in patients with deletions, and schizophrenia is more common in those with duplications, underlying mechanisms are not clear. Several molecular pathways from the 16p11.2 region modulate neuronal differentiation, migration, axonal development, and synapse formation, as well as energy and lipid metabolism. Studies of 16p-animal models have suggested deficits in the KCTD13-RhoA pathway activation, neuronal migration, axonal development, and behavior. In turn, disruption of ceramide homeostasis due to 16p11.2 CNVs at FAM57B locus altered lipid abundance, cell membrane dynamics, synaptic protein expression, and synaptic transport, suggesting that lipidome dysregulation could contribute to neuronal function and activity. However, the exact molecular mechanisms underlying these neuronal dysfunctions in excitatory versus inhibitory neurons are lacking. Moreover, contradictory results have been reported from different animal and human cell models. To address this gap of knowledge, we developed human iPSC-derived neuronal models of 16p11.2 CNVs and demonstrated that i) KCTD13 regulates RhoA pathway activation, and increased RhoA expression leads to hyperactivity of the 16pdel human cortical neuron networks, and ii) there are significant changes in key mitochondrial and lipid enzyme transcripts, including decrease in FAM57B-mediated ceramide synthase expression, that directly correlate with observed changes in the metabolome and lipidome. These data suggest that 16pdel leads to complex metabolic disruptions and deficient ceramide expression that might contribute to the observed functional neuronal network phenotypes. These data have led us to hypothesize that 16p11.2 CNVs cause dysregulation of ceramide abundance in glutamatergic and GABAergic neurons that in turn promotes deficits in synaptic development and function leading to network disorganization and hyperactivation. Here, we will investigate this hypothesis and the effects of 16p11.2 CNVs on cortical neuron development and function in human iPSC-derived 2-dimensional excitatory-inhibitory neuron co-cultures and human iPSC-derived 3-dimensional forebrain organoids. To reduce variability caused by different genotypic backgrounds, we will study CRISPR-Cas9 induced 16p11.2 CNV iPSC lines in addition to iPSC lines derived from patients and healthy controls. We will utilize state- of-the-art molecular methodologies to uncover mechanisms underlying the synaptic dysfunction of the excitatory- inhibitory neurons in 16p11.2 CNVs, including single cell transcriptional gene expression profiling and lipidome/metabolome profiling. Finally, we will investigate the excitatory-inhibitory network function, connectivity, and oscillation patterns with multi-electrode arrays and patch clamping. We anticipate that this study will uncover new molecular targets related to cortical neuron dysfunction in 16p11.2 CNV disorders.

NIH Research Projects · FY 2026 · 2023-03

ABSTRACT Hemoglobin disorders, such as sickle cell disease and β-thalassemia, comprise the most common monogenic diseases of the world and yet, current treatments remain largely supportive and inadequate. Induction of fetal hemoglobin (HbF) could bypass the fundamental genetic defects of adult hemoglobin that cause these diseases. Recent gene therapy successes provide proof-of-concept that understanding the molecular control of adult-stage HbF silencing can identify rational therapeutic targets. However, gene therapy cannot be scaled up globally to match the scope of the clinical problem for the foreseeable future. Therefore, novel pharmacotherapies are needed to induce HbF. The major HbF regulators BCL11A, ZBTB7A, and NuRD each have on-target liabilities that could make therapeutic targeting challenging. Recently ZNF410 was discovered to be a novel transcriptional repressor of HbF level during adult-stage erythropoiesis. ZNF410 has a narrow biological action, which is to enhance the expression of CHD4. CHD4 possesses a unique array of 27 reiterated ZNF410 binding motifs at its promoter and upstream enhancer, an assemblage without comparison in the rest of the genome. This study aims to investigate the: mechanisms whereby ZNF410 controls the expression of CHD4 through homotypic motif clusters; requirements for ZNF410 and its orthologs throughout development, homeostasis and hematopoiesis; and potential of targeted protein degradation of ZNF410 by IMiD congeners as a therapeutic approach. Near-term goals are to define the role of protein-level cooperativity and chromatin accessibility in binding to CHD4 by ZNF410 and the relationship between ZNF410 binding events and CHD4 expression. These mechanistic studies will help identify vulnerabilities in this regulatory axis that might be targeted therapeutically. Furthermore, the roles of ZNF410 throughout mouse development and adulthood as well as in human hematopoiesis will be investigated. Constitutive and conditional alleles of Zfp410 and its cognate regulatory elements at Chd4 in mice will be generated and characterized. Requirements for ZNF410 throughout human erythropoiesis and hematopoiesis will be identified by bulk and single cell gene expression and chromatin profiling in vitro and in vivo. Finally, tool compounds will be generated to validate targeted protein degradation of ZNF410 by small molecules as a promising therapeutic approach. Structural evaluation and systematic exploration of structure-activity relationships will be leveraged to obtain instructive compounds to evaluate in ZNF410/Zfp410 sufficient and deficient cellular and animal models the therapeutic premise that ZNF410 is a favorable therapeutic target for HbF induction in the β- hemoglobinopathies. The long-term goal is to promote the development of drug-like small molecules that ultimately could be used in clinical trials.

