Yale University

NIH Research Projects · FY 2025 · 2023-09

Project Summary This proposal's objective is to develop synchrotron-based X-ray imaging technologies to enable high-resolution imaging of brain-wide neuronal circuits. Comprehensively mapping brain-wide circuits is not currently feasible, even in small mammalian model systems, because light microscopy (LM) lacks sufficient resolution and electron microscopy (EM) cannot be applied over large volumes. Leveraging the unprecedented qualities of the new 4th generation synchrotron source at the European Synchrotron, we will develop X-ray nano-holography (XNH) imaging techniques for large-scale imaging of brain circuits. Taking advantage of improvements in source coherence and brightness, we will improve imaging resolution to allow direct visualization of synaptic connections between neurons, and develop imaging protocols that allow imaging of centimeter-scale circuit volumes within a typical beamline experiment. We will combine non- destructive XNH with EM and LM imaging techniques to rigorously and quantitatively validate the accuracy of XNH- based circuit reconstruction. We will then use this correlative workflow to study the relationships between long-range sensory inputs, local synaptic micro-circuitry, and single-neuron activity, investigating how circuits in the posterior parietal cortex (PPC) support perceptual decision-making. Lastly, we will apply XNH circuit-mapping over an entire cortical hemisphere, and utilize deep-learning based machine vision algorithms to obtain a comprehensive atlas of cortical connectivity. This atlas will in principle resolve all long-range connections between cortical areas at single-axon resolution, lending insight into how distinct cortical areas achieve specialized function, and how distributed cortical networks support cognition and are affected by psychiatric disorders.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Autosomal dominant polycystic kidney disease (ADPKD) is the most common, potentially lethal, monogenic disease. Affecting 1:400 to 1:1000 people, mutations in PKD1 and PKD2 genes encoding polycystin-1 (PC1) and polycystin-2, respectively, lead to development of fluid-filled cysts that progressively expand from renal epithelial cells. This expansion damages surrounding kidney tissue, causing fibrosis, and leads to worsening kidney function that ultimately requires dialysis or transplant. Half of the ADPKD population will experience renal failure by the age of 50, and there are few treatment options to prevent such pathogenesis. These preventative solutions are lacking due to limited understanding into the process of cystogenesis, meaning why cysts form and what makes them worsen over time. Immune cells may contribute to this process. Prior literature has suggested a role for macrophages in cyst initiation and expansion, and CD8+ T cells have recently emerged as exerting a potential anti-cystogenic role. Using a novel transgenic suppression model of ADPKD in which a portion of the C-terminal tail of PC1 is expressed, I will compare the immune cell populations present in a non-suppressed disease model and the suppression model with non-cystic controls. I hypothesize that the differential immune cell landscapes between disease models will reveal pro-cystogenic and anti-cystogenic factors that regulate renal epithelial cells in ADPKD pathogenesis. With the expertise in ADPKD from the Caplan Lab and in immunological investigation from the Craft Lab, I will investigate the role of immune cells in ADPKD pathogenesis in murine models using in vivo interventional strategies, and analyze these models by flow cytometry, immunofluorescence, and single-cell sequencing approaches. I seek to define immune cell signaling in ADPKD and to elucidate the implications of this signaling for ADPKD pathogenesis, expecting to identify immune- mediated factors implicated in cystogenesis. In addition to elucidating cellular signaling that modifies the cystic and fibrotic manifestations of ADPKD, this proposal simultaneously aims to identify immune cell properties that could serve as disease biomarkers and provide insight into treatment and monitoring strategies.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT The mitochondrial protein sulfite oxidase (SUOX) has surprisingly emerged as a potential regulator of ribosome biogenesis in a genome-wide screen. Ribosome biogenesis, which occurs in the nucleolus of eukaryotic cells, is a highly regulated process essential for cell function. Despite its importance and relevance to diseases such as cancer and ribosomopathies, the regulation of ribosome biogenesis in human cells is not fully understood. To identify novel regulators of this process, the Baserga laboratory pioneered a genome-wide siRNA screen using the number of nucleoli per nucleus as an endpoint. MCF10A breast epithelial cells have an average nucleolar number of 2-3, and a decrease to one indicates aberrant ribosome biogenesis. SUOX is a mitochondrial protein that has unexpectedly surfaced as a hit from this screen. SUOX oxidizes toxic cellular sulfite to sulfate, and some SUOX variants are known to cause the severe, fatal developmental disease Isolated Sulfite Oxidase Deficiency (ISOD). Validation shows that SUOX depletion reduces nucleolar number and ribosomal RNA (rRNA) biogenesis. I have collected further data demonstrating ribosome biogenesis disruption upon SUOX depletion, and proteomics data strongly supporting a role for SUOX in making ribosomes. The similarity of the presentation of ISOD to that of known ribosomopathies, along with our intriguing preliminary results, has raised compelling questions about the involvement of ribosome biogenesis in ISOD pathogenesis. Drilling down to the mechanistic level, our preliminary metabolomics data reveal that SUOX depletion causes a decrease in the methyl donor required for the nucleolar methyltransferase fibrillarin. Fibrillarin methylates an rDNA-specific histone and rRNA, regulating rDNA transcription and rRNA processing, respectively. To date, no work has investigated the role of SUOX in ribosome biogenesis nor of ribosome biogenesis in ISOD pathogenesis. In Aim 1, I will establish the precise role of SUOX in ribosome biogenesis, taking advantage of established assays measuring multiple stages of the process. I will describe effects of SUOX depletion on rRNA methylation using the recently developed RibOxi-seq method, and histone methylation using established antibodies. I will further validate our results using the auxin-inducible degron version 2 (AID2) system. In Aim 2, I will determine the effects of disease-associated SUOX variants on human ribosome biogenesis by rescuing the defects that occur upon siRNA-mediated SUOX depletion with translationally silent and disease-associated loss-of-function SUOX variants. The experiments proposed will clearly define a role for SUOX in human ribosome biogenesis and describe the effects of known disease- causing variants on this essential cellular process. Our unique approach based on an unbiased screen for nucleolar function, combined with both well-established and novel methodology to study ribosome biogenesis, gives us the opportunity to take the field of SUOX research in an entirely new direction that will open avenues for understanding the function of SUOX in cellular metabolism and human genetic disease.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Placental development is critical in the early stages of pregnancy, as dysregulation of this process can lead to intrauterine growth restriction of the fetus, preeclampsia, and miscarriage. A fundamental step in placental development is the invasion of extravillous trophoblast cells into the endometrium of the uterus. A key mediator of the invasive trophoblast phenotype is cytoskeletal remodeling driven by the activity of the small GTPase Rho. Dynamic changes in the cytoskeleton are indispensable for initiating cell polarity and forming cell protrusions and adhesions that drive cell migration, and the tightly coordinated regulation of these changes is critical for successful placental development. However, the specific signaling mechanisms that regulate Rho GTPase cytoskeletal remodeling during trophoblast invasion remain unclear. Previously published data have suggested that the c-Jun-N-terminal kinase (JNK) subfamily of mitogen-activated protein kinases (MAPKs) may be involved in trophoblast invasion, however the mechanism by which it accomplishes this is not well understood. Recent screens from our lab have identified the Rho GTPase activating proteins (Rho GAPs) SYDE1 and SYDE2 as novel JNK substrates based on kinase-substrate docking interactions. Existing literature indicate roles of SYDE1 and SYDE2 in migration in trophoblasts and cytoskeletal remodeling. It is therefore likely that SYDE1 and/or SYDE2 may be involved in a JNK signaling axis that regulates Rho GTPase cytoskeletal rearrangement and migration during trophoblast invasion. The objective of this proposal is to elucidate how JNK modulates SYDE1 and SYDE2 to control migration and invasion of trophoblasts. This proposal will determine the functional consequences of JNK-mediated regulation of SYDE1 and SYDE2 in trophoblasts by examining the effects on cell morphology, cytoskeletal organization, and migration in human trophoblast cells harboring SYDE1 or SYDE2 deletion or overexpression. Placental tissue samples will also be analyzed to examine clinical relevance of JNK signaling through these proteins. GTPase biosensor assays will be used to determine whether JNK phosphorylation of SYDE1 and SYDE2 positively regulates their activity as Rho GAPs. In vitro GTPase assays will determine whether this mechanism is accomplished by affecting GAP enzymatic activity. In order to establish whether any observed effects are dependent on JNK phosphorylation, JNK inhibitors and docking-site mutant SYDE1 and SYDE2 will be used to ablate JNK-specific phosphorylation. Ultimately, identifying JNK and small GTPase regulators as a key molecular players in trophoblast signaling, as well as understanding the mechanisms by which they act, will allow them to be explored as molecular targets to treat severe pregnancy complications related to aberrant trophoblast migration.

