Grants

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY The face is at the center of our physical identity. Common genetic variants can lead to changes in gene expression dosage impacting normal-range facial differences among human populations. Additionally, deleterious genetic variants can negatively impact gene expression and lead to a craniofacial disorder which collectively account for approximately one-third of birth defects. Genetic studies have revealed that several sequence-specific transcription factors (TFs) are associated with both normal-range facial variation and craniofacial disorders with hallmarks of haploinsufficiency and distinct facial regional changes. For example, facial development is sensitive to changes in SOX9, a key facial TF, over a broad range with smaller changes in TF levels associated with more subtle but increased specificity in phenotypes. Besides affecting normal-range facial variation, 30-50% SOX9 gene dosage reduction in facial progenitor cells leads to a set of craniofacial features termed Pierre Robin sequence (PRS) characterized by underdevelopment of the lower jaw (micrognathia). These findings indicate the importance of precise regulation of TF expression and dosage during facial development. However, it remains unknown how TF changes directly link to gene regulation and ultimately translate into facial morphological consequences in vivo. The proposed study will use SOX9 as an example to start to address this knowledge gap with the newly generated Sox9 dTAG mouse model utilizing the dTAG degron system allowing stage-specific, reversible, and dosage-dependent protein perturbation. Using the Sox9 dTAG mice, this proposal will determine the stage-specific (Aim1) and dosage (Aim2) effects of SOX9 perturbation during facial development. Aim1 will aim to determine the consequences of transient complete SOX9 depletion in the developing face from molecular to morphological levels using single cell sequencing and quantitative morphometric analysis. Genome-wide SOX9 binding region dynamics will also be identified at key facial development timepoints. Aim 2 will aim to determine how in vivo SOX9 dosage titration at key facial development windows leads to acute gene expression changes and final morphology outcomes. Successful completion of these aims will advance the fundamental knowledge about how our faces are shaped and these findings will also shed light on future preventative therapies of the associated craniofacial disorders. My long-term career goal is to lead my independent research program investigating the transcriptional and epigenetic mechanisms of facial structure development and regeneration. To reach this goal, the proposed research and training plan will combine my scientific background in mouse developmental genetics and “web- lab” techniques and postdoctoral training in genomic and “dry-lab” techniques. I will be mentored by Dr. Joanna Wysocka in a rigorous scientific environment (Stanford Chemical and Systems Biology) with additional support from my collaborator Dr. Nathanael Gray. I believe this fellowship award will facilitate the successful execution of the proposed research and training plan for my transition into an independent craniofacial research scientist.

NIAID - National Institute of Allergy and Infectious Diseases

1 PROJECT SUMMARY 2 3 Apicomplexan parasites are a major cause of mortality and morbidity in humans and animals. As a 4 model apicomplexan parasite, Toxoplasma gondii is highlighted by the CDC as the 2nd leading cause of 5 foodborne illness in the US and is classified as an NIAID Emerging/Re-emerging Pathogen. While life- 6 threatening diseases can occur in immunocompromised individuals and fetuses, infections caused by clonal 7 strains frequently found in Europe and North America are mostly benign in healthy humans. However, severe 8 toxoplasmosis outbreaks with fatal consequences among healthy individuals have been documented in South 9 America, where a variety of non-clonal genetically heterogeneous Toxoplasma strains with distinct virulent 10 features are prevalent. To date, the virulence characteristics and parasite genes responsible for the 11 pathogenicity of such Toxoplasma strains in humans remain largely unknown. 12 This project focuses on the virulence traits of human-pathogenic, atypical Toxoplasma strains in the 13 laboratory rat model and aims to understand the genomic basis of pathogenicity and identify genes 14 determining the parasite virulence. The proposal is organized into two specific aims: 1) to elucidate the 15 pathogenic potential of these atypical Toxoplasma strains in the rat model in vivo enabling downstream 16 phylogenetic analysis of known virulence factor and facilitating comparastive genome analysis to detect 17 additional virulence determinants; 2) to utilize a CRISPR/Cas9-based functional genomic screening approach, 18 both in vivo and in vitro, to uncover novel virulence factors in one atypical Toxoplasma strain associated with 19 human fatality. Collectively, the completion of this project will provide novel insight into the molecular basis of 20 high-pathogenic Toxoplasma outbreaks in the human population and contribute to building the scientific 21 framework for the development of therapeutic strategies against acute toxoplasmosis.