NIH Research Projects · FY 2026 · 2023-02

Project Summary/ Abstract Sepsis remains to be associated with a high mortality of 20 to 30% with annual cost of $24 billion, accounting for nearly one-fifth of the total aggregate costs in all the hospitalizations in the United States. Current sepsis management is supportive. Therefore, identifying therapeutic approaches is an urgent task to improve the outcome of sepsis. Neutrophils eradicate microbes as the first-line defense innate immune cells. In sepsis, exaggerated de novo neutrophil production called emergency granulopoiesis occurs mainly via G-CSF production. However, immature neutrophils are also released into a circulation to meet a high demand for neutrophil number, but they have less antimicrobial defense functions, leading to worse host defense in septic patients. G-CSF itself does not trigger full neutrophil maturation. Thus, an intervention to attain the enhancement of neutrophil maturation is critical in sepsis for better host defense. Integrin CD11c was considered a sensitive marker to differentiate sepsis from systemic inflammatory response syndrome. We previously showed that CD11c KO mice had worse survival in the polymicrobial sepsis induced by cecal ligation and puncture (CLP) surgery, indicating the critical role of CD11c in sepsis. There has been a paucity of research about its functional role in vivo. We unexpectedly identified that CD11c was expressed in the bone marrow (BM) neutrophils (largely intracellular) and its deficiency was associated with less BM neutrophil maturation. In a mouse model to recapitulate emergency granulopoiesis, mature neutrophils were released in significantly less quantity in CD11c KO mice compared to wild type (WT) mice, further suggesting its importance in neutrophil maturation. We created CD11c constitutively active knock-in (KI) mice, which demonstrated to have more mature neutrophils in the BM in a steady-state condition and during infection with better bacterial eradication and outcomes. In addition, our in vitro neutrophil maturation experiments using primary murine CD11c KO neutrophils or HL-60 cells devoid of CD11c by CRISPR/Cas9 technology showed less neutrophil maturation. Based on these results, we hypothesized that CD11c activity would significantly regulate the degree of neutrophil maturation in a steady state and emergency granulopoiesis in a cell-intrinsic manner. Our preliminary data suggested that CD11c would also regulate a subset of neutrophil effector functions irrelevant of maturation. Thus, we will determine the role of CD11c in neutrophil maturation and effector functions in a steady state and sepsis in Aim 1 and Aim 2. Because IQGAP1 was suggested to be a binding partner for CD11c, its role will also be studied to delineate the mechanism of how CD11c regulates neutrophil maturation and effector functions. Studies will be done by using human sepsis subjects and murine models. Because there are no CD11c small molecule agonists and antagonists available, we will screen them in Aim 3. Upon the completion of the proposal, we expect that we would solidify that CD11c would be a critical regulator of neutrophil maturation and effector functions for a therapeutic intervention in sepsis.

NIH Research Projects · FY 2026 · 2023-02

Abstract Fifteen years ago, genetic studies identified an association between TCF7L2 and diabetes; more recently an association with non-alcoholic fatty liver disease (NAFLD) was identified. Yet, the role of TCF7L2, particularly in the liver, remains controversial. We think that progress to date has been hindered by (1) use of dominant negative strategies to elucidate TCF7L2 function; (2) a restricted focus on glucose metabolism; (3) a lack of appreciation of hepatocyte heterogeneity. That is, hepatocytes differ in transcriptional profile and function depending on the anatomic zone in which they reside. The overarching goal of this proposal is to determine the role of TCF7L2 in regulating hepatic gene expression and metabolism. Our preliminary studies using mice with acute deletion of Tcf7l2 in the liver show (1) TCF7L2 has zone-specific effects on gene expression; (2) glucagon inhibits TCF7L2; and (3) disruption of TCF7L2 leads to alterations in amino acid, bile acid, and lipid metabolism. Consequently, in response to a Western diet, mice with hepatic deletion of TCF7L2 show a marked disruption in the size and zonation of lipid droplets, as well as an increase in hepatocyte injury. We therefore hypothesize that TCF7L2 is a zone-keeper in the liver, necessary for maintaining homeostasis during fasting/feeding transitions as well as the safe storage of lipids during overnutrition. To test this hypothesis, we will use single-nuclei sequencing, novel mouse models to examine TCF7L2 chromatin binding in different zones, and metabolomic approaches. We expect that these studies will broaden our understanding of diabetes, and forge the way for developing personalized genotype-based strategies to prevent NAFLD and other diabetic sequelae.

