University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The American Consortium of Early Liver Transplantation-Prospective Alcohol-associated liver disease Cohort Evaluation (ACCELERATE-PACE) study is a prospective longitudinal cohort of patients with severe alcohol- associated liver disease (ALD) evaluated for early liver transplantation (ELT). The cohort leverages the ACCELERATE consortium with 4-linked R01s and 5 additional recruitment sites in the South/Southeast, Mid- Atlantic, Midwest, and West, and will refine and identify best practices in the selection and management of patients with severe ALD considered for ELT across their continuum of care. ALD is now the most common indication for liver transplantation (LT) in the U.S. Historically, LT centers required at least 6 months of alcohol abstinence before LT referral and evaluation, though empiric evidence to support minimum sobriety periods was limited. ELT, defined as LT before 6 months of abstinence, is increasingly performed but with significant practice variability. There is no consensus on optimal ELT candidate selection, and selection criteria vary widely, contributing to disparities in access to lifesaving care. ELT is also controversial due to the potential for liver recompensation with abstinence, which would obviate the need for LT—accurate prediction of recompensation has the potential to increase organ utility and stewardship. Detailed evaluation of the efficacy of alcohol use disorder treatments and improved risk scores based on pre-LT psychosocial factors to predict return to alcohol use are needed to refine selection criteria, optimize post-LT care, and effectively treat AUD. Short- and intermediate-term survival after ELT is excellent, but the incidence and predictors of post-LT complications are poorly defined. To fill these key knowledge gaps, we will enroll and prospectively follow 770 ELT candidates and 270 ELT recipients for 3 years at 9 socio-demographically diverse centers. The proposed Aims will: (i) inform ELT selection criteria and investigate potential sources of bias in ELT evaluation and healthcare disparities in ELT access; (ii) develop risk prediction scores for LT-free survival and recompensation; (iii) identify effective treatments (medical, behavioral) for alcohol use disorder among patients with severe ALD and post-ELT; (iv) evaluate clinical outcomes among ELT candidates and recipients, including mortality, transplantation, post-LT complications (e.g. cancer, cardiovascular events, graft rejection/failure), and quality of life. A comprehensive data repository will include sociodemographic, clinical, geospatial, psychosocial, behavioral, and patient-reported outcome variables. LT documents, checklists, recordings of selection meetings, direct observations of LT procedures, and clinician interviews will identify best practices and pitfalls in candidate selection. A biorepository of blood, urine, explant/donor tissue, pre- and post-LT liver tissue, peripheral blood mononuclear cells, and cross-sectional radiologic imaging will inform future ancillary studies.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT Oxidative alkene functionalization reactions are a fundamental class of organic reactions. These reactions are valuable because they transform readily accessible alkene starting materials into diverse polar functional groups. However, the design strategies that underpin conventional alkene oxidation reactions require electrophilic reagents that serve as both the oxidant and the source of the newly installed functional group. This substantially limits the chemical diversity accessible using these methodologies. A modular approach to alkene oxidation that directly leverages abundant nucleophiles is a long-standing challenge with no general solutions. This proposal is based on a recent discovery from my group that electrochemistry can generate a new class of dicationic adducts between alkenes and thianthrene that are exceptionally selective dielectrophiles. We will study how these adducts can be exploited to develop a suite of alkene oxidation reactions that are otherwise infeasible with modern synthetic tactics. The three specific aims of this research explore distinct but interwoven aspects of this new reaction platform. Aim 1. We are advancing a strategy for strained ring synthesis from abundant precursors Aim 2. We are advancing a modular platform for oxidative alkene heterodifunctionalization Aim 3. We are advancing a strategy for allylic amine synthesis from abundant precursors The methods developed through this work each address long-standing challenges in a fundamental class of organic reactions, alkene oxidations. These new reaction protocols will offer an expanded and diversified pool of building blocks from which the next generation of drugs and molecular probes will be discovered.