NIH Research Projects · FY 2025 · 2023-09

Project Abstract Alcoholic hepatitis (AH) is characterized by intense liver inflammation and injury in the setting of excess alcohol ingestion. Cytokine and chemokine upregulation leads to immune cell infiltration and drives inflammation in AH. Liver sinusoidal endothelial cells (LSEC) are an important source of chemokine expression in the liver and participate in paracrine signaling to attract immune cells in a process termed “angiocrine signaling”. Pathway analysis of AH liver RNA-sequencing suggests a potential role of IL17 in mediating LSEC angiocrine signaling. IL17 is a cytokine involved in many autoimmune and inflammatory disorders. Our preliminary data shows that IL17 synergically stimulate CXCL chemokine production with TNF, but the underlying mechanism is not clear. The regulation of IL17 production from T cells also requires further study. Super enhancers are DNA regulatory elements that complex with target gene promoters to drive gene expression. Our previous work has highlighted the role of super enhancers in AH inflammation response. Here, we hypothesize that LSECs increase inflammatory chemokine expression and enhance immune cell transmigration into the liver parenchyma in response to T cell IL17 upregulation by super enhancer activation. To test our hypothesis, we will employ complementary cell biologic and in vivo approaches to study the following specific aims: Aim 1. LSECs enhance CXCL1-dependent neutrophil transendothelial migration in response to IL17; Aim 2. Therapeutic targeting of a super enhancer in T cells downregulates IL17 and ameliorates inflammation in AH. In Aim 1, we will explore the role of IκB𝜁, a transcription factor previously implicated in IL17 signaling, in mediating the interaction between IL17 and TNF signaling pathways. We will also assess the transcriptomic changes of IL17 and TNF stimulation and IκB𝜁 silencing on LSECs by RNA-sequencing. Using microfluidic devices to simulate liver microvascular circulation, we will directly observe the effects of IL17/TNF stimulation and IκB𝜁 inhibition on neutrophil chemotaxis. In Aim 2, we will identify the IL17 super enhancer by chromosome conformation capture assays. We will assess the feasibility of CRISPR-mediated sequence-specific suppression of the IL17 super enhancer. We will use a Cre dependent dCas9-KRAB knockin mice with AAV6 viral delivery of single guide RNA to target the IL17 super enhancer in vivo. We aim to achieve T cell-specific suppression of IL17 expression and assess its effect on AH inflammatory response in a murine model. Indeed, in vivo CRISPR gene-editing has already been applied clinically to treat hereditary disorders, highlighting the translational promise of precision genome-targeting therapies. Better understanding of the IL17 mediated angiocrine signaling process and IL17 super enhancer regulation may reveal novel therapeutic targets for treatment of AH. Therefore, our overall aims and approaches are aligned with the mission of NIAAA to further understanding and treatment of alcohol- associated liver diseases.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT The development of broadly neutralizing antibodies (BNAbs) remains an ambitious objective for effective vaccine responses. Methods to reliably elicit BNAbs are not known, nor are the mechanisms for their natural development fully understood. Prior work, including research from our lab, has identified a handful of germline immunoglobulin (Ig) variable (V) gene alleles more likely to become broadly neutralizing antibodies for HIV, influenza, and autoimmunity. Unfortunately, this barely scratches the surface of the over 500 documented alleles broadly distributed across populations. We will work closely with experimental collaborators to develop computational methods accelerating Ig characterization with the long-term goal of BNAbs discovery. Success in these endeavors will contribute to the development of personalized healthcare and vaccination strategies, and increase our understanding of this critical component of the adaptive immune response. This proposal will take advantage of the growing quantity of single cell (SC) data being produced, which allows RNA expression to combine with Ig repertoires to confer a host of useful biological context to analyze. Despite the advantages of SC data and its potential to immunology analysis forward, it remains either missing or represents a fractional percentage of adaptive immune receptor repertoire (AIRR) datasets in public repositories due to a lack of analysis tools appropriate to the challenges of SC. To address this problem, we will develop Ig analysis methods leveraging SC data. Our goals are to improve coverage and accuracy of Ig V gene analysis and identify germline alleles associated with differential Ig expression and immunological outcomes. This is an early critical step in promoting broad use of SC data in AIRR analyses, understanding the complex interplay between Ig and factors not captured within the allelic sequence, and linking AIRR analysis to multi-omics data. Results will additionally guide future studies with experimental collaborators and advance novel methodology suited for Ig genes and B cell biology of diseases.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Chromosome segregation errors such as persistent DNA bridges accelerate genome instability, a hallmark of cancer. These toxic DNA lesions occur at a higher frequency in hereditary breast and ovarian cancer (HBOC) cells with DNA repair deficiencies, and thus defective genome maintenance. PARP inhibitors (PARPi) cause targeted tumor cell death in BRCA-mutant HBOC and have been suggested to further increase the frequency of DNA bridges in cultured BRCA-mutant cells. Despite the therapeutic promise of PARPi, BRCA-deficient cancers develop acquired resistance to this therapy. Recent studies show that immunotherapies can prevent or delay PARPi resistance in HBOC tumors; however, the mechanisms by which PARPi synergizes with immunotherapies remain poorly defined. My preliminary data shows that PARPi treatment leads to coating of persistent DNA bridges with the innate immune DNA sensor cGAS in BRCA-mutant cells, which coincides with increased type I interferon signaling. I hypothesize that PARPi-induced persistent DNA bridges are central to the mechanism of PARPi cytotoxicity and synergy with immunotherapies, therefore requiring rigorous further study to identify strategies that maximize the therapeutic potential of PARPi treatment in DNA repair-deficient tumors. The overall objective of this proposal is to thus define the mechanisms by which PARPi-induced persistent DNA bridges lead to tumor-intrinsic immune activation in HBOC. The proposed research will investigate this objective in two specific aims. Aim 1 will define the structure and nuclear integrity of PARPi-induced persistent bridges by use of transposase-mediated fluorescence imaging, immunofluorescence, live-cell imaging, and correlative light and electron microscopy. How these factors affect cGAS-STING activation will be assessed using immunoblotting, RT-qPCR, and enzyme immunoassays. Aim 2 will establish persistent DNA bridges and bulky anaphase bridges as potential predictive biomarkers that reflect DNA repair defects, chromosome segregation failure, and subsequent cGAS-STING activation in PARPi olaparib-treated BRCA- mutant breast cancer. I will leverage digital images of hematoxylin and eosin (H&E)-stained samples and perform immunohistochemistry staining on a tissue microarray of de-identified, paired clinical samples before and during olaparib treatment, which will be interrogated in the context of ongoing phospho-STING staining. Defining the interplay between chromosomal instability-driven immune activation and tumor response to PARPi will be a first step towards improved HBOC treatment. This proposal will be completed in a supportive, collaborative, and interdisciplinary environment that will allow me to perform fundamental and translational cancer biology research. This training will advance my experimental design, rigorous data analysis, and technical skills in molecular biology, high-resolution microscopy, and cytopathology. My training plan also provides ample opportunities to improve oral presentation, writing, teaching, and mentorship skills to prepare me for a successful career as an academic cancer biologist leading a research group to mentor and train the next generation of scientists.