NIA - National Institute on Aging

An estimated 6.9 million Americans are currently living with Alzheimer’s dementia, a number expected to reach 12.7 million by 2050. People with dementia, along with others who have decisional limitations, face threats to their legal and autonomy rights to manage health care, residential, and other personal decisions following assessments of decision-making capacity. Capacity refers to a person’s ability to make informed decisions about whether to accept or reject care and is presumed under the law unless a clinician determines otherwise. After a finding of impaired capacity, legal, ethical, and clinical guidance typically directs clinicians to obtain informed consent from a surrogate decision-maker. However, concerns have been raised regarding the effectiveness of surrogate decision-making and its potential for abuse. Additional concerns have been raised about how capacity has been defined and assessed since the 1990s, including the difficulty of making dichotomous judgments about capacity, poor interrater reliability even among experts, and the risk of bias in assessments. At the same time, the legal and policy landscape related to autonomy rights is shifting toward inclusion and supported decision-making rather than exclusion and reliance on surrogate decision-makers. With some exceptions, however, research has focused primarily on the performance of assessment tools and the characterization of capacity deficits in patient populations, rather than on whether approaches to assessing capacity align with evolving legal and policy frameworks for decision-making with people who have decisional impairments. There is limited research on how capacity is assessed in clinical practice overall, and there is little evidence that clinical care has shifted in response to changes in the legal and policy landscape. The specific aims of this project are: (Aim 1) to use qualitative methods to explore experienced clinicians’ current capacity assessment and decision-making practices, experiences, attitudes, and willingness to change across multiple specialties, settings, and geographic regions; and (Aim 2) to develop and pilot test a survey measuring key aspects of capacity assessment and decision-making, enabling a subsequent large-scale quantitative study of clinicians’ practices across diverse specialties and settings. Results from this study have the potential to (1) provide in-depth insight into current capacity assessment and decision-making practices in clinical care that is not currently available; (2) generate empirical evidence regarding how clinicians assess capacity and make decisions for individuals with decisional impairments; and (3) lay the groundwork for future intervention studies aimed at aligning clinical practices with the legal and ethical autonomy rights of people with decisional limitations. Consistent with the goals of R21 grants, this study explores an understudied area, represents an initial step toward a larger research program, and will inform future intervention studies to benefit a population whose fundamental rights currently receive limited protection.

U.S. National Science Foundation

Decision, Risk and Management Sciences

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NYC Department of Cultural Affairs

Decoda, Inc.

NIEHS - National Institute of Environmental Health Sciences

Project Summary Environmental exposure can alter pseudouridine (Ψ) landscape in cellular RNA. Ψ is the most abundant modified nucleoside in nature and it regulates many RNA processes. Similar to N6-methyladenosine (m6A), whose functions in RNA are modulated in part by its reader proteins, we reason that environmental exposure-mediated changes in RNA pseudouridylation and the resulting changes in RNA-protein interactions constitute a major regulatory mechanism of RNA processes that may underlie disease pathogenesis. Yet up to now, few Ψ reader proteins are known, and our knowledge is limited in the mechanisms through which environmental exposure- mediated aberrant RNA pseudouridylation and the ensuing perturbations of RNA-protein interactions contribute to the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Our proposed study aims to bridge this knowledge gap by uncovering the mechanistic link between environmental exposure and ALS pathology through the lens of RNA pseudouridylation and Ψ-protein interactions. Our preliminary data showed that: (1) at physiological concentration, profilin-1 (PFN1) binds directly and preferentially to Ψ-containing RNA; (2) ALS-linked point mutations in an ALS-associated protein, i.e., PFN1 reduce its affinity for Ψ-containing RNA, and (3) Ψ modification reduces RNA affinity for another ALS-associated protein. We organize our proposed research into three specific aims: Aim #1, to examine how RNA pseudouridylation is modulated by xenobiotic exposure in human neuronal cells; Aim #2, to investigate the impacts of RNA pseudouridylation on the transcriptome-wide occupancy of an ALS-associated protein; and Aim #3, to explore the impacts of RNA pseudouridylation and Ψ-protein interactions in xenobiotic and ALS pathological contexts. To accomplish these research aims and facilitate my transition to an independent academic career, I have assembled a mentoring committee with outstanding expertise in environmental toxicology, bioinformatics and neurobiology. The proposed research is built upon my extensive research skill sets, strong preliminary data, and the proposed mentoring/training plan. The outcome of the proposed research will unveil how environmental exposure-elicited aberrant RNA pseudouridylation alters RNA-protein interactions and contributes to ALS, thereby potentially leading to new biomarkers and therapeutic interventions. Moreover, the proposed career development and research plans will bridge gaps in my training and collect robust data for my independent publications and research grant applications, thereby transitioning me to an independent career at the intersection of environmental health science and neurodegeneration.