NIH Research Projects · FY 2024 · 2023-08

Abstract: Respiratory infections have been among the top three leading causes of global deaths for decades. Their importance is reinforced by the emergence of novel highly transmissible respiratory pathogens, as witnessed in the current SARS-CoV-2 and past influenza pandemics. Current influenza and SARS-CoV-2 vaccines are focused on eliciting antibodies to highly mutable viral surface proteins, and frequent vaccine reformulations are needed to match the antigenicity of constantly evolving viral strains or variants that evade vaccine-elicited antibodies. Therefore, elicitation of lung tissue- resident memory T cells (TRMs), which recognize epitopes that are conserved across viral variants is critical to elicit broad anti-viral immunity. We have developed combination adjuvant-based subunit mucosal vaccine formulations that elicit exceptionally strong and functionally diverse lung/airway CD8 and CD4 TRMs and provide effective and broad protection against influenza A virus (IAV) and SARS-CoV-2 in specific-pathogen-free (SPF) mice. However, a central question is whether vaccine efficacy studies in SPF mice are translatable to humans, who are exposed to diverse microbial species. In recent years, Dirty mice (SPF mice cohoused with pet store mice), have been used to model human immune responses. Significantly, TRM numbers are greatly increased in Dirty mice, but the underlying mechanisms are unknown. We have exciting preliminary data that the lungs and spleen of Dirty mice have markedly elevated number of Granzyme BHI/CD44HI CD8 T cells with transcriptional attributes (T-betLO/EOMESLO/TCF-1LO) reminiscent of precursor TRMs, which are poised for a TRM cell fate. The overarching goal is to exploit the high resolution of our combination adjuvant-based vaccine approach and the Dirty mouse model to elucidate the effects of diverse microbial exposure on the development of pre-TRMs and their subsequent differentiation into TRMs that protect against respiratory viruses. Specific Aim 1 will test the hypothesis that diverse microbial exposure influences the development and protective functions of lung TRMs against IAV and SARS-CoV-2. Here, we will compare the development and transcriptional programming of lung TRMs induced by two combination adjuvant vaccine formulations and protective immunity to IAV and SARS-CoV-2 in SPF and Dirty mice. Specific Aim 2 will test the hypothesis that diverse microbial exposure promotes the conditioning of circulating/lymphoid pre-TRMs, leading to enhanced differentiation of TRMs in lungs of vaccinated Dirty mice. Here, in Dirty and SPF mice, we will incisively dissect whether diverse microbial exposure enhances the pre-conditioning of naïve CD8 or CD4 T cells prior to vaccination and/or antigen-activated effector T cells during vaccination, to a TRM cell fate. Impact:. Proposed studies will leverage microbial exposure to improve the rigor of mouse models to predict human immune response to vaccines, and provide mechanistic insights into the development of TRMs in the lung under conditions of diverse microbial exposure. Hence, this exploratory ‘high pay off’ R21 application blends significance and innovation to lay the conceptual framework for further mechanistic investigations that will pave the way for the development of a biologically relevant and translatable pre-clinical animal model to learn how we can leverage microbiota to enhance vaccine- induced T-cell immunity to IAV and SARS-CoV-2, which are human respiratory viruses of public health importance.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Environmental genotoxins such as oxidation agents, alkylating agents, aromatic amines, crosslinking agents, polycyclic aromatic hydrocarbons, and natural toxins induce a full spectrum of DNA lesions including abasic sites, interstrand crosslinks, and bulky DNA base adducts. These environmental genotoxins are found in our waterways, food, industrial and agricultural chemicals, and air pollution and have the potential to induce mutagenesis and genomic instability if genetic lesions are not repaired. Mutagenesis and genomic instability can lead to developmental disorders, aging, and cancers. HMCES is a replication-coupled repair protein that responds to single-strand DNA abasic sites and prevents their cleavage by AP-endonucleases. Abasic sites are a common lesion caused by environmental genotoxins. My preliminary results suggest that HMCES prevents both genomic instability and mutagenesis, and I hypothesize that it promotes a more faithful repair mechanism such as template switching or fork reversal. For the K99-phase of this proposal I will learn to perform short and long-term mutagenesis assays and DNA deep sequencing methods to understand in detail how HMCES prevents mutagenesis and genomic instability in human cells and promotes more error-free repair. This work will create a technical foundation and blueprint for studies (R00) characterizing the strand-specific replication stress response and how strand-specific obstacles and environmental genotoxins contribute to leading and lagging strand mutagenesis. There are core differences between replication on the leading and lagging strands. DNA replication occurs continuously on the leading strand and discontinuously on the lagging strand through a series of repriming events. I hypothesize that strand-specific lesions and obstacles generate a differential replication stress response, and potentiate mutagenesis differently. I will characterize the lagging and leading strand stress responses using unbiased approaches. Further, I will determine the consequences of strand-specific stress and genotoxins on mutagenic strand bias using deep sequencing-based mutagenesis assays. Ultimately, this proposal will advance the environmental toxicology and DNA repair fields leading to paradigm shifting discoveries.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The long-term goal of my research program is to understand neutrophil heterogeneity and functions in diverse tissues and biomedical contexts. We have combined high-dimensional and single-cell profiling with integrated bioinformatics approaches to defining context-specific neutrophil landscape. Neutrophils, the most abundant human immune cell type, play crucial, first-line roles in regulating swift responses against infections and pathological responses. Emerging evidence has shown that neutrophil response is systematic and context- specific with distinct phenotypes and functions. Burn injuries are among the most common traumatic injuries worldwide. In severe cases, patients can die of infection, shock, and/or organ failure, requiring expeditious clinical responses. Proper wound healing requires a coordinated time-dependent balance of pro- and anti- inflammatory immune pathways to prevent irreversible systemic damage. Despite being the first to mobilize to sites of injury, there remains little known about neutrophil heterogeneity, their fates, and which factors control the plasticity of neutrophils in wound healing. We hypothesize that neutrophils' cellular and molecular landscape change over time during the wound healing process, and that blood neutrophils could be a marker to predict complications and outcomes in severe burn injury. To this end, we will focus on two important discoveries in neutrophil biology over the past decade: (i) neutrophil heterogeneity and adaption to specific tissue environments revealed by high-dimensional flow cytometry and single-cell transcriptome data and (ii) neutrophil reverse migration phenotypes, shown in model organisms demonstrating that neutrophils migrate back to the vasculature in response to inflammation. In this MIRA application, I plan to 1) identify transcriptional and spatial landscapes of neutrophils and their interactions with other immune cells in human burns using multimodal single-cell profiling approaches, 2) define reverse migration neutrophil phenotypes in human burns by performing cross-species single-cell transcriptome analyses of neutrophil heterogeneity in wound healing using clinical samples and pre- clinical zebrafish burn models, and 3) develop analysis pipelines and methods to predict neutrophil behaviors defined by imaging using single-cell genomic data in humans and publicly available data from zebrafish and mouse models. In the next five years, I will establish a research program to study neutrophil heterogeneity and plasticity in wound healing using multidisciplinary approaches. Our approaches are innovative because we will employ: (i) state-of-the-art single-cell technologies, (ii) novel integrated bioinformatics method development and analysis, and (iii) profiling data cross-tissues in humans with in vivo validation models using model organisms. These studies will generate comprehensive data and provide cellular and molecular landscapes of neutrophil heterogeneity to the community studying wound repair and neutrophil biology.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Signaling metabolites control various cellular processes, including cell cycle, differentiation, and adaptations to environmental stimuli. Intracellular trafficking of signaling metabolites is crucial for maintaining cellular homeostasis and integrate metabolic and transcriptional responses. Defects in metabolite transport and distribution may lead to multiple diseases, including cancer, immunological, inflammatory, and metabolic disorders. Subcellular compartmentalization allows the same molecules to partake in distinct biological processes. Signaling metabolites generally act as second messengers for specific proteins or ligands for sensors and nuclear receptors (NR), ligand-activated transcription factors that sense environmental signals and drive cellular response. Because of their intrinsic reactivity, the intracellular levels of NR ligands, along with their subcellular localization, are tightly controlled and may oscillate greatly depending on nutritional states and pathophysiological conditions. Despite our understanding of their functions, our knowledge of how nuclear receptor ligands travel across organelles remains limited due to the lack of specific tools to target such mechanisms. We propose to integrate chemoproteomics, metabolomics, and cellular assays, to develop novel chemical tools to interrogate the protein interactomes of NR ligands and identify their intracellular chaperones. Leveraging these technologies, we intend to reveal the molecular and functional basis of intracellular trafficking of signaling metabolites and identify dedicated protein chaperones that bind NR ligands at their site of synthesis or entry into the cell, transport them to the nucleus, and deliver them to NRs. A driving finding of our preliminary work was the discovery of PGRMC2 as an intracellular heme chaperone that transports heme from mitochondria to the nucleus and regulates the transcriptional activity of heme-responsive transcription factors such as Rev- Erb and BACH1. We will use the experience acquired from this initial work to extend our studies to the identification of other transport mechanisms for known NR ligands, such as fatty acids, that activate PPARs, a family of ligand-activated transcription factors that regulate metabolism and systemic energy homeostasis. The second major goal of this proposal is to develop spatial- and time-resolved protein-metabolite maps, which we expect to go beyond the identification of intracellular trafficking mechanisms and have a broader impact on the field by providing a powerful strategy to study metabolite-protein crosstalk. Lastly, this project uniquely combines our multidisciplinary expertise in transcriptional regulation, metabolism, and chemical biology to lead the exploration of a new exciting findings in cell biology.