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Developing effective cancer therapies remains a significant challenge due to the heterogeneity of tumor physiology, cytotoxicity towards non-cancer cells, and the emergence of drug resistance, all of which hamper positive patient outcome. Thus, there is a critical need for novel anticancer agents with alternate mechanisms of action. Targeting the DNA replication initiation pathway is an underexplored strategy for cancer therapy. Origin licensing, the first step of DNA replication initiation during which the replicative helicase Mcm2-7 is loaded onto replication origins, is of particular interest because its inhibition is selectively cytotoxic towards cancer cell lines. However, no origin licensing inhibitors have been developed for clinical therapy yet, which is in part due to an incomplete mechanistic understanding of DNA replication initiation in metazoans. Contrary to S. cerevisiae, which has been used extensively to study replication initiation both at the biochemical and structural level, no structures of metazoan origin licensing intermediates containing Mcm2-7 on-pathway to helicase loading have been solved. How small-molecule origin licensing inhibitors bind and inhibit Mcm2-7 loading factors is likewise unknown. These gaps in knowledge have impeded the development of effective drug candidates targeting replication initiation. The goal of this proposal is to uncover structural insights into metazoan origin licensing. A deep understanding of metazoan origin licensing will aid in the development of small-molecule inhibitors targeting DNA replication initiation complexes and in defining the mechanisms by which these inhibitors act. These results will contribute to the long-term goal of developing origin licensing inhibitors as a new class of cancer drugs.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Our current understanding of in vitro protein folding is due to decades of experimental and computational research that provided high-resolution characterization of protein structure, identification of folding principles, and development of folding algorithms. However, proteins often participate in new and unexpected functional and pathological behaviors in vivo. Because protein processes involve a large network of interactions that strongly depend on the environment, understanding how proteins work inside cells requires knowledge of protein structure, stability, and dynamics in vivo. While evidence that the cellular environment perturbs protein behaviors emerged over half a century ago, we still have limited fundamental information about the effects of these cooperative cellular interactions on protein properties. The gap in knowledge is largely attributable to the weak transient nature of interactions in the cellular milieu and challenges associated with studying protein functions in living cells. This limitation is concerning because proteins in cells and organisms are continuously interacting with other biomolecules, which may disrupt the ability of a protein to fold and assemble properly and results in loss of function and eventually disease. To address these gaps, our research group leverages groundbreaking in vivo spectro-microscopy methods, in combination with functional biochemical assays, in vitro biophysical spectroscopy, and numerical analysis solutions to characterize protein structural dynamics in living cells and tissues. This platform will transform our ability to examine unexplored layers of protein complexity and regulation in cells and tissues, specifically: (1) Do classic in vitro protein principles translate to cells? How accurate are the in-cell predictions of folding theory and molecular dynamics simulations? (2) Can we develop methods to visualize the spatial distribution of metabolism and associated metabolic protein structural dynamics in living cells? (3) How does thermal adaptation and acclimation by organisms change the stability, folding, and aggregation of proteins in differentiated tissues? Overall, this work will lead to a greater understanding of how remodeling of the cell interior during development, environmental stress, and disease contributes to protein homeostasis. Unraveling these interactions will improve our molecular-level understanding of essential processes in human health and disease.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Alcohol and illicit drug use during the past month increased from 2019 to 2020 among pregnant women. Of women abstaining from substance use during pregnancy, up to 51% relapse by 3 months postpartum. Importantly, maternal substance use continues throughout childhood and adolescence, with 1 in 8 children under the age of 17 in the United States living with at least one parent with a substance use disorder. While previous work has evidenced prenatal substance exposure effects on child neurocognitive outcomes, prior assessments have overlooked the impact of being raised in households where substance use continues after delivery and throughout childhood. Further, this prior work has focused on prenatal substance use exposure in isolation without considering other psychological risk factors highly comorbid with substance use. Therefore, it is critical to identify the impact of substance use during pregnancy and beyond on child neurocognitive functioning, and determine whether substance use, or the myriad of psychological risk factors associated with substance use, shape child neurocognitive development. Here, I will analyze data from the Adolescent Brain Cognitive Development (ABCD) Study to assess the impact of prenatal and household substance exposure independently and in concert with maternal psychological risk on child executive functioning during early adolescence through measures of neural and behavioral correlates of executive functioning. Together, this work will provide further evidence of prenatal substance exposure effects on child neurocognitive development, highlighting the importance of continued household exposure to substance use and broader maternal psychological risk to child outcomes. Further, this approach will recognize the importance of psychological symptoms beyond substance use and provide an empirical foundation for future lifespan assessments of maternal psychopathology and its impact on other domains of children’s neurocognitive functioning. Finally, clinical implications include informing intervention and treatment programs for families affected by substance use by necessitating parenting-specific support across the lifespan and recognition of co-morbid psychological symptoms.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Genetic variants in the TRIO gene increase risk for neurodevelopmental disorders (NDDs) including schizophrenia, autism, and related disorders. TRIO encodes a large protein with two guanine nucleotide exchange factor (GEF) domains for Rho family GTPases: GEF1 activates Rac1 and RhoG, and GEF2 activates RhoA. We found a cluster of variants associated with autism and intellectual disability that selectively activate or inhibit TRIO GEF1 activity. While our findings highlight the central importance of this enzyme activity for proper brain development, the molecular mechanisms by which TRIO GEF1 activity is regulated, the downstream targets of TRIO GEF1 signaling, and how these processes are disrupted by GEF1-targeting variants remain fundamental, yet unresolved questions. Answering them will reveal how variants in TRIO lead to NDDs and may inform new therapeutic interventions. Our proposal will address these questions in three Aims: Aim 1. To elucidate the mechanism of TRIO GEF1 activation. We discovered that spectrin repeats 6-9 in TRIO bind and autoinhibit its GEF1 activity and that NDD-associated variants in spectrin repeat 8 relieve this autoinhibition. A short list of receptors and kinases has been identified as known or likely TRIO GEF1 regulators, but the mechanisms by which these activators engage TRIO to activate