NIH Research Projects · FY 2025 · 2023-01

Project Abstract Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia worldwide. Despite decades of research, there are few therapies that can delay or prevent AD progression. Retrograde trafficking through retromer-dependent cargo recognition has emerged as a critical cellular process that is mutated or disrupted in patients with AD and other forms of dementia. Conditional knockout of retromer genes in neurons leads to increased secretion of Tau and Amyloid β (Aβ), hallmark protein pathologies linked to AD. This milieu of neuronal-secreted factors leads to inflammation in microglia and astrocytes, two glial cell types thought to influence the progression of neurodegeneration. In this proposal, I aim to study the cascade of events linking neuronal retromer disruption to glial inflammation, characterizing the specific cell state changes involved, and identify the key factors that mediate this effect. I will address this aim using genetically engineered stem cell- derived models of human neurons, microglia, and astrocytes. Microglia also express retromer components and upregulate these factors in AD, yet there are few studies of retromer function specifically in microglia. In Aim 2 I will therefore explore the effects of retromer-related mutations specifically on microglia in the context of early aging in mice, a comparable time point to when dementia manifests in patients with these mutations. I will additionally utilize stem cell models to dissect the functional and signaling changes that are induced in microglia with retromer disruption. Finally, although there have been several studies looking at the effects of retromer on specific receptors, little is known about the systems-level effects of retromer mutations on protein trafficking to the endosomes. To identify retromer-dependent signaling pathways that may be pathogenic, I have developed novel proteomics tools to quantify endosomal proteome changes and will use these tools to compare the effects of different retromer mutations on neuronal and microglial endosomes. The ultimate goal is to understand how retromer disruption affects brain cell states and leads to pathogenic signaling changes.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY/ABSTRACT Neural tube defects, including spina bifida and anencephaly, are common and severe birth defects that occur when the neural plate, the embryonic precursor to the brain and spinal cord, fails to close during early gestation. Folic acid fortification and supplementation have helped to reduce the global burden of neural tube defects; however, less than 50% of neural tube defects are estimated to be attributed to known risk factors. Arsenic contamination of drinking water continues to be a major public health threat worldwide and has been shown to induce neural tube defects in animal models. An emerging hypothesis is that arsenic acts via the epigenome, the multitude of compounds that affect the expression of genes without changing the underlying DNA sequence. In this ViCTER application, we have established a team of experts in child neurology, neurosurgery, epigenetics, developmental and molecular epidemiology, functional genomics and biostatistics to test the hypothesis that DNA methylation mediates the neurotoxicity of arsenic on the developing nervous system. Better understanding of the interplay of epigenetics, nutrition, and environmental arsenic exposure will inform strategies to prevent and treat neural tube defects. In this proposal, we establish a new basic science-clinical science consortium to assess whether arsenic induces recognizable DNA methylation changes at loci critical for normal neural tube closure. We will utilize in vitro methods and biological samples collected from an epidemiological study in Bangladesh to measure DNA methylation patterns using cutting-edge methodologies, including whole genome bisulfite sequencing and the Illumina EPIC/850K BeadChip technology. Additionally, we will investigate DNA methylation changes in genes associated with arsenic exposure and folate status following a short course of high-dose folic acid supplementation in women who previously conceived a child with a neural tube defect. This interdisciplinary and collaborative study, which includes research activities spanning basic to applied research, will seek to identify genes involved in neural tube closure that are differentially methylated in relation to arsenic exposure and folate status. This consortium effort will accelerate the translation of findings into folic acid-based preventive strategies to reduce the global burden of neural tube defects, particularly in areas with high arsenic exposure.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT This Mentored Research Career Development Award (K01) proposal includes a coordinated training plan and research project that will facilitate the candidate's transition to independent investigator at the intersection of adolescent substance use, in particular marijuana use, sleep disturbances and chronic pain. Adolescent marijuana exposure predicts many negative outcomes, particularly for early, heavy users. Chronic pain is an under-recognized, yet highly prevalent adolescent health problem. Evidence suggests that chronic pain potentiates risk factors for substance use, in particular those related to sleep disturbances and diminished inhibitory control. However, the interplay among sleep, pain, and inhibitory control, as it relates to marijuana use in adolescence, remains minimally explored, which is significant given the unique risk for frequent and heavy marijuana use in this population. The proposed research project utilizes mobile health (mHealth) measurement techniques, combining mobile phones and wearable devices, to enhance our understanding of the longitudinal interplay among these mechanisms in patient’s natural settings. The long-term goal of this K01 award is for the candidate to establish an independent research career aimed at developing and implementing mechanistically informed interventions for marijuana use disorders in youth with chronic pain and sleep disturbances using mHealth approaches. To do so, specific short-term training is required in: 1) conducting adolescent substance use research, with a focus on marijuana use; 2) conducting randomized controlled trials; 3) sleep and circadian biology and sleep measurement techniques; 4) the use of mHealth technologies and protocols and 5) the responsible conduct of research. Two independent, yet related studies are proposed to characterize the relation among sleep, pain, inhibitory control, and marijuana use in an adolescent chronic pain population. A first study will interrogate a large cross-sectional sample of youth with chronic pain to understand the association that particular pain dimensions (i.e. intensity, frequency and interference) show with marijuana use, both directly and through associations with sleep and inhibitory control. Given the potentially complex interplay among factors, a longitudinal assessment is vital to map the unfolding of pathophysiological processes. The second study will therefore combine ecological momentary assessments, mobile app-based cognitive tests, and passive collection of sleep data to provide time-sensitive and ecologically valid models of the longitudinal interplay among risk factors within the context of the individual’s daily life. Such methods are vital for mapping temporal sequences of events and developing specific interventions to interrupt this perpetual cycle and reduce risks, which will be the goal of a subsequent R01 grant.

NIH Research Projects · FY 2026 · 2023-01

Modified Project Summary/Abstract Section Respiratory RNA viruses can induce very different host outcomes. While we have made progress in understanding clinical, cellular, and molecular correlates of disease severity, few studies have assessed if or how specific factors present at baseline may induce severe disease. There is a tremendous knowledge gap in whether correlates of disease severity represent causal factors (i.e. if presence at baseline lead to more severe infection), or may actually represent generally beneficial attempts at restoring tissue function (i.e. a resilience mechanism), that are detrimental only in select host contexts. Despite distinct biology of coronaviruses and influenza, epidemiological studies have noted that overweight and obese individuals are at greater risk for severe infection, implicating lipid metabolism, and further genetic studies have found mutations in the Type I/III interferon system in severe cases. Importantly, treating the underlying causes of severe viral respiratory diseases will require a deeper understanding of the epithelial cell states that contribute to different outcomes to design host-directed therapies that avoid long-lasting damage to the respiratory and cardiovascular systems. Recently through single-cell RNA-sequencing (scRNA-seq) of nasopharyngeal swabs, we have discovered that a muted interferon antiviral response combined with an increase in intracellular cholesterol biosynthesis potential in respiratory epithelial cells characterizes severe vs. mild-moderate viral-induced disease. In this same study, we also revealed subsets of secretory and goblet cells with uncharacterized functional potential, overlapping with subsets we had previously identified in a study of seasonal influenza. Our published data, together with that of our colleagues, mandate further investigation into how pre-existing antiviral and cholesterol biosynthetic cell states in human respiratory epithelial cells dictate host outcomes to respiratory viral infection. In light of these findings, we hypothesize that baseline cholesterol biosynthesis in respiratory epithelial cells is a critical host resilience mechanism which becomes pathogenic in the absence of effective antiviral resistance mechanisms. This overarching hypothesis can only be tested through a shift in the conceptual and experimental approaches we traditionally deploy (New Research Direction). Successfully testing our hypothesis will address (Aim 1) whether cholesterol biosynthesis dictates the maximum potential interferon response in airway epithelial cells, or whether a muted interferon response underlies enhanced cholesterol biosynthesis in mice. Furthermore, it will identify novel contributions of airway epithelial cells to local and organismal lipid metabolism. Our work will also test (Aim 2) the stability of metabolic and antiviral cellular phenotypes in human epithelial progenitor cells. Successful completion of our plan will lead to the development of non-invasive screening approaches to better ascertain risk of susceptible populations to respiratory viruses, and of prophylactic and therapeutic strategies to achieve optimal balance of host defense strategies in the respiratory tract.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Post-hemorrhagic hydrocephalus (PHH) is a leading cause of morbidity in premature infants. PHH is triggered by germinal matrix intraventricular hemorrhage (IVH) that results in accumulation of cerebrospinal fluid (CSF) in the brain compression of surrounding brain tissue, and permanent neurological deficits. While PHH is clearly caused by an altered balance of CSF production and removal, the mechanisms are poorly understood, limiting our ability to guide rational therapies. Here, we propose to examine two processes that could be manipulated therapeutically to alleviate PHH: (1) ion and fluid transport by the choroid plexus (ChP), and (2) ventricular blood clearance by macrophages. In adults under normal physiological conditions, sheets of specialized ChP epithelial cells secrete CSF via an incompletely understood set of membrane proteins including NKCC1, a phosphorylation activated bi-directional Na-K-Cl cotransporter. Strikingly, we recently discovered that NKCC1 participates in CSF removal rather than CSF secretion during early stages of brain development. CSF-K+ levels are significantly higher in embryos than adults, likely explaining this opposite direction of NKCC1 water transport8. Experimental introduction of blood into the ventricles during development appears to further elevate CSF-K levels, and to drive intracellular calcium activity in ChP epithelial cells, expression of the immediate early gene c-fos, and increased expression/phosphorylation of NKCC1. Our findings suggest a novel counter-regulatory response to IVH in premature infants: ChP absorption of CSF via NKCC1, driven by K+. We will test this hypothesis by determining if NKCC1 activation either worsens or mitigates hydrocephalus in our mouse IVH model (Aim 1; preliminary data suggests the latter). We also found that following IVH, blood products linger in the developing ventricles and may account for the persistence of PHH. The brain's ventricles and the apical surface of the ChP are home to specific macrophages known as Kolmer cells. While Kolmer cells have been implicated as responders to brain hemorrhage, their scavenging and other functions have remained elusive. Our data suggest that during early stages of brain development, ventricular macrophages/Kolmer cells are activated and recruited to the site of blood leakage within the ventricle (Aim 2A) and that these macrophages are necessary and sufficient to clear blood and/or inflammatory signals from the ventricles (Aim 2B, C). Collectively, our data suggest that the ultimate severity of PHH depends on a developmental stage-specific interplay between blood products, ion concentrations (e.g. [K]), immune and inflammatory reactions, and NKCC1 expression levels. An estimated 20% of infants that experience intraventricular bleeds develop PHH. We suspect this is due to insufficient endogenous compensatory responses. The ultimate goal of this proposal is to improve outcomes by laying the groundwork for development of clinical treatments that boost endogenous removal of CSF and blood that drive the pathogenic processes that lead to PHH. This proposal should also guide therapies for adult IVH and other conditions with disrupted extracellular ionic homeostasis.