NIH Research Projects · FY 2026 · 2023-08

A powerful technology for characterizing subcellular proteomes is “proximity labeling” (PL), in which a catalyst is localized to a specific cellular location, followed by promiscuous tagging of endogenous proteins in the vicinity. The tagged proteins are then isolated and identified by mass spectrometry. Although PL catalysts are powerful, new PL catalysts are needed to enhance the sensitivity and specificity of spatially resolved proteomic mapping. Genetically encoded enzymes can be conveniently targeted to cellular locations of interest, but they are limited in their mechanisms of tagging, which hampers control over the labeling radius (limiting specificity) and restricts which amino acids can be tagged (limiting sensitivity). Synthetic PL catalysts have recently introduced a greater diversity of chemical labeling mechanisms, but new approaches are needed for selective activation of these catalysts in highly specific subcellular regions of interest. We propose that hybrid biological-abiotic PL catalysts can achieve improved sensitivity as well as specificity in spatially-resolved proteomic mapping. We are pursuing this hypotheses through the development of three classes of hybrid PL catalysts, which offer complementary advantages. In Aim 1, we have used directed evolution to discover heme peroxidase enzymes capable of generating highly reactive radicals, which exhibit a shorter diffusion radius and label chemically diverse amino acids, in contrast to the APEX approach that almost exclusively labels tyrosines. This ability to react with more amino acids will enhance sensitivity for detecting proximal proteins. In Aim 2, we have developed hybrid DNA- synthetic PL catalysts that become activated only in highly specific subcellular locations. We are applying these switchable catalysts for activation of PL selectively at protein–protein interactions (PPIs) on the surface of cancer cells, and we will extend this approach for activation at intercellular PPIs in neuronal synapses. In Aim 3, we have developed hybrid DNA-synthetic catalysts that tag proteins through contact-dependent mechanisms, instead of generating diffusible reactive species. We will attach these contact-dependent catalysts to DNA nano- rod structures with tunable lengths and rigidities, enabling precise control over the labeling radius in the range of ~1–50 nm. We are applying all three classes of PL catalysts for proteomic mapping in living mammalian cells, in collaboration with Prof. Lloyd Smith, an expert in high-resolution biomolecular mass spectrometry. Additionally, we are collaborating with Prof. Edwin Chapman to employ these PL tools in cultured neurons to benchmark their performance against existing tools. Throughout the next five years, my laboratory will continue to develop new mechanisms for PL using hybrid abiotic-biological catalysts. I envision that these technologies will be employed not only in my laboratory, but also in the broader community, to elucidate novel protein functions in a variety of biological contexts.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract For the millions of bilingual parents around the world, and in the US specifically, one of the most pressing questions in the face of the Autism Spectrum Disorder (ASD) diagnosis is how to reconcile it with bilingualism. The choice is most frequently framed in terms of whether to maintain the home language or to switch to the community language (English in the US). Existing studies on bilingual children with ASD take the approach of comparing them to monolingual children with ASD in order to establish whether bilingualism carries additional risks to the development of an autistic child10,22,28,62,110,116,121,122,126,131,141,143,152. However, comparisons between bilinguals and monolinguals can be deeply biased, and equally importantly, such a group-comparison approach says little about the best way to support dual language development in young bilingual children with autism. In this proposal we ask: What are the patterns of dual-language development in young bilingual children with ASD, and how does dual-language input shape their language outcomes? An especially significant and innovative aspect of this proposal is its combination of tightly-controlled experimental tasks testing early English and Spanish skills via highly sensitive eye-gaze measures and ecologically-valid measures of language input, obtained via parent-child interactions. Our participants are young children (18-36 months) exposed to Spanish in the home, with and without ASD. We will provide the diagnosis of ASD in-lab, using the family’s preferred language, thus shortening the evaluation timeline, and yielding an immediate benefit to Hispanic and Latine families that are drastically underserved, both in terms of service access and in terms of research participation. Aim 1 is to examine the development of Spanish and English skills in young bilingual children with and without ASD, defining trajectories of dual-language development. Aim 2 is to examine whether dual-language input can be optimized in young bilingual children with and without ASD. Studies under this Aim will offer first evidence for the need to separate dual-language input into distinct streams to optimize language outcomes in bilingual children with ASD. Aim 3 is to characterize input parameters in the linguistic environment of bilingual children with and without ASD over time, and to test the effects of dual-language exposure on language outcomes. This will be the first prospective longitudinal study to examine how families adapt their language practices when they receive a diagnosis of ASD and to investigate how dual-language input shapes language outcomes in bilingual children with and without ASD. Proposed studies are highly innovative in their focus on very young children, use of dynamic processing measures that can capture language skills in children with varying levels of ability, and longitudinal approach. Recruitment of a neurotypical group that is distribution- matched to children with ASD allows for important insights into the effects of ASD diagnosis vs. maturation on input and language outcomes in bilingual children with ASD. Together, the proposed studies are foundational to any future efforts aimed at developing intervention approaches for this high-needs population.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract We have recently discovered dehydroamino acids (DHAAs) in the capsid and matrix proteins that make up HIV-1 virions. These dehydroalanine (DHA) and dehydrobutyrine (DHB) residues result from posttranslational modification of serine, threonine, or cysteine residues. We propose here to investigate the importance and origin of these fascinating protein modifications. Their high prevalence in these viral proteins, in marked contrast to their low levels in the human proteome generally, raises two key questions: a) why are they there? and b) how did they get there? We hypothesize that DHAAs are present in virions because they play important roles in the HIV virion assembly or capsid maturation. We hypothesize further that DHAAs are formed by a presently unknown enzymatic activity in viral or host proteins. To test these hypotheses, we will conduct mutation and inactivation studies of the amino acids that convert to dehydroamino acids and study the effects on the viral replication cycle and infectivity. We will use quantitative proteomics to determine how much of this modification is generated and whether it is before or after viral maturation. We will seek to discover intra- or intermolecular protein crosslinks, which dehydroamino acids are known to be able to form, using innovative proteomics approaches. Further, we will seek to discover the enzyme responsible for the formation of these modifications. The discovery of the enzyme responsible for DHAA formation would be interesting as fundamental biology, offer new insights into HIV replication, and potentially reveal a new therapeutic pathway for the treatment of HIV infection.