GEF1 activity are unclear. We will use purified recombinant proteins to test how these receptors’ cytoplasmic domains and kinases impact TRIO GEF1 activity. We will also use a FRET-based activity biosensor and morphological measurements to reveal how these mechanisms contribute to Rac1/RhoG activation and neuronal development induced by receptor activation. Aim 2. To identify and characterize the neuronal signaling events regulated by TRIO GEF1 activity. We have generated mice bearing TRIO variant alleles with reduced (K1431M) or elevated (R1078Q) TRIO GEF1 activity. We will use comparative proteomics and phospho-proteomics in samples from wild-type mice versus those bearing TRIO GEF1-inhibiting or activating alleles to identify proteins, signaling events, and TRIO- interaction partners impacted by changes in TRIO GEF1 activity. We will systematically test how manipulation of these GEF1-mediated events impacts neuronal development and synaptic connectivity. Aim 3. To measure how selective changes in TRIO GEF1 activity impact neuronal development and synaptic transmission. Heterozygosity for the GEF1-defective TRIOK1431M allele causes reduced brain size and behavioral defects, consistent with our hypothesis that selective alterations in TRIO GEF1 activity compromise normal neuronal development and synaptic function. We will use quantitative histopathology and electron microscopy in mice bearing the K1431M and R1078Q variants to reveal how altered TRIO GEF1 activity impacts axon, dendritic arbor, and synapse development. Whole-cell electrophysiology and optogenetic manipulation will enable us to identify the consequences of changes in TRIO GEF1 activity on neuronal excitability, synaptic function, and circuit connectivity.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT: In cancer cells, exposure of self DNA to the cytosol is driven by a variety of genomic instabilities such as micronuclei, chromatin bridges, and nuclear ruptures. This cytosolic DNA can be recognized by cytosolic DNA sensors such as cGAS (cyclic GMP-AMP synthase), which triggers a downstream innate immune response. Interestingly and confoundingly, the activation of the cGAS/STING innate immune pathway can protect or sensitize tumors to immunotherapeutic interventions depending on the specific context. Therefore, insight into the ways in which cGAS/STING signaling is regulated in cancer can inform targeted intervention. Sources of cytosolic DNA in cancer cells arise primarily from defects in mitosis that lead to the enclosure of chromosomes in micronuclei that are prone to rupture. These ruptured micronuclei recruit cGAS and nuclear envelope reformation (NER) factors—such as LEM2, CHMP7, and BAF—but it remains unknown how, or if, these NER factors impact cGAS/STING signaling but there is emerging evidence in published and in our preliminary data that there is potential crosstalk between cGAS/STING signaling and NER proteins. The goal of this proposal is to provide key insights into the regulation of the innate immune response to cytosolic DNA in cancer cells by nuclear envelope reformation factors. In order to achieve this goal, I will use transfected herring testes (HT) DNA and transfected DNA-coated beads as models for cytosolic DNA as this can be more readily controlled compared to the stochastic formation of micronuclei, only some of which are unstable and prone to rupture. With this model, I will use CRISPR/Cas9 gene-editing and the auxin-inducible-degron (AID) conditional degradation system to probe the roles of NER factors in cGAS/STING signaling in response to transfected HT DNA and DNA beads. This proposal will address fundamental aspects of cell biology and innate immune signaling that will shed light on immunotherapeutic targets for cancer.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT In Nepal, the prevalence of HTN among adults is 25% is similar to the global prevalence. In Nepal, however, a comparatively larger proportion of adults (44%) are unaware of their HTN status, 33% of HTN patients are receiving treatment, and only 12% of the patients have their BP under control. Despite the availability of proven effective lifestyle changes and low-cost anti-HTN treatment in preventing major vascular events and total mortality, these recommendations have not been translated into practice. In Nepal, the Package of Essential Non Communicable Diseases (PEN) was adopted that includes protocols to detect and manage HTN at the basic health facilities However, major implementation barriers at multiple levels exists: (a) Individual level: low perceived susceptibility, low health literacy, misconceptions; (b) Interpersonal level: peer pressure; (c) Community level: norms supporting unhealthy eating and low medication adherence; and (d) Organizational level: unfilled human resource positions, overburdened health staff, interrupted medical supplies and medicines; inefficient recording and reporting, and inadequate provider-patient interaction. In response to these multi-level implementation barriers, we propose to implement and evaluate a new task-shifting strategy to community health workers (CHW), leading to improved HTN prevention and control. CHWs will : (a) engage with and educate clients more frequently, for longer periods, and in their homes, hence building clients' self- efficacy; (b) improve health system efficiency by providing quality provider-client time to modify lifestyle, monitor blood pressure; and (c) CHWs will directly connect the HTN patients with health care providers at health facilities through time referral. We will conduct a type III hybrid effectiveness-implementation study to implement and evaluate a CHW led HTN prevention and control (CHPC) implementation strategy to deliver increased uptake and sustainment of healthy diet, physical activity, and antihypertensive medication use; leading to lowering of blood pressure.Aim 1 will assessimplementation outcomes of CHPC implementation strategy using the RE-AIM framework at the patient, provider and health system levels. We will utilize mixed methods to measure the Reach, Effectiveness, Adoption, Implementation and Maintenance outcomes for sustained implementation of CHPC. Aim 2 will assess the effectiveness of the CHPC implementation strategy compared to facility-based PEN on systolic BP via a cluster randomized controlled trial. We will recruit 2432 participants with high blood pressure in 171 geographic clusters randomized to assess CHPC on systolic BP (primary outcome). Aim 3 will evaluate the economic sustainability of CHPC. We will collect primary cost data from facilities and participants and use the effectiveness estimate from aim 2 to model the costs and cost- effectiveness and household out of pocket expenditure impacts. If successful, this study will provide the governments of Nepal and other LMICs a HTN prevention and control strategy to mitigate the burden of HTN in low resource settings.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Lipid droplets (LDs) are cellular energy reservoirs in the form of triglycerides and steryl esters and are central to the maintenance of membrane structure and energy homeostasis. They serve as the primary organelle for energy storage both in cells and in organisms (as adipose tissue). To regulate cellular and organismal energetics, lipids must be trafficked to other organelles where they are consumed by fatty acid oxidation or enzymatically altered to maintain membrane structure. Dysregulation of cellular lipid metabolism is a common feature of metabolic disorders such as obesity, diabetes, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). The VPS13 or repeating beta groove (RBG) family of proteins- containing ATG2A/B- comprises proteins thought to partake in pipe-like, bulk lipid transport between two membranes. Deletion of ATG2 and two of its known binding partners lead to massive accumulation of LDs, defects in lipoprotein biogenesis, and full-blown NASH. ATG2 localizes primarily to LDs, and our group has recently demonstrated that the protein can facilitate bulk lipid transfer in vitro. Loss of ATG2 blocks fluorescent fatty acid movement from the LD to mitochondria through yet undefined mechanisms. However, whether ATG2 directly participates