NIH Research Projects · FY 2026 · 2022-12

SUMMARY Intercalated disks (ICDs) connect the termini of adjacent cardiomyocytes (CMs) physically, electrically, and chemically. The structural role of ICDs to preserve CM integrity in the face of billions of cycles of forceful con- traction and relaxation is well appreciated; however, the function of ICDs as essential CM signaling hubs is only now emerging. Arrhythmogenic cardiomyopathy (ACM) provides a unique window into the function of ICDs and specifically desmosomes. ACM is a potentially lethal disorder characterized by high arrhythmia bur- den, loss of contractile myocardium, and replacement by fibro-fatty tissue. Mutations of desmosome genes (PKP2, DSG2, DSC2, DSP, JUP) occur in approximately half of ACM patients. Despite growing knowledge about ACM disease pathogenesis, the mechanistic links between desmosome mutations and arrhythmias, my- ocardial dysfunction, and fibrofatty replacement remain poorly understood. The overall goal of this proposal is to gain insights into the mechanisms by which desmosome mutations cause arrhythmia and myocardial dysfunction; Our overarching hypothesis is that desmosomes are inte- gral for maintaining normal cardiomyocyte homeostasis through both their structural and signaling activities. ACM mutations disrupt these activities to cause both loss of structural integrity and aberrant signaling. We will test these hypotheses through four parallel but complementary Specific Aims: (1) We will examine cell composition and gene regulation of human ACM myocardium, using concurrent single nucleus RNA-seq and ATAC-seq, and spatial transcriptomics (snMulti-seq) with massively parallel single molecule fluo- rescent in situ hybridization (MERFISH); (2) We will use mosaic, adult, cardiomyocyte specific inactivation of Dsp to probe the cell autonomous functions of desmosomes. This model will be studied using snMulti-seq and MERFISH, followed by interrogation of key predicted regulators using in vivo gain- and loss-of-function ap- proaches; (3) Using proximity proteomics of ICD component N-cadherin, we identified novel ICD components and ICD components that are altered by Dsp ablation. We will use in vivo gain- and loss-of-function ap- proaches to study the function of selected candidates identified by this screen; (4) Define the contributions of WNT and GSK3 signaling to ACM phenotypes in DSP mutant hiPSC-CMs. Using genetic approaches in bioen- gineered hiPSC-CMs, we will dissect the involvement of GSK3 and WNT signaling to ACM pathogenesis. Impact: This proposal will advance our understanding of the function of desmosomes and ICDs in CM homeostasis and the molecular pathogenesis of ACM. This knowledge will accelerate efforts to develop targeted ACM therapies.