NIA - National Institute on Aging

Abstract Frontotemporal dementia (FTD) is a heterogeneous neurodegenerative disorder characterized by progressive frontal and/or temporal lobe atrophy. In approximately 50% of cases, FTD is associated with TAR DNA-binding protein 43 (TDP-43) pathology, including nuclear depletion and cytoplasmic aggregation, which can lead to the generation of cryptic exons (CEs). Emerging evidence suggests that these cryptic peptides may be recognized by the immune system, contributing to disease pathology. CD8+ T cell infiltration has been observed in FTD patient brains, and C9orf72-related ALS/FTD mouse models show CD8+ T cell accumulation alongside microglial activation. However, the antigen targets, functional phenotypes, and temporal dynamics of these T cells remain poorly characterized. Our preliminary data, using cryptic peptides validated by mass spectrometry, show that CD8+ T cells from ALS and IBM patients recognize these cryptic peptides, undergo clonal expansion, and exhibit activation markers. Furthermore, CD8+ T cells transduced with cryptic-specific TCRs induce cytotoxicity toward astrocytes with TDP-43 knockdown, demonstrating the potential for cryptic peptides to be naturally processed and presented in neuronal cells. Given the shared pathology of cryptic exon production across ALS, IBM, and FTD, we hypothesize that clonally expanded CD8+ T cells in C9-FTD patients recognize cryptic peptides, with greater expansion in symptomatic compared to pre-symptomatic patients. In collaboration with Dr. Corey McMillan at the Penn FTD Center, we will test this hypothesis and establish a link between CD8+ T cells and FTD progress. This study will provide crucial first insights into the possible role of antigen-specific CD8+ T cells play in FTD pathology, paving the way for potential immunotherapeutic strategies targeting cryptic epitope-specific T cells.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Bariatric surgeries, such as Vertical Sleeve Gastrectomy (VSG) and Roux-en-Y Gastric Bypass (RYGB), are highly effective interventions for obesity and related metabolic disorders. While they lead to weight loss and improved glucose metabolism, the neural mechanisms underlying these effects are not fully understood. These data suggest disparate and spatially distinct activity profiles of arcuate Neuropeptide Y/Agouti-related Peptide (NPY/AgRP), Proopiomelanocortin (POMC), and leptin receptor (LepR) expressing neurons that may contribute to the metabolic benefits of RYGB and/or VSG. A critical question is whether the changes in arcuate neuron activity precede or are required for the observed metabolic benefits of bariatric surgery. To address this, we will employ electrophysiological techniques to study alterations in neuronal excitability and synaptic physiology, providing insights into the activity patterns of these neurons before the weight loss effects are observed post- bariatric surgery. Additionally, we will measure metabolic parameters while silencing or inhibiting the activity of arcuate POMC, NPY/AgRP, and LepR to investigate if these neurons contribute to the RYGB and/or VSG- induced metabolic benefits. Furthermore, we aim to explore molecular alterations in the differential responses of POMC, NPY/AgRP, and LepR based on the type of bariatric surgery. In pursuing these research objectives, our study seeks to understand the intricate mechanisms and cell populations involved in the metabolic benefits induced by bariatric surgery. Together these data have the potential to inform the development of targeted therapeutic interventions for obesity and associated metabolic disorders.

NIGMS - National Institute of General Medical Sciences

The overarching objective of this project is to develop computational methods to infer cellular history from single-cell sequencing data, enabling insights into disease progression in tissues that are not longitudinally accessible, such as the brain to study Alzheimer’s disease or the heart to study cardiomyopathy. Current single-cell approaches excel at capturing static snapshots of cellular states but fall short in reconstructing earlier unobserved cell states and dynamic histories critical for understanding disease mechanisms. To address these gaps, this project proposes a three-pronged approach: 1) leveraging lineage-tracing single-cell RNA sequencing to quantify how far back cellular history can be inferred; 2) using single-cell RNA sequencing data from large donor cohorts to decode human-specific disease dynamics while accounting for biological and environmental variability; and 3) integrating single-cell multi-omics (e.g., RNA and ATAC-seq) to model regulatory mechanisms that drive cellular transitions over time. Each aim will be validated using systems where longitudinal sequencing is possible to ensure methodological rigor and biological relevance. By building upon cutting-edge statistical and artificial intelligence ideas, this research will uncover hidden cellular histories not observable in the collected tissue and identify critical early events in disease progression. These insights will advance fundamental knowledge of complex biological processes, inform therapeutic strategies, and align with the mission of NIGMS to enhance health and reduce disease burden by supporting innovative and impactful biomedical research.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract/summary The factors and mechanisms driving robust respiratory mucosal immunity, particularly respiratory IgA responses, post-infection or vaccination are largely unknown. This represents a significant gap in our understanding necessary for designing future vaccination strategies aimed at providing enhanced mucosal protection against respiratory viral infections including new SARS-CoV-2 variants. This RO1 grant proposal aims to address this critical knowledge gap. Our central hypothesis is that the generation of mucosal IgA and respiratory protective immunity is contingent upon the localized interactions among pulmonary macrophages, CD4 T cells, and B cells within the respiratory tract. Three specific aims (SA) are proposed. Aim 1: Identify the associated mechanisms by which respiratory CD4+ T cells promote IgA production in situ. Aim 2: Elucidate TGFβ-dependent macrophages and B cell interactions in mucosal IgA production. Aim 3: Define the molecular and functional signatures of mucosal cross-reactive IgA-producing B cells. We believe that the insights obtained will be crucial in developing next-generation mucosal vaccines designed to effectively counter SARS-CoV-2 variants and other respiratory pathogens, significantly enhancing public health prevention strategies against respiratory infections. .