in lipid transport at the LD and how other organelles and protein machinery partake in this process is not known. In this proposal, I outline a strategy to directly test ATG2 mediated lipid transport at the LD, identify the membranes that participate in lipid transport at the LD, and mechanistically define the proteins that cooperate in lipid transport. This project is structured to maximize progress toward my training goals in in vitro biochemistry, assay design, and quantitative image analysis, thereby equipping me with a full set of technical and intellectual skills to generate mechanistic explanations to complex biological questions. Accordingly, I place a strong focus on in vitro biochemistry, cell-based assays, and automated image analysis. In aim 1, I draw heavily on the expertise of the Melia and Reinisch labs to directly test for lipid transport activity of ATG2 at the LD using a combination of newly published and newly engineered in vitro lipid transport assays and cell-based assays of LD accumulation. In aim 2, I build on an APEX proximity labeling approach validated in the Melia lab to identify proteins and membranes that ATG2 links to the LD. In aim 3, I employ a wide range of cell-based assays, binding assays, and automated image analysis to determine which proteins mechanistically cooperate with ATG2 to move lipids at the LD. This project will pioneer new approaches to test lipid transport in VPS13/RBG family proteins and it will elucidate novel mechanisms of lipid transfer at the LD.

NIH Research Projects · FY 2025 · 2023-09

Abstract Data-driven evaluation and interventions have improved emergency care across an array of acute care conditions including acute myocardial infarction, stroke and sepsis -- yet, similar systems to support and advance emergency care for OUD are lacking amidst worsening patient outcomes. Our team completed foundational data infrastructure work revealing gaps in ED data systems as well as identifying opportunities to utilize the American College of Emergency Physician's Clinical Emergency Department Registry (CEDR) as a national data backbone of over 1000 Emergency Departments (EDs) for OUD care. We propose to create new data processes that will generate a collection of data products embedded within the CEDR ecosystem to improve the timeliness, quality, accessibility, and usefulness of CEDR data to address the overdose epidemic. To address these critical needs, we propose an acceleration project that will leverage our prior work developing and refining electronic health record OUD data elements both within and outside of the CEDR registry. This proposal will: 1) automate OUD-related data extraction from participating sites, 2) map ED data to a standardized, scalable Observational Medical Outcomes Partnership (OMOP) Common Data Model (CDM) 3) Translate relational data within the CDM into research ready datasets with algorithmic extension of important OUD concepts, and 4) create site and public facing ED OUD overdose prevention dashboards for care improvement and surveillance. We hypothesize that the automation and integration of electronic health record and administrative substance use disorder data into CEDR will improve the ability to identify opportunities for improvement in ED OUD care and advance future research initiatives. During this study we plan to assess digital readiness within CEDR and design an enhanced ED OUD data infrastructure suitable for large scale observational research, support of future real-world clinical trials and benchmarking dashboards (R61 phase) and develop and deploy an ED OUD digital infrastructure across clinical, research and surveillance dimensions (R33 phase). Upon study completion, we anticipate an improved national ED data registry using a standardized common data model, automated site level ED OUD dashboards to guide near-real time local quality improvement initiatives, the existence of an independent ED OUD Registry Research dataset, and a public facing, web based near-real time ED OUD care dashboard.

NIH Research Projects · FY 2026 · 2023-09

Nearly 2 million persons aged 65 years or older are admitted to an intensive care unit (ICU) each year; of those who survive, half will not achieve functional recovery over the subsequent months. To date, prior post-ICU in- terventions targeting functional outcomes have not been successful; moreover, no prior post-ICU interventions targeting functional recovery have focused on older adults, who are more vulnerable to poor functional out- comes than their younger counterparts. Our long-term goal is to develop interventions to improve functional outcomes among older ICU survivors. Our preliminary data suggest that unmet needs across four domains (home environment, skilled rehabilitation, hearing and vision, and informal and formal care) may adversely af- fect functional recovery among older adults who have returned home (either directly or after short-term rehab [STR]) after an ICU hospitalization. The overall objective of this application is to elucidate the unmet needs of older ICU survivors, evaluate the association of these unmet needs with disability, hospital readmissions, and mortality over the subsequent 6 months, and assess barriers and facilitators to addressing these unmet needs. The central hypothesis is that older ICU survivors have unmet needs in the aforementioned domains that rep- resent potential targets for intervention, and that these unmet needs are associated with disability burden, hos- pital readmissions, and mortality. The rationale for the proposed research is that this work will directly inform the development of an intervention to address unmet needs and facilitate functional recovery among older ICU survivors. The central hypothesis will be tested by enrolling a new cohort of older ICU survivors to achieve the following specific aims: 1) To identify unmet needs in multiple domains after return home from an ICU hospitali- zation, evaluate whether these unmet needs are associated with disability burden in the subsequent 6 months, and ascertain whether these associations are moderated by initial discharge destination (home or STR); the domains include: the home environment, skilled rehabilitation services, sensory needs (in hearing and vision), and informal (unpaid) care and formal (paid) home care services; 2) To evaluate the association of unmet needs in these multiple domains with hospital readmissions and mortality over the subsequent 6 months; and 3) Through qualitative interviews with a subset of older ICU survivors (and their caregivers, if applicable), to explore barriers to addressing unmet needs and to gather patient and caregiver input about facilitators in ad- dressing unmet needs to inform a future intervention. The proposed research is innovative because it will use a geriatrics lens to rigorously evaluate unmet needs across multiple domains, which in turn will directly inform the development of a future intervention to facilitate functional recovery among older ICU survivors by addressing these unmet needs. The research proposed in this application will provide a strong evidence base for actiona- ble targets to reduce disability, hospital readmissions, and mortality among older adults who have survived a critical illness, in line with NIA’s strategic priorities.