NIH Research Projects · FY 2026 · 2022-12

In cardiomyocytes, dyads are nanoscale structures formed by the juxtaposition of T-tubules, a network of tubular invaginations of the plasma membrane, and regions of the endoplasmic reticulum specialized for Ca2+ release, known as the junctional sarcoplasmic reticulum (jSR). Dyads are positioned adjacent to Z-lines, such that sarcomere Z-lines, jSR, and T-tubules co-localize in a regular, transverse, linear pattern. Dyads mediate excitation-contraction (E-C) coupling, which converts rapidly propagating plasma membrane electrical signals into coordinated Ca2+ transients throughout the cardiomyocyte, resulting in synchronized, forceful sarcomere contraction. A hallmark of failing cardiomyocytes is disorganization of dyads, which disrupts Ca2+ handling and results in decreased contraction and increased risk of arrhythmia. The molecular mechanisms underlying dyad architecture and positioning have remained a mystery, despite their importance to heart homeostasis and disease. Our preliminary data establish a hierarchy for dyad formation in which a little studied protein, CMYA5, tethers jSR to sarcomere Z-lines, and T-tubules associate with jSR to form dyads. We further show that CMYA5 is required for normal dyad architecture, fidelity of E-C coupling, and regulation of RYR2 Ca2+ release activity. Mice lacking CMYA5 had dilated cardiomyopathy and were sensitized to develop severe cardiac dysfunction in response to pressure overload. In failing human hearts, loss of T-tubule and jSR organization were coupled to perturbed CMYA5 localization. Our studies establish CMYA5 as a novel entry point to study mechanisms responsible for dyad architecture and positioning adjacent to Z-lines, and implicate abnormalities of CMYA5-dependent mechanisms in the disorganization of dyads in human heart failure3–5, which contributes to heart failure pathogenesis. Building on these novel observations, we will pursue the following Specific Aims to gain further insights into the function of CMYA5 in regulating CM Ca2+ release and E-C coupling: (1) Investigate CMYA5 regulation of RYR2 activity. We will test the hypothesis that CMYA5 interaction with RYR2 regulates RYR2 Ca2+ release by controlling RYR2 channel activity and RYR2 channel clustering. (2) Identify mechanisms by which CMYA5 tethers RYR2/jSR to Z-lines. We will test the hypothesis that CMYA5 anchors RYR2/jSR at Z-lines through interaction with currently unknown bridging proteins. (3) Evaluate contribution of CMYA5 mislocalization to dyad disruption in human and experimental heart disease. This proposal will reveal novel mechanisms responsible for the subcellular organization of dyads, hallmark nanostructures of CMs that are essential for normal E-C coupling. Elucidation of these mechanisms will provide insights into the mechanisms that perturb dyads in human heart failure, exacerbating contractile dysfunction and arrhythmia, and may lead to avenues to protect E-C coupling in inherited and acquired forms of heart disease.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY PA-21-048 Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship, NOT-MD- 19-001 (Notice of Special Interest in Research on the Health of Sexual and Gender Minority (SGM) Populations): The coronavirus disease 2019 (COVID-19) pandemic has had a profound negative impact on population mental health in the United States, especially for marginalized populations such as sexual minorities (SMs). Emerging research suggests that this disparity is driven by minority stress processes (e.g., stigma) and structural vulnerabilities (e.g., institutional oppression) that systematically expose SMs to more pandemic-related stressors and exacerbate their effects. However, critical knowledge gaps remain regarding the intersectional distribution and upstream (i.e., social and structural) determinants of COVID-19-related disparities in mental health. To address these gaps, the current project will draw on minority stress, intersectionality, and ecosocial frameworks to examine how multiple dimensions of social identity/position and upstream pandemic-related stressors have jointly impacted population mental health for SMs over the course of the pandemic. Leveraging unprecedented data from the COVID-19 Pandemic Sub-Study (a population-based longitudinal cohort study embedded within the Nurses’ Health Study 2 & 3 and the Growing Up Today Study with N>57,000) and novel analytic methods from social, spatial, and legal epidemiology, the project aims are to: 1) estimate the time-varying prevalence of mental health symptoms (i.e., depressive, anxiety, and eating disorder symptomology) over the first year of the COVID-19 pandemic across groups jointly defined by sexual orientation, gender, and race/ethnicity; 2) evaluate whether the prevalence patterns observed in Aim 1 are related to the spatiotemporal distribution characteristics of COVID-19 morbidity and mortality (e.g., county-level mortality rate); and 3) evaluate whether the prevalence patterns observed in Aim 1 are related to the broader pandemic policy environment (e.g., lockdowns/stay-at- home orders, with or without concomitant economic relief efforts). These aims are consistent with the stated priorities in the NIH FY 2021–25 Strategic Plan to Advance Research on the Health & Well-Being of Sexual & Gender Minorities, and importantly, are of urgent relevance to public health. Ultimately, the proposed project will provide a more nuanced and contextualized understanding of SM mental health during the ongoing COVID-19 pandemic, with the intent of generating knowledge that can inform the development and implementation of much- needed mental health equity efforts. A tailored mentored training plan accompanies this proposal and outlines the steps required to advance the Applicant’s career as an independent investigator with expertise in conducting methodologically-rigorous and theoretically-informed SM mental health disparities research.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY The goal of this grant application is to understand how mutations in tetratricopeptide repeat domain 7a (TTC7A) affect formation of the polarized apical membrane; and how these mutations cause human disease. TTC7A loss of function mutations result in severe infantile-onset gastrointestinal disease, with phenotypes related to both gut epithelial and immune cell dysfunction. Previous studies suggest that loss of function in TTC7A results in altered apico-basolateral polarity and lumen formation in intestinal epithelial cells, although how this occurs remains unclear. Prevailing models suggest that initial organization of the apical membrane occurs at an initiation site (AMIS), which is enriched with distinct phosphoinositides compared to the basolateral membrane. The putative function of TTC7A is to serve as a chaperone and scaffolding protein for the phosphoinositide (PI) kinase – PI4KIIIα - at the plasma membrane. This kinase is principally responsible for the generation phosphoinositide PI4-phosphate (PI4P), which is one of the major precursors to the apically enriched PI(4,5P)2 and basolaterally enriched PI(3,4,5)P3. The role of PIs in specialized compartments is thought to partly direct vesicular cargo to the correct subcellular compartment – also responsible for proper polarity and lumenogenesis. Given the known function of TTC7A in coordinating PI4KIIIα localization, we hypothesize that TTC7A mutations perturb PI4KIIIαs normal functionality in cells, resulting in disordered spatiotemporal production of PI(4,5)P2 and PI(3,4,5)P3, thus driving improper cellular polarity, lumenogenesis and endosomal trafficking. To test this idea, we investigate the role of TTC7A in cell polarization, epithelial lumen formation, and endosomal trafficking. We propose studies using patient-derived enteroids, intestinal epithelial cell (Caco2) monolayers and Caco2 cyst cultures to determine the contribution of TTC7A to cell polarity and lumenogenesis. In Aim 1 of this proposal, we will further develop novel technology to monitor the formation of the early apical membrane, protein and lipid movement during apical membrane formation (and lumenogenesis), and how the proper subcellular localization of TTC7A contributes to these processes. We will carry out high resolution confocal and lightsheet imaging of phosphoinositide and apical and basolateral proteins localization in the polarization and lumen formation of Caco2- cysts and enteroids. In Aim 2 of this proposal we further translate our recently established high- throughput, quantitative endosomal trafficking assays for studies on primary patient derived enteroids. Further, we develop novel imaging to spatially probe endosomal trafficking in live enteroids. Our studies seek to identify if the membrane scaffolding function of TTC7A is important for correct spatial orientation of epithelial cells, and how the loss of TTC7A induces abnormal intestinal lumen formation. The results of these experiments may plausibly reveal general rules underlying epithelial development and suggest therapeutic approaches for TTC7A deficiency as well as other more common GI pathologies. Further, successful completion of these aims will provide extraordinary training towards my goal in becoming an epithelial biologist.