NIH Research Projects · FY 2025 · 2023-08

Abstract Dysphagia is a major consequence of Alzheimer’s disease (AD) that is understudied and thus undertreated despite high prevalence and high cost to heath care systems. Pathology in AD (inflammation, amyloidosis, phosphorylated tau) occurs in the central and peripheral nervous systems early in disease progression and in brain regions and muscle systems associated with swallowing functions. Barriers to effectively treating dysphagia in AD are the lack of: (a) an understanding of central and peripheral pathology associated with dysphagia, and (b) controlled studies of interventions at crucial timepoints, including early versus later in the disease process. The proposed research is significant in addressing these barriers and rigorous in that we will apply established translational research approaches currently used in our labs in models of aging and Parkinson disease. Our scientific premise is that pathology in AD occurs in the central and peripheral nervous systems early in disease progression and that exercise interventions can mitigate deficits induced by the presence of pathology and potentially change the course of sensorimotor decline in function. Because tongue muscles are primary actors in the swallowing process, our central hypotheses are that pathology manifests in tongue muscle and brainstem, subcortical, and cortical regions associated with oropharyngeal swallowing and that early implementation of tongue exercise leads to better swallowing outcomes. We will gain insight into mechanisms by using the well-established TgF344-AD rat model and conducting physiological, morphological, bioenergetic, neuroimaging, and behavioral assays in the brain and tongue muscles. Feasibility data show the oromotor and swallowing dysfunction, evidence of inflammation in the brainstem, and increased beta-amyloid in brain regions associated with swallowing in 12-month-old TgF344-AD rats. The tongue exercise intervention is modeled after those used in clinical practice. However, these clinical protocols are not optimized due to barriers in human research, such as presence of co-morbidities, adherence confounds, and limited access to tissues. Aim 1 will test the hypothesis that TgF344-AD rats demonstrate deficits in oromotor and swallowing behaviors and manifest pathology in tongue muscles and brain structures critical to swallowing function. Aim 2 will test the hypothesis that early implementation of tongue exercise improves oromotor and swallow function and modulates pathology in TgF344-AD rats. This research is innovative and will provide a new understanding of mechanisms that underlie swallowing deficits in AD, query relationships among relatively unexplored AD pathology and physiological function in swallow-related systems, and establish the effectiveness of early versus late tongue exercise intervention for AD. Rehabilitation is often not provided to patients with AD due to uncertain benefit. To advance evidence-based treatment, we must provide preclinical data. This foundational work has a high impact because of the large and increasing population of people with AD-associated dysphagia who can benefit from treatments optimized in the proposed studies.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Genome editing is an exciting avenue for treating common and rare genetic diseases, and the advent of CRISPR/Cas technologies has accelerated the development of this therapeutic option. These therapies rely on cellular DNA repair machinery to install their edits, so changes in the balance between DNA repair pathways result in different editing patterns. This dependency on endogenous DNA repair has led to considerable variability in editing efficiency between cell types or even among targets in the same cell. Thus, a long-term goal of my laboratory is to investigate factors that modify the efficacy of genome editing and to develop strategies that address these shortcomings. Histone modifications play a role in many cellular processes, including transcriptional regulation and DNA repair. Recent evidence found that histone modifications correlate with biases for specific DNA repair pathways. However, given their role in multiple processes, interpreting the effects of individual histone modifications has been challenging. I will dissect these effects to define the role of histone modifications in the repair of double-strand breaks by innovating a platform that recruits histone modifiers to thousands of break sites in parallel. This work will determine whether histone modifications influence DNA repair and uncover properties of the target site that predict these effects. For genome editing to reach its therapeutic potential, precise control of DNA repair is required. I will identify peptides from the human proteome that alter the repair of double-strand breaks. To do this, I will adapt a peptide screening platform I developed to investigate DNA repair. The peptides will reveal critical protein-protein interactions and other methods for altering DNA repair. The completion of these projects will significantly advance our understanding of mammalian DNA repair and has the potential to improve genome editing therapies.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Non-typhoidal Salmonella are successful foodborne pathogens in part because of their ability to utilize diverse nutrient sources to support disease in many hosts. Within the mammalian gut, catabolism of organosulfur compounds by the host and/or the microbiota can lead to production of the electron acceptor dimethyl sulfoxide (DMSO). The genome of Escherichia coli K12, a closely related gut-commensal bacterium, encodes a single co- transcribed operon dedicated to the anaerobic reduction of DMSO (dmsABC) and has been used as a model system to study DMSO respiration in Enterobacteriaceae. In contrast, Salmonella serotypes that cause enteric disease encode three operons homologous to dmsABC, suggesting this pathway is important to support fitness within the gut. Our prior work demonstrates that DMSO reduction is a biologically relevant pathway to support Salmonella fitness during acute intestinal colonization. In vitro phenotyping suggests STM0964 is the dominant homolog of dmsA, the catalytic subunit of a DMSO reductase, while STM4305 acts an alternate dmsA homolog during anaerobic growth. However, there is a critical gap in our understanding of how the bacterium regulates the use of each DMSO reductase and how each DMSO reductase contributes to fitness during enteric infection. Genetic redundancy in anaerobic respiration pathways is a common theme in Enterobacteriaceae that allows bacteria to benefit from changes in nutrient availability to regulate fitness in the gut. My preliminary data shows that DMSO increases the promoter activity of the alternate dmsA homolog, STM4305. The promoter activity of the dominant dmsA homolog, STM0964, is not activated by DMSO akin to E. coli dmsA, suggesting that Salmonella possesses a novel mechanism for transcriptional regulation by DMSO. I hypothesize that differential activation of DMSO reductases supports Salmonella fitness within the gut. In Aim I, I will establish a mechanism for DMSO-mediated transcriptional regulation of the alternate DMSO reductase using biochemical, genetic and RNA sequencing approaches. In Aim II, I will utilize fluorescence microscopy and competitive infections to elucidate the contribution of each DMSO reductase during enteric infection of the bovine host. The proposed work will integrate my veterinary training with large animal modeling of enteric disease and advanced gene expression analysis to establish how Salmonella benefits from apparent genetic redundancy in DMSO reduction. At the completion of fellowship training, I will be poised for success in a career as an independent clinician-scientist with expertise in genetic approaches and animal modeling to study infectious diseases of One Health significance.