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Capillary leak is common in acutely critically ill children. Although no gold standard definition exists, it is clinically recognized as new or worsening organ failure despite appropriate cardiovascular resuscitation. Unfortunately, little has been learned of the pathophysiologic processes despite decades of struggling at the bedside of volume overloaded children with multiple organ dysfunction syndromes. Treatment is limited to intensive supportive care for failing organ systems. Less confusion exists in vitro, where leak around cultured human microvascular endothelial cells (EC) is identified as disruption of intercellular tight junctions (TJs) with functional changes in monolayer permselectivity. Such changes may be modeled in the EC response to cytokines, including those known to be elevated in the plasma of critically ill children. However, targeting specific cytokines has repeatedly failed to improve patient outcomes. Our overarching hypothesis is that while a great many leak-producing cytokines may be elevated in acute critical illness, there are only limited EC responses and that final common signaling pathways result in leak either between (para-) or through (trans-) ECs are therapeutic targets. However, the relative contributions of trans- and paracellular leak to the clinical manifestations of leak (i.e., organ dysfunction) and the pathways that cause them are incompletely understood. We will focus on targets we identified upregulated in ECs isolated from critically ill children (collected from vascular access insertion equipment and immediately analyzed by single-cell RNA-sequencing). This unique data set has identified candidate regulatory molecules associated with paracellular leak and oncostatin M (OSM) as a novel mediator of transcellular leak. We will test the contribution of these targets to leak in our culture models of TJ-forming human microvascular ECs from a healthy donor (both male and female) skin and lung using trans-endothelial electrical resistance, macromolecular flux assays, morphological analyses, molecular engineering, and immuno- chemical tools. Aim 1 will utilize tumor necrosis factor (TNF) to model paracellular leak to test the hypotheses that ArhGEF15, ArhGAP21, and -26 regulate RhoB activity, promoting junctional disassembly via downstream kinases directly acting on TJs and amplified new gene transcription. We have also discovered that formoterol, but not other β2-adrenergic agonists, inhibits TNF-induced leak and will investigate potential mechanisms. Aim 2 will test the hypothesis that OSM induces vesicle-associated JAK3/STAT3 signaling resulting in transcellular leak, which also depends on new gene expression, and investigate how formoterol reduces OSM-induced leak. Finally, in Aim 3, we will determine if the specific pathways identified in Aims 1 and 2 are recapitulated in intact human capillaries in vivo using human skin xenografts in mice and ex vivo using machine-perfused human lungs. The proposed research will advance our fundamental understanding of how capillary leak occurs in acutely critically ill children and evaluate an FDA-approved therapy re-targeted to the endothelium to prevent or reverse capillary leak, greatly improving the care of our sickest patients.

NIH Research Projects · FY 2025 · 2023-09

Morphogenesis requires careful regulation across multiple dimensions to ensure proper positioning and identity of differentiating progenitor cells. Ultimately, differentiating cells must coordinate multiple physical parameters - their environment, position, proliferation and shape - and use these cues to inform their final state and function. One such cellular shape change broadly utilized to produce complex tissue architectures is apical constriction: the shrinkage of the apical domain of cells to become wedge-shaped. Apical constriction cumulatively drives tissue shape changes during various key developmental processes, including gastrulation and branching morphogenesis. The majority of what we have learned about the physical and regulatory features of apical constriction come from non-mammalian model organisms, primarily invertebrates. Whether mammalian tissues use conserved or distinct apical constriction mechanisms and machinery has not been elucidated, nor is it clear how this conserved biophysical phenomenon can be so flexibly utilized across multiple disparate developmental transitions. Although apical constriction machinery generally converges on the same conserved proteins, their spatiotemporal dynamics vary widely across contexts and species. We aim to identify the proteins that direct mammalian apical constriction, define their spatiotemporal dynamics, and connect the induction of this pathway to core genetic drivers. We will uncover the general principles that ensure robustness and mechanical integrity by examining diverse developmental contexts where apical constriction is a fundamental morphogenic feature. Over the next five years, we will address how cell shape changes initiate and control morphogenesis by answering the following questions: 1) What force-generating mechanisms drive mammalian apical constriction? 2) What molecular mechanisms regulate cortical reorganization during apical constriction? 3) How do developmental cell fate transitions license physical aspects of cell shape? To ensure our findings can be broadly generalized, we will investigate multiple systems where apical constriction is essential, including primary intestinal cell culture and the early mouse embryo. Specifically, we model apical constriction in intestinal crypt formation, primitive streak formation and neural tube formation. These systems will allow us to investigate the dynamics of mammalian apical constriction, localization of key machinery components, protein-protein interactions, and the transcriptional networks that control the timing of their induction. These studies will produce deep insight into the mechanisms driving one of the most widely used morphogenetic cell shape changes, identify novel factors that direct the process, and connect the regulation of cell state to essential physical features. Together, this knowledge will establish a fundamental understanding of how mammalian tissues coordinate morphogenesis. The proposed project aligns with my research group's long-term vision to define cellular mechanisms controlling tissue patterns to understand how architecture regulates cell fates and behaviors.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Cells within populations act collectively, allowing them to behave in ways that exceed the capability of a single cell. At the same time, even a genetically homogenous population exhibits phenotypic diversity, allowing the population to adapt in unpredictable environments. Both of these features complicate treatment of cancer, infections, and diseases. It is of critical importance to understand how phenotypic diversity modulates a population’s behaviors, yet this interaction is not fully understood. The proposed project will address this knowledge gap by studying the collective migration of commensal and pathogenic bacteria. Groups of bacteria (and eukaryotic cells) can migrate collectively by consuming attractants in the environment and chasing the moving gradient that they have created. This allows cell populations to travel over much longer distances than what can be achieved by an individual cell following external gradients. Our lab found that bacterial cells within migrating groups spatially organize themselves by their chemotaxis abilities, or the speeds at which they climb chemical gradients. This spatial organization within the migrating group allows cells with diverse motility behaviors to migrate together because it places high performers at the front, where the traveling gradient is shallower, and low performers near the back, where the traveling gradient is steeper. Here, I will examine the consequences of this spatial organization for populations that are migrating in different environments and performing pathogenesis. Aim 1 will determine how the spatial organization of motility behaviors in a bacterial population is altered when the physical properties of the environment are changed. Then, Aim 2 will explore to what extent a population of a human pathogen utilizes the spatial organization of secondary messenger levels and motility behaviors to co-sort virulence phenotypes. Our findings will be used to develop a mathematical model that describes how a migrating group of cells alters the spatial organization of its phenotypes to migrate across different environments. They will also provide insights on how the spatial organization of swimming behaviors can lead to co-organization of secondary messenger levels and virulence traits, allowing pathogens to perform multiple infection-related tasks simultaneously during migration. Because some of the basic mechanisms involved – e.g. phenotypic diversity in motility and the degradation or consumption of an external attractant to drive the collective behavior – are also present during the collective migration of immune cells and cancer cells, the findings in the project will be relevant beyond microbiology.