NIH Research Projects · FY 2025 · 2022-09

Chronic ocular pain is a highly distressing symptom as its occurrence results in high morbidity without effective treatment. Patients with dry eye (DE) commonly have painful ocular symptoms, yet the neural mechanisms underlying this type of pain may include inflammatory (as in Sjogren's syndrome) and/or neuropathic contributors (as in neuropathic ocular pain [NOP]). The cornea is innervated by the trigeminal nerve, which conveys peripheral input to the central nervous system. Using neuroimaging to evaluate peripheral nerves to supraspinal structures, we propose to define the structural and functional differences in the trigeminal circuit that differentiate inflammatory and neuropathic pain. In aim 1, we will determine trigeminal nerve pathology in persons with Sjogren's vs. NOP vs. controls using quantitative sensory testing (QST), in vivo corneal nerve microscopy (IVCM), and diffusion tensor imaging (DTI). In aim 2, we will define differences between Sjogren's vs. NOP in the central nervous system by comparing functional responses to light-induced pathways associated with photophobia using fMRI, and structure using MRI and DTI. This study is likely to yield (1) structural and functional diagnostic markers to differentiate inflammatory and neuropathic ocular pain with neuroimaging, and (2) evince a neurological source of pain symptoms with neuropathic ocular pain. Data generated from this investigation may be used to improve the diagnosis and monitoring of patients suffering from chronic ocular pain, and provide an objective marker to base clinical decision making.

NIH Research Projects · FY 2025 · 2022-09

Corneal neovascularization greatly increases the risk for corneal graft rejection, and thus contributes to severe vision loss, risk of endophthalmitis, and life-threatening meningitis. It afflicts up to 1.4 million new patients annually and together with other pathological angiogenesis is the leading cause of blindness in the United States. Angiogenesis also contributes to diseases that range from cancer to arthritis. A wide range of protein growth factors (e.g. VEGF, bFGF, PDGF) stimulate angiogenesis, but only VEGF is currently targeted for antiangiogenic therapy in the eye. CMG2 is an integrin-like transmembrane protein with an extracellular domain that binds ECM proteins and an intracellular tail without homology to domains of known function. We find that targeting CMG2 via the protein inhibitor PASSSR, CMG2-binding small molecules, or CMG2 knockout profoundly inhibits corneal neovascularization, but the mechanism underlying this effect is unknown. Angiogenesis requires endothelial cells to migrate towards growth factors. This migration has both a movement component (motility = chemokinesis) and a directional component (chemotaxis). However, these components cannot be distinguished using traditional (wound scratch or transwell) assays. Using a microfluidic migration assay that tracks individual cells over time, we recently discovered that CMG2 targeting completely disrupts chemotaxis, but not chemokinesis. This effect is observed with multiple growth factors (bFGF, VEGF, PDGF) and all targeting methods tried thus far (CRISPR knockout, PASSSR, blocking peptide). Thus, we hypothesize that CMG2 is a key intermediary in a pathway required for growth-factor induced chemotaxis and efficient angiogenesis. We will test this hypothesis by: 1) identifying intracellular interactions required for CMG2-mediated chemotaxis; 2) identifying extracellular interactions required for CMG2-mediated chemotaxis in response to growth factors, and 3) evaluating the contribution of RhoA to CMG2-directed chemotaxis. Our working model of CMG2 signaling is based on preliminary data from our lab and others that indicates that CMG2 localizes near RhoA and several Rho pathway members. Thus, CMG2 is positioned to directly regulate the cell polarity required for directional migration (chemotaxis). Indeed, we observe that inhibiting RhoA phenocopies CMG2 inhibition. Finally, we can bypass CMG2 signaling by activating RhoA via the S1P receptor, so that chemotaxis is no longer sensitive to CMG2 targeting. Thus, RhoA is downstream of CMG2. Successful completion of proposed work will identify the mechanism underlying the strong antiangiogenic effects observed upon CMG2 targeting in vivo and accelerate exploitation of this potential target for broad- spectrum antiangiogenic therapy. In addition, this work will enable the development of pharmacodynamic assays to rapidly evaluate drug leads. Finally, key aspects of this proposal are designed to produce possible therapeutic leads. Drugs arising from these studies could supplement anti-VEGF therapies to treat blindness caused by ocular neovascularization as well as many other angiogenesis-dependent diseases.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT The diagnostic process unfolds across multiple settings over time. Risk factors for error in each setting may vary, but for the patient, once a serious diagnostic error occurs, the specific clinical area where it happened is unimportant. Outpatient and inpatient settings have similar rates of diagnostic harm. Interactions within and between clinical teams and settings may either create resilience or increase risks for failure. Resilience engineering, or Safety II, is based on the concept that safety is a consequence of adaptations to the changing conditions of a system’s function. Robust communication supports a shared mental model that may create diagnostic resilience. Currently, clinician-patient/family communication along the diagnostic journey is haphazard. For example, pediatricians consistently fail to tell parents about “red flags” which are signs of a serious complication requiring immediate attention. Among children with chronic conditions at home, we found that 14% had serious diagnostic delays caused by parental misunderstanding of instructions. Pediatric diagnostic safety is understudied, and overall rates and types of outpatient pediatric diagnostic errors is unknown. To support robust communication with families of hospitalized children, we developed a structured communication intervention (PFC I-PASS) which reduces medical errors by 38%. I-PASS has been pilot tested for use during hospital discharge with similar success. Secondary analyses suggests that I-PASS may be effective at reducing diagnostic errors. PFC I-PASS has not been used in the outpatient setting and its impact on diagnostic safety has not been tested. Among children with multiple chronic conditions, we aim to: 1. Characterize the diagnostic journey, focusing on successes, errors, and patient/family and clinician communication; 2. Adapt PFC I-PASS to create Outpatient PFC I-PASS, a structured communication intervention for patients/families and clinicians in the outpatient setting; 3. Evaluate the effectiveness of PFC I- PASS (outpatient and discharge) to improve patients/family and clinician communication and experience, and to reduce errors and harm. The proposed Diagnostic Center of Excellence is comprised of two cores: a Methods Core and an Education and Dissemination Core. Cores include expertise in the diagnostic safety, Safety I and II, communication, medical education, and health disparities. The cores will work with Patient and Parent, Clinician, and Health System Leader Advisory Panels. At Boston Children’s Hospital, Cincinnati Children’s Hospital Medical Center and Children’s Hospital of Philadelphia, to address aims, we will employ observations, interviews, simulation, surveys, chart review, using S1 and S2 approaches. We will evaluate the impact of adapted PFC-IPASS on diagnostic errors using interrupted time series analysis. Methods will be immediately available to other Centers of Diagnostic Excellence and, through several networks, to over 200 health systems. Combining S1 and S2 approaches to characterize the diagnostic journey and test interventions has the potential to transform patient safety science.