NSF

Massive stars are born in thick clouds of gas and dust. Within these environments, radio telescopes have revealed compounds known as “complex organic molecules” (COMs). These carbon-based molecules are precursors of larger molecules that are needed for life. Understanding how COMs form and evolve in space is key to uncovering the origin of complex chemical species on Earth and on planets orbiting other stars. Led by a team at the University of Virginia, this project combines cutting-edge astronomical observations and computational chemical simulations to investigate how and why the chemical content varies across different star-forming environments. This research will help astronomers to understand the chemistry of our galaxy and to trace the building blocks of life across the universe. In addition to expanding our knowledge of space chemistry, the project also focuses on training future scientists. It will support students at every level, from elementary school through graduate school, and increase learning opportunities for students with limited exposure to astronomy and space science. Public events will help bring science education and excitement to local communities. Understanding the origin and evolution of space chemistry, particularly the disparities in chemical content observed between star-forming regions, requires a comprehensive study of a large sample of star-forming objects spanning a wide range of masses, ages, and environments. This project combines (i) multi-wavelength observations of molecules in massive star-forming regions with (ii) state-of-the-art simulations that provide a theoretical picture of the spatial distribution of molecules in such regions at different moments. From here, the team will build a detailed chemical evolutionary sequence for massive star formation, guided by observations that resolve molecular emission across various sources at different scales and evolutionary stages. Synthetic spectra and emission maps generated from the simulations will be directly compared with observational data to identify the chemical pathways leading to various important molecules and to determine how environmental factors shape the chemical content of new stars and planets. This project will directly inform the interpretation of data from radio-telescopes such as the NSF’s ALMA and Green Bank Observatory (GBO). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NCI - National Cancer Institute

Project Summary (Abstract): Glioblastoma (GB) remains one of the most lethal cancers, characterized by a profoundly immunosuppressive tumor microenvironment (TME) that limits the effectiveness of current immunotherapies. While research has heavily focused on traditional regulators of immune cells, nociceptors—pain-sensing sensory neurons known to regulate immune responses in the periphery—have not been studied in GB, creating a significant gap in our understanding of immune regulation in the disease. Of note, in the cranial region, nociceptors are densely concentrated in the dural layer of the meninges but are absent from the brain parenchyma. This anatomical separation from GB tumors has likely contributed to their historical neglect in GB research, overlooking their potential as critical regulators of anti-tumor immunity. Our preliminary data provide compelling evidence that nociceptors play an active role in GB pathogenesis. In syngeneic orthotopic GB mouse models, we observed heightened activation of dural nociceptors in the presence of tumors, marked by increased production of calcitonin gene-related peptide (CGRP), a neuropeptide with known immunomodulatory functions. Furthermore, cerebrospinal fluid (CSF) from GB-bearing mice promotes pronounced axonal elongation in cultured primary trigeminal nociceptors, indicating that tumor-derived factors can directly modulate these neurons. Strikingly, nociceptor ablation in GB-bearing mice leads to transformative changes: prolonged survival, a shift in the TME from an immune-suppressive ‘cold’ state to an immune-activating ‘hot’ state and enhanced responsiveness to immune checkpoint blockade (ICB) therapy. These findings demonstrate that nociceptors, despite their physical separation from the tumor, can remotely regulate GB progression by modulating the immune landscape. To elucidate the mechanisms underlying nociceptor-mediated immune regulation in GB, we are employing methodologies including ELISAs, in vitro neuronal culture assays, single-nucleus and single-cell RNA sequencing (snRNA-seq, scRNA-seq), multi-dimensional flow cytometry, and survival studies under conditions of immune perturbation. These approaches will elucidate the tumor-derived factors that modulate nociceptors and define the bidirectional interactions between nociceptors and immune cells within the TME, revealing the mechanisms by which these neurons regulate anti-tumor immunity. Beyond advancing fundamental understanding, this work holds significant therapeutic potential. By targeting nociceptor-driven immune regulation, we aim to develop strategies to reverse immune suppression in GB, including repurposing existing nociceptor-targeting therapies to enhance efficacy of immunotherapies. Ultimately, our goal is to uncover new therapeutic avenues for improving survival outcomes in patients with this devastating disease.

NIGMS - National Institute of General Medical Sciences

Project Summary Rational enzyme design holds the promise to transform biological research and therapeutic development by enabling the creation of efficient biocatalysts. However, current methodologies in rational enzyme engineering remain limited; computationally designed enzymes often fall short of natural enzymes and require extensive experimental optimization. A fundamental barrier to progress is the incomplete understanding of enzyme sequence-activity relationships. In fact, even the most advanced data-driven and physics-based models cannot reliably and systematically predict mutation effects on enzyme activity. Such a prediction is critical for designing optimal enzyme sequences for efficient catalysis. This limitation underscores the need for novel approaches. Generative AI offers a compelling solution to overcome the barrier, analogous to how language models like ChatGPT interpret semantics in human language. Generative AI can learn from sequences evolved in nature to predict protein function. In particular, the PI has pioneered the application of generative AI to predict enzyme activity using sequence data. Our research has demonstrated that natural sequence information can reliably predict the impact of mutations on enzyme catalytic activity, especially in active sites, enabling the successful optimization of multiple enzymes in biochemical assays. Additionally, we found that loop sequence within TIM- barrel scaffold enzymes can predict novel activity when integrated into de novo designs. These insights represent a new paradigm for studying enzyme catalysis, highlighting how different enzyme regions are evolved for catalytic efficiency in nature. This research program aims to further advance our understanding of enzyme sequence-activity relationships through generative AI, with the long-term goal of achieving fully rational enzyme design. In Direction 1, we will evaluate the generalizability of these relationships across diverse enzymes, laying the groundwork for future sequence-based enzyme engineering. Directions 2 and 3 will overcome the current limitations of black-box AI models by incorporating physics-based modeling to elucidate the sequence-activity relationships identified by generative AI. Specifically, Direction 2 will investigate how active sites are preorganized for efficient catalysis. Direction 3 will explore the role of loops in TIM-barrel enzymes and develop strategies to enhance catalytic activity by engineering these loops. Overall, this proposal aims to introduce innovative methodologies to link enzyme sequences with their catalytic activities, deepen our understanding of enzyme catalysis, and pave the way for fully rational enzyme design.