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract In this grant application we propose to develop and validate an optimal 18F-labeled sigma-1 (σ1) receptor radioligand for translational research in Alzheimer's disease (AD) to further elucidate the role of σ1 receptor in AD pathogenesis and progression, to probe longitudinal changes in σ1 receptor in AD animal models, along with the synapse biomarker synaptic vesicle protein 2A (SV2A), and AD pathologic biomarkers b-amyloid (Ab) and tau, and to explore the potential of σ1 receptor imaging for early diagnosis of AD. AD is a progressive degenerative disorder that afflicts 6 million people in the USA. From a diagnostic perspective, AD is increasingly viewed along a continuum from preclinical AD, to mild cognitive impairment (MCI), and to AD-dementia. The clinical dementia of AD is coupled to a distinct pathology, with plaques composed of b-amyloid (Ab), neurofibrillary tangles of hyperphosphorylated tau protein, and synaptic loss. However, the molecular mechanism(s) of AD pathogenesis is complex and remains elusive. Several hypotheses have been put forward, including the b-amyloid hypothesis, the misfolded tau protein hypothesis, the cholinergic hypothesis, and the involvement of oxidative stress and calcium dyshomeostasis. Before the accumulation of plaques and tangles, the biochemical and morphological changes, such as altered calcium, cholesterol, and lipid metabolism, altered mitochondrial dynamics, and reduced bioenergetic interaction, are all closely associated with functions localized to the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs). The σ1 receptor is situated at the MAM. The most recent research has located the earliest Ab generation in AD to neurons in the MAM, and its critical regulation by the σ1 receptor, thereby confirming the central role of σ1 receptor in AD pathogenesis. As such the σ1 receptor is considered an important target for AD therapeutic development. As a surrogate marker for mitochondria function and regulator of Ab production on the MAM, it also holds great promise as a biomarker for diagnosis of AD at its earliest stage. The research proposed in this application will bridge an important gap in the understanding of the σ1 receptor in AD pathogenesis and progression by leveraging the unique expertise and experience at Yale in novel PET radioligand development, AD mechanism study, and therapeutic target identification. Investigation of the σ1 receptor in AD animal models longitudinally in relation to biomarkers for synaptic density, Ab, and tau, is a natural extension of our ongoing research. When carried to completion, this project will provide further insights into the etiology of AD, and help identify a most sensitive and effective biomarker for early AD diagnosis, and for monitoring of disease progression and the efficacy of emerging AD therapies.

NIH Research Projects · FY 2025 · 2023-09

Abstract This project will use PET imaging and machine learning to relate Mu and Kappa receptor levels in Alcohol Use Disorder (AUD) to clinical outcomes during a quit attempt. Over 14 million adults in the US suffer from AUD and there are few effective pharmacotherapies. Rates of lapse and relapse are remarkably high and this remains the biggest hurdle to successful recovery. Development of new treatments must be based on an understanding of the relationship between neurobiology and behaviors involved in recovery from AUD. Existing evidence suggests that the opioid system, through a balance between euphoria (Mu-Opioid Receptors; MOR) and dysphoria (Kappa-Opioid Receptors; KOR), regulates key clinical outcomes (e.g., alcohol craving, anhedonia, and withdrawal) which play critical roles in alcohol lapse/relapse behaviors in AUD. The balance between these OR receptors in healthy animals is disrupted by the consumption of alcohol and the progression of addiction. There is limited data in humans to characterize this disruption. Developing an understanding of the imbalance between MOR and KOR in individuals with AUD, and the associations of this imbalance with craving, mood, withdrawal, and time to lapse (first drink) during a quit attempt, will facilitate the development of tailored therapies to target the imbalance and improve clinical outcomes. PET studies of people with AUD have examined MOR and KOR availability, separately. Previous studies measured MOR in ventral striatum (VS) with an MOR-selective tracer and found higher MOR in VS of people with AUD compared to healthy subjects (HS). We used a KOR-selective PET tracer and observed that people with AUD had significantly lower KOR availability in amygdala and pallidum vs. HS. KOR availability in key regions (e.g., whole striatum) also predicted a reduction in drinking in participants treated with the opioid antagonist naltrexone. It seems that upregulation of MOR and downregulation of KOR occur in the course of AUD and that both are related to clinical outcomes. No one has ever imaged both targets in the same people. We will use PET to image both MOR and KOR availability in HS and AUD. We will quantify the relationships between MOR and KOR of AUD patients, separately and jointly, to key clinical outcomes during their subsequent quit attempt. We will use linear models to relate regional values of binding of each tracer to clinical outcomes. We will also use advanced clustering techniques to identify distinct groups of participants according to their imaging data. Our goal is to understand the interactions of the two major opioid receptor systems and how they encode the behaviors of AUD sufferers during a quit attempt. The results could guide development of new targeted therapies. Machine learning (Spectral Clustering) will also help us identify features in the images that may be useful in the future for prediction of clinical outcomes.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, affecting up to 0.5% of the general population. HCM confers an increased risk of morbidity and mortality but remains clinically underrecognized. Traditionally, the diagnosis of HCM has relied on comprehensive assessment by echocardiography or magnetic resonance imaging, modalities which are not available for screening of the general population. As novel disease-modifying therapies emerge, there is a need for efficient strategies to improve HCM screening outside specialized centers. The research proposed in this post-doctoral fellowship will leverage advanced computational methods and the expanding availability of wearable and portable technologies to adapt machine learning algorithms for the efficient, point-of-care screening of HCM. In Aim 1, the applicant proposes to use a large electrocardiographic (ECG) library to adapt ECG signals for use with wearable devices and fine-tune those signals for the detection of HCM. Noising-denoising algorithms and cross-modal pre-training with corresponding echocardiographic and cardiac magnetic resonance videos will ensure that the models are robust to noise and learn key representations of the HCM phenotype, respectively. In Aim 2, single-view, two- dimensional echocardiographic videos will be extracted, pre-processed, and augmented to simulate point-of- care image acquisition. Through a self-supervised, contrastive pre-training approach, the applicant will build data-efficient computational models to screen for HCM based on echocardiographic videos reflecting the quality and unique challenges seen with point-of-care use. In Aim 3, the applicant proposes a prospective case-control study of patients with and without HCM, who will undergo point-of-care electrocardiography and echocardiography, to test the feasibility and real-world performance of a two-stage HCM screening protocol based on Aims 1 and 2. The proposal is supported by strong mentorship from experts in biomedical machine learning, computer vision, and implementation science. The Yale School of Medicine offers the facilities and computational resources necessary to accomplish the research goals, whereas the Yale-New Haven Health electronic health record and well-phenotyped echocardiographic and ECG libraries ensure access to a diverse and representative population. The proposed period of mentored research will support the applicant’s training in computer vision, advanced analytics, and medical informatics. The experience, data, and skillset acquired during this period will further support the applicant in preparing for a successful career in the implementation science of cardiovascular artificial intelligence technologies.