NIH Research Projects · FY 2026 · 2022-09

Two primary features of visual coding—spatial frequency and contrast perception—are abnormal in neurophysiological and behavioral measures of amblyopia. Mounting evidence indicates that interocular inhibition may drive amblyopic deficits, and this has led to the employment of varying gain control models to explain abnormal binocular interaction amblyopia. Psychophysical measures of contrast perception are commonly used to validate these models; however, a single gain control model has been unsuccessful in explaining amblyopic performance. This 5-year mentored training award seeks to address this problem by using novel neuroimaging and visual psychophysics to investigate whether spatial frequency and contrast deficits in anisometropic amblyopia are indeed secondary to interocular inhibition. Each aim of the study corresponds to specific training goals, which will map to competency in four main areas: (1) fMRI experimental design and model-based analyses; (2) computational modeling of both neuroimaging and psychophysical data; (3) clinical research design incorporating the use of fMRI, psychophysics, and computational modeling in clinical populations; and (4) career development. Such training will transform the applicant into an independent translational clinical scientist who can utilize both neuroimaging and psychophysics to examine underlying deficits in pediatric vision disorders. Training will be implemented at the reputable environments of Boston Children’s Hospital, Boston University, and Harvard Medical School with the expert guidance of Dr. Sam Ling (primary mentor), Dr. David Hunter (co-mentor), and Dr. MiYoung (advisor). Specifically, the mentor team will train the applicant to design, implement, and analyze measures of population spatial frequency tuning and contrast response in participants with anisometropic amblyopia. This work will enhance our understanding of interocular inhibition and spatial frequency and contrast-dependent deficits in amblyopia. Our data will guide and constrain models of binocular interaction in amblyopia. Furthermore, this additional neural characterization of the response of the amblyopic visual system to unbalanced dichoptic stimuli will explain variability in the efficacy of alternative therapies and identify new treatments that specifically target deficits in spatial frequency and contrast coding of the amblyopic eye. Through this training, the candidate will gain considerable mentorship and training in advanced neuroimaging techniques to quantify binocular visual function in amblyopia, providing a foundation to build a career as an independent clinician-scientist.