NIH Research Projects · FY 2026 · 2023-08

The co-occurrence of age-related cognitive and sensory organ impairments exacerbates negative effects on the health, lifestyle, and quality of life of older adults. These age-related multimorbidities include Alzheimer’s disease and Alzheimer’s disease related dementias (AD/ADRD), in addition to sensory declines such as loss of vision, hearing, and olfaction (sense of smell). We hypothesize that these multimorbidities share common neurotoxicant risk factors that are present in the home environment. With the recent increase in time spent at home, the human exposure to home environment neurotoxicants is changing. However, the effects of these changes on the risk of developing neurocognitive-sensory aging and AD/ADRD are unknown and will take many decades to understand in humans. We propose to study pet (companion) dogs as an efficient sentinel model for the human health impacts of home environment-derived neurotoxicants. Pet dogs are promising sentinels as they share the human indoor and outdoor home environment, share similar exposure to toxicants in the home, yet have shorter latency of onset of toxic effects. Dog lifespan is significantly shorter than humans, yet both species experience similar multimorbidities of neurocognitive-sensory aging. Therefore, epidemiologic studies in dogs could help us quickly understand the human health implications of changes in risk factor exposure. We will determine shared human/dog neurotoxicant exposure and dog neurocognitive-sensory outcomes related to a common group of neurotoxicants found in the home environment, the toxic heavy metals As, Cd, Cr, Hg and Pb. These metals accumulate in neurologic tissues and have well-defined neurotoxic effects, but their effect on age-related neurologic decline is unclear. In Aim 1 we will establish shared risk of multimorbidity of neurocognitive-sensory aging in cohabiting humans and dogs. We will determine the cognitive and sensory function declines associated with aging in dogs and relate the trajectory of dog declines to cohabiting human declines. In Aim 2 we will validate companion dogs as sentinels for human and environmental heavy metal exposure by comparing drinking water and house dust concentrations of heavy metals with dog and human blood concentrations of heavy metals. In Aim 3 we will determine the impact of high heavy metal burden on dog neurocognitive-sensory aging multimorbidities, and on metabolism in the blood. The outcomes of this work will establish the companion dog as a relevant, expedient surrogate model of human age-related neurocognitive-sensory decline and AD/ADRD risk and confirm that humans and dogs share home environment heavy metal risk factors that increase the risk of metabolic dysfunction and neurocognitive-sensory decline in aging. Future studies could utilize this sentinel dog model to test promising therapeutics prior to translation to humans. This work is relevant to the mission of multiple NIH institutes as it encompasses the important topics of aging, cognitive decline, sensory decline, and toxicant risk factors for neurocognitive-sensory aging and AD/ADRD risk in a relevant animal model with an accelerated timeline.

NIH Research Projects · FY 2025 · 2023-08

Project Summary: Dementia due to Alzheimer’s disease (AD) affects 1 in 8 Americans over the age of 65, and is currently not well treated. While therapeutic development has largely focused on clearing brain amyloid via antibody approaches, brain metabolism is also known to be substantially altered in the disease. Altering the metabolic state—for example, via ketogenic diet—can improve cognition through incompletely understood mechanisms. Previous studies indicate that acute supplementation with the metabolite β-hydroxybutyrate (BHB), one of the ketone bodies produced as a result of ketogenesis, improves cognitive function both in people with AD dementia and in mouse models of AD. However, the factors—apart from diet—that impact BHB levels, as well as the specific mechanisms by which BHB may exert positive impacts on the brain are unknown. Our research team has generated several important leads that better inform the factors that impact BHB levels, as well as discovering that BHB impacts AD pathology through inhibition of the inflammasome in microglia. While previously underappreciated in studies of ketogenic diet, gut microbiome has a significant impact on BHB levels. Using gnotobiotic mice, we provide preliminary evidence brain levels of BHB can be altered by precise manipulation of the gut microbiota. We have also found that modifying the abundance of BHB through long-term direct administration in the drinking water results in remarkably diminished plaque burden and microgliosis in 5XFAD mice. Further, in our studies of tissue from individuals in the Wisconsin ADRC with AD dementia who came to autopsy, we found that brain levels of BHB levels were lower compared to individuals without AD dementia at death. In the proposed study, we will follow up these findings to determine how BHB modulates disease progression and address knowledge gaps that would facilitate therapeutic use of this metabolite. Answering these questions has immediate translational implications and is expected to lead to novel strategies to prevent or slow the course of AD. Here, we hypothesize that BHB protects against AD-associated pathology by inhibiting Nlrp3 inflammasome activation through activation of Hcar2 in microglia. We will determine the features of the inflammasome that mediate the effects of BHB on AD pathology in the 5XFAD mouse models of amyloid β plaque deposition, determine the extent to which gut microbiome impacts BHB levels via butyrate producing bacteria, and finally, using human metagenomic and biomarker data we will determine the extent to which gut microbiome composition and BHB are associated with AD pathology using fluid biomarkers. The work proposed here will provide a deeper understanding of the interplay between the innate immune system, gut microbes, and metabolism in AD, generating the needed data that will support the development of novel strategies to prevent or slow the course of AD.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The tumor microenvironment (TME) in essentially all epithelial cancers is associated with significant biochemical and structural changes in the extracellular matrix (ECM). Many tumors including those of the breast, pancreas and ovary are characterized by profound changes in the collagen architecture. ECM changes (~micron scale) are below the resolution of conventional imaging modalities but analysis of this structure is critical for understanding carcinogenesis and metastasis. We have used the collagen-specific modality of Second Harmonic Generation (SHG) optical microscopy to discriminate cancer specimens from normal tissues based on changes in supramolecular structure, fibril structure, and fiber morphology, where we have focused on high grade serous ovarian cancer (HGSOC). However, SHG cannot identify the specific molecular alterations, which could provide critical information on disease etiology, prognosis, and response to therapy. Now we will develop a novel method that combines spatially registered SHG and surface enhanced mid-infrared spectral imaging (SE-MIRSI) correlating morphometric and chemometric information to elucidate tumor-promoting ECM alterations. The latter spatially probes specific molecular signatures from vibrational spectroscopy and provides increased sensitivity using nanophotonic substrates, allowing rapid and large-area chemical imaging of whole tissue sections. Specifically, SE-MIRSI can quantitatively identify specific changes in isoform distribution, posttranslational modifications and altered crosslinking of the collagen fibers. Spatial registration of SHG and SE-MIRSI then will provide a comprehensive, ultrasensitive, label free, non-destructive, high-resolution structural and biochemical imaging platform to investigate the role of ECM alterations in promoting tumor carcinogenesis and metastasis. Here, we will develop a multivariate data processing workflow that identifies the specific signatures of collagen and other ECM components from the two modalities establishing the basis of an accurate classifier. We will validate the multimodal characterizations on HSGOC tissue samples. At the end of this project, we will have developed a multimodal imaging platform that will uniquely identify collagen and other ECM biochemical alterations in the TME. We will establish performance measures based on imaging speed and throughput, sensitivity and classification accuracy. These structural and biochemical analyses will provide new insight into carcinogenesis and disease progression in several carcinomas. We propose these Aims: Aim 1. Identify specific structural and biochemical signatures of in vitro ECM models through the combined use of SHG and SE-MIRSI. Aim 2. Validate spatially registered SHG/SE-MIRSI method on high grade serous ovarian cancer and identify specific associated structural morphology and biochemical signatures.