NIEHS - National Institute of Environmental Health Sciences

Project Summary This project tackles a crucial public health concern: human exposure to nano- and microplastics (NMPs) released directly from everyday plastic food containers, specifically reusable plastic cups, single-use water and soft drink bottles, and takeaway coffee cups, made from four major polymer types, i.e., Polypropylene (PP), Polyethylene Terephthalate (PET), Polyethylene (PE), and Polylactic acid (PLA). Growing evidence suggests that microwaving, high-temperature storage, and other common uses of plastic containers can release billions of microplastics and trillions of nanoplastics into beverages. This study aims to quantify these releases and assess their potential health risks. The research design includes three primary aims: • Aim 1 will measure the release of NMPs from plastic containers under a variety of real-life conditions, generating critical data on NMP concentration and particle characteristics in common use scenarios. This aim will reveal how polymer properties and usage conditions influence release quantities. • Aim 2 will use in vitro and in vivo models to evaluate the biological effects of NMP ingestion on human health, specifically looking at impacts such as inflammation, oxidative stress, and toxicity to cells and organs. This aim will clarify how non-uniform particle shapes and sizes impact cellular and organ responses, with potential biodistribution variations across tissues. • Aim 3 will evaluate how everyday behaviors—such as microwaving, storing, and drinking from plastic containers—affect NMP exposure, establish dose-response relationships for different plastic materials, and assess health risks linked to long-term NMP exposure from water and beverage consumption. This project is innovative in its approach to assessing human exposure to NMPs released directly from plastic food containers, a previously underexplored pathway. By using an advanced laser-based technique to generate NMPs that reflect real-world variability in particle size and shape, the study aims to produce more accurate data on NMP release, health impacts on cell toxicity and organ function, and consumer behaviors that increase exposure. The outcomes will guide practical recommendations for manufacturers and consumers to reduce health risks associated with NMP ingestion from everyday plastic use.

NIDCR - National Institute of Dental and Craniofacial Research

7. PROJECT SUMMARY/ABSTRACT Although recent advancements in single cell and spatial transcriptomics have provided unprecedented resolution to study intercellular communication, there are currently still no comprehensive methods to fully decipher the complexity of truly functional cell-cell communication and to investigate its implications for human health and disease. The long-term goal is to reveal the complex networks of cell-cell communication to better understand their roles in maintaining tissue homeostasis, contributing to disease mechanisms, and ultimately guiding the development of novel therapeutic strategies. The overall objectives in this application are to 1) develop computational methods to systematically decipher functional cell-cell communication, and 2) investigate the regulatory roles of cell-cell communication in cellular states, tissue organization, and disease genetics. The rationale for this project is that while cell-cell communication is recognized as a fundamental aspect of tissue biology, its full complexity, especially in the context of disease, remains incompletely understood. By advancing computational methodologies and integrating multi-dimensional data, we can provide deeper insights into the mechanisms governing intercellular communication, which are crucial for both normal tissue function and disease progression. The central challenges will be addressed by pursuing three specific aims: 1) Develop innovative computational approaches to accurately define and categorize active cell- cell communication networks within tissues; 2) Systematically explore the impact of these communication networks on cellular states and tissue organization; and 3) Elucidate the functional contributions of cell-cell communication in the context of disease genetics. The research proposed in this application is innovative, in the applicant’s opinion, because it leverages cutting-edge computational and experimental techniques to integrate spatial and single-cell data, providing a multi-layered understanding of cell-cell communication. This approach allows for the creation of detailed communication maps and the identification of key regulators that could serve as targets for novel therapeutic interventions. This research is significant because it addresses a critical gap in our understanding of how cell-cell communication influences tissue function and disease systematically. By providing a comprehensive framework to study these communications, this project has the potential to transform our approach to understanding complex genetic diseases and to identify new therapeutic targets. Ultimately, such knowledge has the potential to offer new opportunities for the development of innovative therapies to treat genetic diseases, contributing to the broader field of precision medicine.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Human rhinoviruses (RV) are a major cause of common cold and exacerbator of chronic lung diseases. The antigenic variability of RV across more than 160 different serotypes has hindered the design of interventions to cure or mitigate infection. Despite possessing memory T and B cells that cross-react with different RV strains, most individuals remain susceptible to re-infection. Moreover, neutralizing antibodies (nAb) induced by RV are strain-specific, wane over time, and do not reliably protect. Thus, there is a critical need to identify reliable and durable immune determinants that will inform novel therapeutic targets. Our work in human RV challenge models reveals variable infection profiles based on viral loads, kinetics of viral shedding, and the propensity to develop serum nAb. These differences likely reflect the degree of success or failure of viral control in the upper airways. Our data also indicate the influence of prior RV (and perhaps non-RV) exposures on the response to RV, as evidenced by the contributions of pre-existing innate and adaptive immunity to improved protection. These findings suggest a complex interplay between “memory” innate and adaptive elements in regulating immunity to RV. Nonetheless, significant knowledge gaps remain regarding the role of these components, and how their coordinated actions dictate the degree of protection to the same or different RV strain. To address this, our exploratory study will leverage existing specimens to perform a comprehensive analysis of immune cells and anti-viral mediators in the blood and nose during sequential homotypic and heterotypic RV challenges performed over a 30-week period. Advanced analytical pipelines designed to maximize discoveries will pinpoint a limited set of biologically relevant targets for follow-up studies. Two complementary and reinforcing aims will test the central hypothesis that distinct immune programs determine the degree of local viral control, and reflect immunity that is shaped by prior RV exposures. Aim 1 will address how the dynamics of innate and adaptive cells determine susceptibility to infection. This will be done using high-dimensional spectral flow cytometry in conjunction with machine learning methods, to identify and quantify the expansion or depletion of numerous cell types in the blood during each RV challenge, define their trajectories, and elucidate their relationships to infection outcomes. Aim 2 will answer whether distinct immune programs dictate protection versus infection, and how these programs are shaped by prior exposures. Here, a multi-modal integrative strategy will build immune programs by connecting large cytometric datasets in the blood to mediators in the nose. Interrogation of immune programs at each RV challenge, will provide an unprecedented view of the interplay between innate and adaptive elements in relation to local viral control versus failed viral containment with broader immune activation, and elucidate the impact of prior homotypic and heterotypic RV exposures on these programs. Outcomes will accelerate the discovery of potential innate and adaptive mechanisms for follow-up studies, while informing how to protect against other common respiratory viruses.