NIH Research Projects · FY 2026 · 2022-09

(PLEASE KEEP IN WORD, DO NOT PDF) This project is rooted in the clinical observation that the blood diseases that affect infants, children, and adults differ. We hypothesize that that is due to developmental and age-related intrinsic differences in the fundamental properties of hematopoietic stem and progenitor cells (HSPCs). Our research group is focused on understanding how temporal differences in HSPCs impact manifestation of blood diseases. The preliminary data supporting this proposal expand our prior studies on the role of the heterochronic Lin28b/let-7 axis in defining the maturation states of definitive HSPCs. Lin28b/let-7 acts as a molecular switch whereby Lin28b is expressed in the fetal state to implement hallmarks of juvenile hematopoiesis such as fetal globin expression, erythroid-biased output, and innate-like lymphocytes. Developmental downregulation of Lin28b releases let-7 microRNAs that target transcripts to establish mature adult myeloid-biased hematopoiesis. We have found that the Polycomb repressor complex 1 (PRC1) component Cbx2 is a target of let-7 microRNAs in the hematopoietic system and that PRC1 controls the expression of master hematopoietic transcription factors (TFs) such as Erg. On the basis of this finding, we hypothesize that histone H2A lysine 119 monoubiquitylation (H2AK119Ub) by PRC1 downstream of Lin28b/let-7 regulates the TF networks that control HSPC self-renewal and differentiation. This proposal aims to understand 1) how Lin28b is normally developmentally downregulated and how it may be aberrantly expressed in blood diseases with oncofetal gene expression such as myelodysplastic syndrome; and 2) how Erg, as a master hematopoietic TF, effects Lin28b/let-7/Cbx2’s control of hematopoietic maturation. Our experience in normal hematopoietic maturation, modeling of blood diseases, and the collaborations that we have established to complete this proposed research position us to answer these key questions, which we believe have immediate applications to better understanding age-biased blood diseases.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Three-dimensional (3D) folding of the genome plays fundamental roles in the regulation of transcription, replication, DNA repair and many other biological processes. Facilitated by Hi-C and related techniques, it is becoming clear that the eukaryotic genome folds at multiple genomic scales to form different types of 3D architecture, including topologically associated domains (TADs) and stripes. Different physical patterns of change may happen to a type of 3D architecture, e.g., a TAD may show change of overall connectivity, or split into smaller TADs. Whereas the existence and functional importance of the genome’s 3D architecture is increasingly recognized, analyzing its dynamic changes is currently a major challenge to biologists. The community urgently needs novel bioinformatics techniques to define potential physical patterns of change for each type of 3D architecture, to systematically detect all changes in the genome, and to statistically determine the significance of each change. Our preliminary data strongly suggest that two physical patterns of change to the genome’s 3D architecture -- TAD splittings and stripe strengthenings -- regulate cell identity transitions. Accordingly, we propose to develop TADsplit and StripeDiff, two bioinformatics toolkits to systematically define these and additionally physical patterns of change to TADs and stripes between samples. As a proof of principle, we will utilize the new techniques to investigate 3D genome alterations during endothelial-to- mesenchymal transition (EndMT), a cell identity transition that plays critical roles in both normal development and many prevalent cardiovascular diseases. We will illustrate new mechanisms by which transcription factors regulate genome’s 3D architectures to oppose EndMT. These investigations have the potential to better guide the treatment of many diseases in which EndMT plays important roles. The novel bioinformatics techniques in TADsplit and StripeDiff will enable researchers to investigate 3D genome changes in diverse biological models of development and diseases.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY / ABSTRACT Children with tracheostomy and home ventilation have an annual mortality rate of 5%, and have the highest healthcare utilization and costs of all U.S. children, with annual hospital charges that exceed $2.5 billion. ARIs are the #1 cause of death and hospitalization in this very high-risk population of healthcare superutilizers. Yet, little is known about the pathophysiology of these ARIs, the mechanisms underlying their severity, and no treatment pathways exist. Our long term goal is to address these knowledge gaps by refining the “one pathogen-one disease” ARI paradigm to a more ecosystem-wide approach to ARI pathobiology in order to develop more precise ARI treatment strategies for this population. The objective of this study is to determine the dynamics – within the airway ecosystem – of the microbiome and host response during viral ARI and their contribution to ARI severity. The rationale is that while most ARIs are viral, viruses infect airways colonized with functional bacteria. In a previous tracheostomy study we found blooms (i.e., higher relative abundance) of a colonizing bacterium during a viral ARI. However, it remains unclear if these blooms represent infections requiring antibiotics or are associated with ARI severity. Our cross-sectional results and those of others show children with dominance of specific microbiota compositions are associated with increased viral ARI severity. We now extend this work by applying metatranscriptomic (microbial function) and transcriptomic (host response) approaches to tracheal aspirates collected longitudinally over an 18-month period from children with tracheostomy and home ventilation. In the first 6 months of our 1-year R56 AI163013 (Mansbach, PI) high- priority award, site teams at 11 U.S. hospitals will complete enrollment of 300 children with a tracheostomy and home ventilation. In late February 2022, these children will begin 6 months of specimen collection. With the expertise of the Emergency Medicine Network (EMNet) and the support of the Pediatric Acute Lung Injury & Sepsis Investigators (PALISI) network, we now seek to complete the remaining 4 years of work, including 12 more months of specimen collection. Using tracheal aspirates collected ~1 week before and at the onset (i.e., day 1) of ARI, we plan to complete 3 Specific Aims. In Aim 1 we will determine if specific bacterial blooms are related to higher viral ARI severity. In Aim 2 we will determine the mechanisms underlying colonizing bacteria becoming pathogenic and how bacterial blooms contribute to viral ARI severity. In Aim 3 we will determine if bacterial blooms are related to the airway host response and viral ARI severity. Our pilot data demonstrate compelling support for our hypotheses. This study has >80% power for all aims, validates the results in a generalizable independent cohort, and creates a robust biorepository from multiple body sites to test future hypotheses. Results from this study will provide fundamental insights into ARI pathophysiology and mechanisms underlying ARI severity including how the airway microbiome relates to bacterial blooms and host responses in this very high-risk population. Ultimately, these results will inform ARI treatment strategies.

NIH Research Projects · FY 2025 · 2022-09

SUMMARY Myeloid cells of the innate immune system (neutrophils and macrophages) interact closely with the vascular endothelium to modulate inflammatory and resolution responses. During early stages of the inflammatory response, inappropriate neutrophils activation causes vascular and parenchymal injury. However, in late stages of inflammation, precisely-controlled neutrophil resolution mechanisms are now considered to be critical to limit tissue damage and initiate regeneration of new vascular channels and parenchymal tissues. Even though neutrophil function in early stages of inflammatory processes is well understood, their involvement in resolution responses is poorly understood. We have found that the sphingosine 1-phosphate (S1P) receptor-1 (S1PR1), a G protein-coupled receptor (GPCR) with well-established functions in vascular and adaptive immune (T and B) cells, regulates neutrophil resolution responses. Specifically, S1PR1 signaling induces a non-inflammatory, long-lived neutrophil phenotype that undergoes efficient phagocytosis. We also found that this novel and unappreciated function of neutrophil S1PR1 signaling axis is critical for efficient recovery from virus-induced lung injury and chemical-induced acute liver failure. To activate this beneficial process, we developed a novel biologic based on our knowledge of S1P chaperones. We hypothesize that local S1PR1 signaling in neutrophils is a general mechanism that resolves inflammatory tissue injury and thus enable vascular and parenchymal regeneration in multiple organ systems. Furthermore, we posit that therapeutic activation of this signaling axis may provide a novel strategy to control chronic smoldering inflammation that lead to fibrotic diseases and organ dysfunction. To test this hypothesis, we will examine GPCR proximal mechanisms and nuclear transcriptional events that are regulated by S1PR1 in tissue neutrophils during resolution responses. Second, we will examine the importance of this signaling axis in resolution responses that are induced after virus-induced lung injury and chemical-induced liver injury in mouse models. Third, we will obtain proof-of-concept data to activate this signaling axis that utilize engineered designer HDL particles that contain ApoA1 and ApoM to stimulate neutrophil resolution responses and enhance vascular endothelial survival and regeneration. These data are anticipated to reveal novel mechanisms of resolution processes and enable innovative therapeutic strategies to control chronic inflammatory diseases.