NIH Research Projects · FY 2025 · 2023-07

Project Summary / Abstract Mechanical signals play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and quality of life. Although the link between mechanical signals and the regulation of muscle mass has been recognized for decades, the mechanisms that control this process remain ill-defined. For instance, most studies indicate that the mechanically induced growth of skeletal muscle is driven by an increase in the size of the existing myofibers rather than an increase in the number of myofibers. Moreover, current models assert that the increase in myofiber size is mediated by an increase in the balance between the rates of protein synthesis and protein degradation which, in turn, leads to the accumulation of newly synthesized proteins (NSPs) and the concomitant structural changes that drive the growth response. For instance, it is well known that an increase in mechanical loading can lead to microstructural changes such as the radial growth of myofibers. Surprisingly, however, the ultrastructural adaptations that drive these microstructural changes have not been defined. Indeed, a number of foundationally important questions such as whether the radial growth of myofibers is driven by an increase in the size and/or the number of myofibrils have not been answered. Likewise, the location(s) in which NSPs accumulate during mechanically induced growth (i.e., the sites of growth) are not known. As such, one of the major goals of this project is to fill these gaps in knowledge. Another major goal is to develop a better understanding of the signaling events that control the different aspects of mechanically induced growth. For instance, our previous work has established that signaling through mTORC1 plays a central role in the process via which mechanical stimuli induce the radial growth of myofibers. However, our preliminary data indicate that the longitudinal growth of myofibers can also make a substantive contribution to the mechanically induced accretion of muscle mass, yet, unlike radial growth, the longitudinal growth of myofibers does not appear to require signaling by mTORC1. In other words, our preliminary data suggest that the radial and longitudinal growth of myofibers are regulated by distinct signaling pathways. Specifically, we propose that the radial growth of myofibers is driven by a mTORC1-dependent mechanism that we have coined as the “myofibril expansion cycle”, whereas the longitudinal growth of myofibers is mediated by a mTORC1-independent mechanism that involves transverse Z-line splitting of sarcomeres at regions called sphenodes. To test the validity of these hypotheses we will use advanced imaging techniques, various genetic interventions, two complementary models of mechanical load-induced growth, and our new state-of-the-art technology that enables us to visualize and quantify (with ≤10 nm resolution) where NSPs accumulate. Collectively, it is anticipated that the outcomes of this project will not only fill major gaps in our understanding of how mechanical stimuli regulate muscle mass, but they will also build the framework for future studies that are aimed at developing a better understanding of this highly important process.