NHLBI - National Heart Lung and Blood Institute

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

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY The studies proposed in this application reflect the Principal Investigator’s long-standing commitment to unraveling the regulatory role of kindlin-3 signaling in thrombosis and inflammation. These investigations aim to address critical conceptual gaps relevant to the NHLBI’s mission, as thrombosis and inflammation are interrelated pathophysiological processes contributing to numerous diseases, including cardiovascular disease, acute and chronic lung injury, venous thromboembolism, autoimmune disorders, and sepsis. Central to these processes are platelets and neutrophils, whose dysregulation can lead to severe complications, such as excessive clotting or tissue damage. Our research focuses on elucidating the molecular mechanisms by which kindlin-3, a critical signaling adaptor expressed in cells of hematopoietic origin, regulates platelet activation during thrombosis and neutrophil activity during inflammation. Kindlin-3 is essential for supporting integrin αIIbβ3 activation in platelets, yet its precise role in regulating integrin bidirectional signaling remains poorly understood. Based on our novel discovery that kindlin-3 interacts with myosin in platelets, we hypothesize that the crosstalk between kindlin-3 and the myosin complex facilitates integrin αIIbβ3 activation during inside-out signaling and promotes myosin activation during outside-in signaling. To test this hypothesis, we will investigate the structural basis of the kindlin-3-myosin complex and assess the functional impact on platelet activation and activity using designed kindlin-3 knock-in mouse models. Kindlin-3 exhibits dual roles in neutrophils, acting as a pro-inflammatory and anti-inflammatory regulator. While kindlin-3 promotes β2-integrin- mediated neutrophil recruitment, our recent findings indicate that it also suppresses reactive oxygen species (ROS) production and neutrophil extracellular trap (NET) release in an integrin-independent manner. We propose that kindlin-3 levels in neutrophils are dynamically regulated across different stages of inflammation to balance these opposing functions. Specifically, we aim to elucidate how kindlin-3 is downregulated in extravasated neutrophils at the late stage of inflammation and the mechanism by which it modulates ROS and NET signaling in cellular and mouse models. Collectively, this program will leverage cutting-edge technology by integrating structural and cellular biology with ex vivo and in vivo models to decode the critical role of kindlin-3 signaling in platelets and neutrophils. These insights will lay the groundwork for advances in understanding and treating blood and vascular disorders, aligning well with the mission of the NHLBI.

NSF

With the support of the Macromolecular, Supramolecular and Nanochemistry Program of the Division of Chemistry, Professor Mesfin Tsige of the University of Akron will carry out research using theory and computer simulation to better understand how large, charged molecules called macroions interact and self-organize in liquid environments. These molecules are important in nature and in materials science, and they sometimes show unusual behaviors such as recognizing similar molecules or forming stable, hollow, shell-like structures resembling blackberries. These behaviors are not well explained by existing scientific models. The research could lead to new ways of designing materials for drug delivery, water purification, and other technologies. The project will also support education and workforce development by involving high school and undergraduate students in hands-on computational research. The research team will also collaborate with local industry to strengthen regional connections between academic research and real-world applications. This project will address a fundamental gap in understanding the physical principles that govern macroion self-assembly by employing a comprehensive suite of multiscale computational methods. All-atom molecular dynamics simulations will be used to calculate solvation shell energies, providing insight into the role of solvent quality and ion interactions. Potential of mean force (PMF) simulations will explore how macroions selectively associate, with a focus on the influence of charge distribution and counterion effects. For systems involving chiral macroions, density functional theory (DFT) will be applied to capture electronic structure contributions to assembly behavior. The final phase of the project will develop highly coarse-grained models, using insights gained from atomistic simulations and structural features identified through data-driven analysis, to enable efficient simulation of large-scale aggregation phenomena, including hollow, blackberry-type structure formation. These efforts are expected to yield a predictive framework for macroion behavior that connects molecular-level interactions to mesoscale self-assembly processes relevant to both synthetic and biological systems. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF

Non-technical summary: Polymer based products penetrate all aspects of our life, from food packaging to energy and medical technologies. This wide use of polymeric materials and their poor recyclability (caused by strong covalent bonds) led to the explosive growth of plastic waste and raises the urgent need for creating recyclable polymers. One of the potential solutions is introducing a few reversible (dynamic) bonds. Polymers with reversible bonds traditionally called Dynamic Covalent Networks (DCNs) are not only easily recyclable but have many unique properties not achievable in traditional polymers, including self-healing and shape memory aspects. However, detailed quantitative understanding of mechanisms controlling properties of DCNs remains limited. This hinders development of novel functional materials with desired properties. The main goal of the research proposed here is to unravel the fundamental mechanisms controlling properties of DCNs. Several experimental techniques will be employed to study dynamics and structure of DCNs on different time and length scales. The proposed research will deepen fundamental understanding of mechanisms controlling properties of polymers with reversible bonds. This will be instrumental in a rational design of easily recyclable polymeric materials with unique viscoelastic and self-healing properties not achievable in traditional polymers. The proposed program will significantly impact the education of specialists for future science and engineering critical for the US competitiveness. Technical Summary: Dynamic Covalent Networks (DCNs) containing reversible (dynamic) bonds provide a solution for creating polymers recyclable by design. Moreover, DCNs are not only easily recyclable but have many unique macroscopic properties not achievable in traditional polymers, including self-healing, shape memory, and time programmable functions. However, detailed quantitative understanding of microscopic mechanisms controlling dynamics and viscoelastic properties of DCNs remains limited, due to additional complexity introduced by the reversible bonds. The goal of the proposed here research is to decode the fundamental mechanisms controlling dynamics and viscoelasticity of DCNs. The project focuses on three main objectives: (i) Bridge quantitatively the segmental and chain dynamics with bond rearrangement processes and viscoelasticity of DCNs with both dissociative and associative bonds; (ii) Develop a predictive understanding of the role of steric factor in bond rearrangement processes for both dissociative and associative mechanisms; and (iii) Unravel the mechanism of viscoelasticity in DCNs with microphase separation of reversible bonds. In the proposed work, the chain and segmental dynamics, bond dissociation and rearrangement, and viscoelastic properties of polymers will be studied by a combination of rheology, dielectric and light scattering spectroscopy, and differential scanning calorimetry. It will be complemented by analysis of structure using small angle X-ray and neutron scattering. The proposed experimental research will deepen fundamental understanding of microscopic parameters and mechanisms controlling macroscopic properties of polymers with reversible bonds. This will be instrumental for a rational design of easily recyclable polymeric materials with unique viscoelastic and self-healing properties which are critical for the sustainability. From a broader perspective, it might also have a strong impact on understanding of many biological materials where reversible bonding and interactions play a critical role. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIGMS - National Institute of General Medical Sciences

The removal of misfolded, damaged, and regulatory proteins by AAA proteases is essential for maintaining cellular integrity and health across all kingdoms of life. Disruptions in this process can lead to cellular dysfunction and the accumulation of cytotoxic protein aggregates. Mitochondrial protein quality control is particularly crucial, as proteins within the inner membrane and matrix are highly susceptible to oxidative damage. To prevent inappropriate protein degradation, substrate selection by AAA proteases is tightly regulated. It is thought that these enzymes primarily recognize unstructured sequences at the C- or N-termini of target proteins, using ATP hydrolysis to unfold substrates and translocate them into the proteolytic chamber of a peptidase for degradation. Adaptor proteins add an additional regulatory layer by either promoting or inhibiting the degradation of specific substrates. However, the precise mechanisms by which these enzymes maintain substrate specificity, interact with adaptors, and target substrates under different cellular conditions remain poorly understood. This proposal seeks to elucidate the mechanisms of substrate recognition and adaptor mediated degradation by soluble human ClpXP and membrane-bound m-AAA and i-AAA proteases, along with their accessory adaptors. Mutations in these proteases have been linked to various neurological disorders, and human ClpXP (hClpXP) has emerged as a promising therapeutic target in cancer treatment. Despite the high sequence similarity between bacterial and hClpXP, the mechanisms of substrate recognition and adaptor interaction in the human complex remain unclear. The mechanism by which membrane-bound AAA proteases recognize and degrade both soluble and membrane protein substrates is notably murky. To address these gaps, we will employ a multidisciplinary approach, integrating structural biology, biochemical analysis, genetics, and mass spectrometry to understand the mechanisms of substrate specificity and processing by these enzymes. Pulse-labeling mass spectrometry and genetic approaches will be used to identify substrates for each AAA protease and quantify degradation rates in vivo. This will be followed by biochemical reconstitution of a subset of these substrates in degradation assays to identify degron sequences and understand how adaptors influence substrate recognition and degradation by AAA proteases. Finally, we will determine the cryo-EM structures of these complexes, along with their cognate adaptors, in substrate-free and during substrate recognition, recruitment, and unfolding. Given that many aspects of these degradation mechanisms are still not well understood, the MIRA award will allow the Pl to allocate more time and resources to bridging this knowledge gap while also focusing on training and mentoring a diverse group of scientists. This research will offer critical insights into substrate specificity and adaptor-mediated proteolysis, carrying significant implications for both fundamental science and therapeutic advancements.