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY/ABSTRACT Acute megakaryoblastic leukemia (AMKL) is a form of cancer most prevalent in children under four years old. Targeted AMKL-specific treatment options are limited, and survival rates remain variable. A major obstacle to improving therapy options for AMKL is the dearth of data regarding the mechanisms that contribute to AMKL leukemogenesis. Of several known causative genomic alterations in AMKL, the t(1;22) translocation, which encodes the RBM15-MKL1 (RM) fusion protein, is considered a neonatal mutation as it is always diagnosed in children younger than 6 months old. RNA-binding motif protein 15 (RBM15) is required for recruitment to RNA of the N6-methyladenosine (m6A) writer complex and subsequent epitranscriptomic modification of the transcripts. Megakaryoblastic leukemia 1 (MKL1) is a transcriptional coactivator and is involved in gene expression and megakaryocyte maturation. RM retains all functional domains of both proteins and, despite our understanding of these proteins, the properties of RM itself remain poorly understood. The goal of this proposal is to investigate the molecular mechanisms by which RBM15’s association with RNA in the context of the RM fusion protein contributes to leukemogenesis. Based on our preliminary data, we hypothesize that RM alters gene expression via binding to RNA and promoting modification of the epitranscriptome which promotes oncogenesis via aberrant Wnt signalling. To test this hypothesis, two Aims are proposed. The first Aim is to identify RNAs bound and m6A modified by RM using enhanced crosslinking and immunoprecipitation sequencing techniques. This will determine which transcripts are targeted by RM and which adenosine residues are modified following RM binding, and functional analysis will determine pathways important to leukemogenesis. Computational integration with RNA-seq will provide insight into the fate of RNAs targeted by the RM fusion protein in contrast to control RBM15. The second Aim is to investigate the role of candidate proteins in RM- mediated leukemogenesis by knocking down select candidates in a physiologically relevant murine megakaryoblastic cell line model. In vitro and in vivo assessment of leukemogenesis will determine the requirement of these candidates for the survival of leukemia cells in vitro and for maintenance of disease in vivo. These experiments will provide insight into the transcriptomic and epitranscriptomic effects of RM that are critical to the mechanistic function of the fusion protein. A better understanding of the mechanisms driving RM-mediated AMKL will significantly broaden knowledge of AMKL leukemogenesis and provide potential novel therapeutic avenues to improve outcomes and survival rates of neonates with this disease.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Regulation and sorting of leukocyte integrins are fundamental questions in cell adhesion and polarity that has great implication for various inflammatory and autoimmune diseases, but it has been poorly studied compared to cell adhesion receptors in fibroblast cells. We propose to investigate the sorting of leukocyte integrins and tetraspanin proteins that play critical role in immune cell adhesion and migration. We propose its dynamic sorting depends on a novel F-BAR-dependent mechanism that not only depends on curvature, but on a specific range of shallow curvature. We also propose that this mechanism is governing the mesoscale pattern these receptors are assembled, including propagating gradients and cortical oscillations, which link trafficking events to cell polarity. Specifically, in Aim 1, we plan to characterize surface expression and dynamics of these potential transmembrane protein cargos including integrin αMβ2 and CD81 after systematically identify and validate membrane cargos for the endocytic pathway mediated by F-BAR protein FBP17 and CIP4 using nonbiased proteomic approach. In Aim 2, we aim to isolate the factor of membrane curvature and tension using well-defined in vitro systems and to determine their effects on F- BAR membrane binding and tubulation. We will employ a nanobar-based supported bilayer system to critically evaluate if F-BAR protein senses curvature. We will also test the hypothesis that membrane tubulation may require lipid sorting and active membrane mechanics. These experiments will provide a quantitative biophysical understanding of how F-BAR proteins tubulate membranes without the hydrophobic insertion mechanisms commonly used by other BAR proteins. In Aim 3, we will dissect the functional consequences of the altered trafficking by examining spontaneous migration or migration under confinement. In particular, we will investigate the mechanism for polarized membrane receptor gradient formation. Collectively, combining advanced single cell imaging, genome-editing, proteomics, and in vitro reconstitution, the proposed research will shed key insights into the regulation and function of leukocyte integrins through sophisticated coordination and regulatory mechanisms operating at multiple spatial and temporal scales that have not yet been investigated.

NIH Research Projects · FY 2024 · 2023-09

This high risk / high reward exploratory R21 project will make breakthroughs in understanding chemically-induced occupational allergy (asthma, hypersensitivity pneumonitis, skin disease). We will identify small molecules (peptides) that mimic the structure of chemical allergens by combining cutting edge technology with unique reagents generated in our laboratory. The results, including newly identified chemical allergen mimics (mimotopes), will lead to new diagnostics, surveillance approaches, and potential therapies. The immunology of chemical-induced occupational allergy has long remained unclear hindering the development of preventative, diagnostic and targeted therapeutic approaches. Chemical allergens are generally too small to trigger an immune response on their own but attain immunogenicity upon reacting with self-molecules. The specific structures (epitopes) of modified self proteins that trigger immune responses are challenging to define. While hapten-like recognition of chemicals (self-molecule conjugate) are possible, chemical allergens can also alter the native structure of self-molecules, creating new structures (neo-epitopes) recognized as foreign by the immune system. We hypothesize that peptide mimics of chemical allergens can be readily identified using contemporary phage-display libraries and bioinformatics with specific antibodies triggered by chemical exposure, either occupationally or in animal studies. We propose to define mimotopes of a model chemical allergen, methylene diphenyl diisocyanate (MDI), one of the most abundantly produced chemical allergens in the US. Mimotopes will be defined by combining SERA/IMUNE (a high-throughput bacteriophage-bioinformatic technology) with unique reagents from our laboratory (i) a panel of anti-MDI mAbs, (ii) IgE/IgA/IgG antibodies from mouse models of MDI asthma, and (iii) serum antibodies from MDI exposed/asthmatic workers. The biological relevance of newly identified mimotopes will be demonstrated through binding assays with serum samples from MDI exposed workers with and without asthma, animal models of MDI sensitization and asthma, and controls. Strong precedence for the proposed studies comes from published literature on mimotopes of chemical food contaminants, human autoimmune targets, and COVID-19 vaccine epitopes. The present investigation would bring similar cutting edge technology to the field of occupational chemical allergy that affects workers in multiple NORA sectors (Construction, Manufacturing, Transportation) and cross-sectors (Respiratory Health Immune, Infectious and Dermal Disease Prevention). r2P outputs and outcomes will be reduced hazardous exposures/immune disease via new diagnostics.