NIH Research Projects · FY 2026 · 2023-07

The giant Drosophila protocadherins Fat and Dachsous (Ds) form a heterophilic, bidirectional signaling pair that regulates proliferation via the growth-inhibiting Hippo pathway, and planar cell polarity (PCP) both through and independently of the “core” PCP pathway. These functions are shared by their mammalian homologs, and human mutations in Fat and Ds homologs cause the neurological and multisystem defects of Hennekam and Van Maldergem syndromes. Despite its importance, only a little is known about how binding between Fat and Ds change cell behavior, and thus how it regulates development and pathology. Fat, Ds and the effectors of the Hippo and PCP pathways are concentrated in the subapical domain of epithelial cells, and the intracellular domain (ICD) of Fat has strong effects on the subapical levels of two critical proteins. The first is the scaffolding myosin Dachs, which binds and inhibits Warts (Lats1/2), the final effector kinase in the Hippo pathway, and which regulates Sple in the core PCP pathway. The second is the FERM scaffolding protein Expanded, which stimulates Warts activity. Using a combination of protein-binding screens, biochemistry and molecular genetics, we established a link from the Fat ICD to Dachs and Expanded levels and localization via the DHHC palmitoyltransferase Approximated and one of its targets, the SH3 domain protein Dlish. However, Approximated must have additional targets in the pathway, and the details of Approximated and Dlish regulation are poorly understood. In this proposal we outline plans to identify characterize new Approximated targets, including Expanded itself, and further plans to analyze the regulation of Approximated activity and target recognition, first through modification of Approximated and its targets, and second through the previously uncharacterized GOLGA7-like adapter protein CG5447. Fat and Ds are also remarkable in their ability to polarize cells along the epithelial plane via their own cell-by- cell polarization to opposite cell faces. We have initiated studies on the intracellular control and amplification of Fat/Ds polarization. We explore previously unsuspected roles for intracellular pathway components in the regulation of Fat and Ds levels and polarization, including the casein kinse 1 Dco, the ubiquitin ligase Slimb, the myosin Dachs, and the intracellular domain of Fat itself.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT This research program integrates concepts of biology, physics, and applied mathematics to produce new understanding connecting cell force generation and transmission to migration. A major area of focus is collective cell migration, which underlies essential processes in development of tissues and progression of disease. The long-term vision of this research program is to apply experiment-informed computational models to predict how biochemical perturbations will affect the collective migration. Such models would enable design of methods to control the collective migration, which would lead to therapies with important impacts on human health, such as healing of chronic wounds, slowing invasion of cancer cells, and engineering tissues of desired size and shape. Achieving this modeling capability requires a biophysical approach, because the motion results from physical forces that are produced by the cells in response to biological signaling and transmitted across the cell layer. Although there exist methods to measure the forces, the common methods used are often uninformative for physics-based models of collective motion or for studies of the biochemical signaling that produces the forces. Thus, there is a need to improve upon current methods and to develop new methods to quantify forces while simultaneously connecting to both the physics-based models and the underlying biology. The goals for this 5-year MIRA award are to advance methods in quantifying cell forces in both in vitro and in vivo systems and to apply those methods to build frameworks that enable modeling the relationships between biochemical signaling, forces, and motion in collective cell migration. To accomplish these goals, the research will take two parallel approaches. One approach will improve upon currently available experimental methods to measure forces produced by each cell, including the variation of those forces in space and time. The other approach will develop a new methodology for quantifying cell forces by integrating methods of data science with physics. Importantly, this new methodology will be able to infer cell forces from only images of the cells, meaning it can be applied in complicated cell culture systems and even in vivo. The two approaches will be used to study the collective migration by organizing the research around two complementary frameworks: the first will study collective motion by focusing on the forces associated with local rearrangements between neighboring cells; the second will determine how motion is coordinated across multicellular groups. Together, these two frameworks will provide a means to organize observations about collective migration into a holistic understanding, which will hint at the underlying biological mechanisms and provide an essential step forward towards achieving experiment-informed computational models that can predict the collective migration in applications such as wound healing, cancer invasion, and tissue engineering.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY Ryanodine receptors (RyRs), are the Ca2+ release channels of sarcoplasmic reticulum (SR) that play an essential role in excitaton-contraction coupling of cardiac and skeletal muscle cells. Mutations in the cardiac RyR gene (RYR2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia and sudden death. CPVT is clearly an arrhythmogenic disorder stemming from intracellular Ca2+ mishandling caused by RyR2 dysfunction. As such, insights gained from CPVT syndromes benefit our mechanistic understanding of other cardiomyopathies where RyR2-linked Ca2+ mishandling plays a pivotal role. RyR2 dysfunction brings about deleterious effects in the heart by inducing SR Ca2+ “leak” and/or spontaneous Ca2+ release (SCR), a sudden, diastolic Ca2+ bolous of critical mass that triggers arrhythmic activity. Thus, reigning in RyR2 hyperactivity to mitigate SR Ca2+ “leak” and avoid SCR is a primary goal of therapeutic regimes for major cardiomyopathies. A group of globular peptides termed calcins target RyRs with high affinity and specificity. Imperacalcin (IpCa), the founding member of the calcin family, engages RyRs by entering through their wide vestibule and binding with exquisite precision to a site deep in the cytosolic “cap” of the channel, adjacent to the transmembrane region, acting as a wedge that “pushes” laterally the transmembrane helices that line the channel pore, causing it to open and giving rise to a long-lived subconductance state. In animal models of CPVT, IpCa penetrates the external membrane of ventricular myocytes and induces a partial depletion of SR Ca2+, thus preventing SCR and in effect relieving the adrenergically-mediated Ca2+ overload that triggers Ca2+-dependent arrhythmias. Thus, IpCa behaves naturally as an agonist of RyRs, with anti-arrhytmic effect in cardiomyopathies where SCR is the primary trigger of arrhythmias, and by rational design of IpCa analogs capable of closing the natural “grooves” it creates with RyRs, it has the tangible potential to become the first high-affinity RyR blocker that may prevent SR Ca2+ “leak”. Using cryo-EM structures of calcin-RyR complexes and a series of functional assays, aim 1 will determine the structural domains of calcins that allow them to bind to RyRs with exquisite affinity and specificity, and to generate calcin analogs capable of blocking RyR ion conduction. Aim 2 will use CPVT mouse lines that readily present SCR during sympathetic stimulation and a novel rabbit CPVT model that presents constitutive SR Ca2+ leak and pathological cardiac remodeling to determine whether native and mutant calcins are capable of preventing RyR dysfunction-triggered arrhythmias. Our multi-disciplnary program, with well-defined deliverables and milestones and led by two PIs experts in structural (Van Petegem) and functional (Valdivia) biology of RyRs, will produce a novel paradigm for the treatment of Ca2+-dependent arrhythmias, an area severely underserved by existent pharmacotherapy.