NHLBI - National Heart Lung and Blood Institute

Project summary Mechanotransduction is the process by which cells convert external mechanical signals into biochemical signals that shape their phenotypic adaptations. In cardiomyocytes, short-term extracellular stiffening induces readily reversible phenotypic adaptations, while sustained exposure to extracellular stiffening induces persistent changes in cellular structure and chromatin architecture: a phenomenon referred to as 'mechanical memory' (MM). We recently reported that stabilization of microtubule (MT) architecture is required for both the formation and maintenance of MM in cardiomyocytes. In this proposal, I focus on the time-dependent nuclear responses to extracardiac stiffening, including changes in chromatin architecture, gene expression and DNA damage responses. My working hypothesis is that DNA damage is a central component of persistent responses to extracellular stiffening and disease-relevant mechanical stresses. In Aim 1, I will determine the molecular conduits and epigenetic regulators of MM using normal adult cardiomyocytes and a novel cell-culture system with bidirectionally tunable stiffness. After defining the temporal and magnitude thresholds for inducing MM, I will perform a whole genome ATAC-seq to determine the distinct epigenetic landscapes associated with transience vs. persistence of the stiffness-induced phenotype in cardiomyocytes (K99 phase). Building on this foundation, I will then determine the role of the DNA damage response (DDR) in determining the reversibility of nuclear responses to extracellular stiffening. These studies will define whether DDR elements may be targets for therapeutics to limit or reverse MM in CMs. In Aim 2 studies, I will translate these experiments to the tissue level using living myocardial slices (LMS) from normal rat and human hearts. I will determine whether pathomimetic increases in afterload evoke the same time-dependent MM responses, mechanotransduction cascades, and DNA damage signals observed in isolated cardiomyocyte (K99 phase). I will then examine whether interventions targeting MT dynamics and the DDR mechanism will attenuate afterload induced MM (R00 phase). Finally, in vivo studies will explore whether time-dependent MM dynamics, and mitigating strategies, are relevant to the myocardial dysfunction observed in the viable myocardium following a large myocardial infarction(R00 phase). Through the proposed work, I will significantly expand my expertise and facility with several powerful and versatile skills (RNA-seq, ATAC seq, the LMS model, and rodent experimentation) while broadening my ability to work with large datasets and manage a multifaceted research program. During this process, I will pursue interactions and scientific connections with centers and collaborators within and outside my institution that will contribute to the advancement and completion of this work and prepare me for success as an independent research scientist.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Cells continuously synthesize, degrade, and redistribute phospholipids. While vesicular trafficking was once considered the mechanism of this redistribution, recent studies have highlighted the critical roles of lipid transfer proteins (LTPs) in various contexts. For example, large quantities of phospholipids must be supplied to repair damaged lysosomal membranes or to form autophagosomes during starvation. However, it remains unclear why cells expend substantial energy to transport phospholipids even under normal physiological conditions. A major barrier to addressing this question has been the lack of methods to directly and comprehensively measure phospholipid transport in vivo without introducing bulky tags that perturb native lipid behavior. To circumvent this challenge, Aim 1 will establish a method to comprehensively profile inter-organelle phospholipid transport, referred to here as a “fluxome profile,” starting with phosphatidylcholine (PC). Using phospholipase D (PLD), I will perform organelle-specific pulse labeling by converting PC into deuterated PC (d- PC). After a chase period, d-PC distribution will be measured by stimulated Raman scattering (SRS) imaging, which detects vibrational modes of subcellular chemical bonds as well as the carbon–deuterium bond. This minimally perturbative label enables the observation of natural PC transport under physiological conditions. In parallel, I will create a “lipoprint map” by reclassifying organelles based on lipid-derived SRS spectral fingerprints. By projecting d-PC pulse-chase data from various organelles onto this map, I will obtain the fluxome profile, a comprehensive landscape of inter-organelle relationships defined by lipid composition and transport dynamics. In Aim 2, I will extend this strategy to other phospholipid classes by engineering PLD to alter its substrate specificity. This will involve three approaches: (i) an in vivo strategy that converts cellular PLD activities toward two different substrates into spectrally separated fluorescent signals for high-throughput screening; (ii) an in vitro method to pulldown PLD by phospholipids to enrich PLD sequences with desired substrate specificity; and (iii) an in silico deep learning model that predicts PLD activity for virtual screening. These approaches will enable selective deuterium labeling of major phospholipid classes and allow construction of fluxome profiles beyond PC. In Aim 3, I will first establish a method for the absolute quantification of organelle phospholipids. These data will enable quantitative comparison of phospholipid transport when different organelles serve as the pulse- labeling origin, providing the basis for a quantitative network model of inter-organelle phospholipid transport. I will then assess the structural robustness of this network model and experimentally validate it through LTP overexpression or knockout and engineered PLD-based perturbations. These analyses will test the hypothesis that the lipid transport network alone is a sufficient and efficient mechanism for maintaining organelle phospholipid compositions, uncovering fundamental design principles by which cells preserve membrane homeostasis under normal physiological conditions.