NIH Research Projects · FY 2025 · 2023-07

Project Summary / Abstract Despite robust evidence that (a) depression rises precipitously during adolescence, (b) mental health during adolescence predicts lifelong trajectories, and (c) meditation-based interventions (MBIs) reduce depressive symptoms, access to and research on MBIs in adolescent populations is limited. Mobile health (mHealth) delivery of MBIs offers the promise of accessibility, affordability, and personalization. Research on mHealth MBIs in adults indicates these programs are feasible, acceptable, safe, and effective in reducing depressive symptoms. Coupled with advances in ambulatory assessment of behavior and psychophysiology (i.e., personal sensing data) through wearable devices, mHealth and personal sensing data represent a new frontier in intervention research and mental health care. Although mHealth technologies are ubiquitous for today’s adolescents, there is virtually no research on mHealth MBIs in adolescents and no research integrating mHealth MBI delivery with personal sensing data. The proposed Career Development Award begins to address this knowledge gap by developing the candidate’s skills to conduct research involving clinical adolescent samples, personal sensing data, and advanced mediation methods to identify mechanisms of change. This new training will be leveraged to study, in a sample of 150 adolescents with elevated depressive symptoms, the impact of a mHealth MBI that has strong preliminary evidence of efficacy. Participants will be randomly assigned to the mHealth MBI or to a wait-list control condition. Aim 1 will assess the feasibility, acceptability, and safety of the 8-week mHealth MBI in depressed adolescents. Aim 2 will preliminarily assess program efficacy in reducing depressive symptoms. Aim 3 will characterize psychological (i.e., perseverative thinking, cognitive distancing, loneliness), behavioral (i.e., social isolation via geolocation data), and psychophysiological (i.e., sleep quality and heart rate metrics via wearable) mediator effects on depressive symptoms using advanced structural equation modeling methods. These methods will provide preliminary evidence of intervention efficacy while potentially identifying mechanisms of change at multiple levels of analysis, providing critical data for planning future, large-scale randomized controlled trials. All aims will be supported by didactic, experiential, and mentored training in the fundamentals of clinical research through the NIH-funded Institute for Clinical and Translational Research (ICTR) and the Center for Healthy Minds. Aim 3 will be facilitated by new training in personal sensing data and advanced mediation methods, supported by a mentor team with extensive expertise in these domains. Combined, the research aims and training goals of this project seek to promote the development of accessible, acceptable, safe, and effective mHealth MBIs for the treatment and ultimately prevention of adolescent depression. This award will enable the candidate to conduct future intervention and mechanism research aimed at reducing the burden of depression in adolescents.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Microphysiological systems have great potential for modeling human disease but advanced in vitro models of parasitic infection and immunity are severely underrepresented. Parasites are major causes of morbidity and mortality globally, infecting millions of people every year, yet, there are no effective vaccines available for any enteric parasitic infection. Oral transmission via contaminated food or water is the most common route of parasitic infection for humans, but our knowledge of parasite/host interactions, including how parasites interact with immune cells to either cause disease or elicit a protective immune response, within the intestinal tract is very limited. There is a critical need to create improved in vitro models of human immune-parasite interactions to capture key features present during parasitic infection establishment and disease progression. To address this need we will develop a microphysiological gut vasculature lumen system based on the LumeNEXT microfluidic device system. This 3-dimensional cell culture device recapitulates the gut architecture and includes a human intestinal epithelial lumen flanked by blood and lymphatic vasculature. With these advanced in vitro models, we will introduce parasites into the intestinal epithelium and human immune cells into the vasculature to monitor parasitic disease and immune response. Our goal is to create a microphysiological system that can be used for the study of any protozoan parasite. For this proposal, we will use the protozoan parasites Toxoplasma gondii (T. gondii) and Entamoeba histolytica (E. histolytica) because 1) they are human pathogens of global importance, 2) they are on distinct branches of the protist evolutionarily tree, 3) they have defined lab growth conditions and genetically tagged marker strains, 4) our lab has recently developed the unique ability to produce large numbers of the highly infectious oocysts and cysts forms of both T. gondii. Our data shows that T. gondii infection of the intestinal epithelial lumen in our in vitro model system elicits an active immune response and migration of human immune cells from the vasculature. In this project, we will use these 3D biomimetic gut-vasculature lumen models to address critical knowledge gaps of the human immune responses to T. gondii and E. histolytica. We will incorporate an anaerobic environment so that the immune responses can be defined under hypoxic conditions. These experiments will provide the foundational understanding of the human innate immune responses to intestinal T. gondii infection that are essential for vaccine development. We will also model E. histolytica invasive disease using nutrient limitation and co-culture with Clostridiodes difficile. The advances we will achieve in this proposal will allow the microbiology and immunology fields to determine the immune responses to the biologically relevant stages of intestinal parasites in human models.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT The retina is comprised of neural circuit ensembles that communicate through connections called synapses to generate visual perception and behavior. Retinal diseases cause signaling deficits that derail this communication and block information flow traveling from the retina to the brain. Severe congenital stationary night blindness is of particular interest because despite complete suppression of signal transmission through the on retinal pathway that signals light increments, the on neural circuitry is anatomically intact. Alpha ganglion cells, the primary output neurons of retinal pathways that code for specific visual features, receive excitatory and inhibitory synaptic inputs, integrate these inputs across their dendritic compartments, and generate and transmit trains of action potentials to the brain. We know that neural circuits can adopt diverse strategies to conduct precise synaptic computations and generate response properties, however, the cellular and synaptic factors prone to alteration during retinal diseases are not well understood. This proposal seeks to address important unanswered questions about the mechanisms of neural compensation that occur in response to specific signaling deficits in well-defined alpha retinal output circuits. Using a set of neurophysiological and anatomical approaches, these experiments will define how intrinsic properties and synaptic computations of alpha retinal ganglion cells are altered when the synaptic inputs and balance of excitation/inhibition that a neuron receives is perturbed. Two CRISPR-edited knockout models of the principal glutamate receptor of the on retinal pathway, mGluR6, will be used to study neural compensation in the inner retina across homozygous (100% block) and heterozygous (50% block) conditions. We will correlate single cell electrophysiology with high resolution imaging and visual behavior assays to complement observations across the cellular, synaptic, and behavioral levels. Together, the proposed experiments stand to significantly deepen our mechanistic understanding of the substrates of neural compensation in the inner retina and define the cellular and synaptic deficits of severe congenital stationary night blindness.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Influenza B viruses (IBV) cause annual epidemics with appreciable morbidity and mortality, but have been understudied compared to influenza A viruses (IAV). Currently available vaccines for IBV and IAV are sub- optimal and must be updated frequently due to the emergence of novel antigenic variants. To date, efforts to develop broadly protective, potentially ‘universal’ vaccines have almost exclusively focused on IAV. RFA-AI-20- 003 therefore calls for the “development and/or characterization of IBV vaccine components that complement existing lead IAV vaccine candidates”. In Aim 1 (R21 phase), we plan to develop broadly reactive influenza B candidate vaccine viruses. Using mutagenesis approaches, we have already generated mutant IBV hemagglutinins (HA, the major viral antigen) whose antigenic properties are in-between those of the two major lineages of IBV. Thus, these antigens may elicit immune responses that confer protection against viruses of both IBV lineages. Here, we plan to develop additional IBV HA mutants with potentially higher cross-reactivity than that of our current candidates. In addition, we will establish an antigenic map for IBV HA to analyze the antigenic properties of IBV HAs (antigenic maps are now widely used for IAV HAs, but have not been developed for IBV HA). In Aim 2 (R33 phase), we will assess the immunogenicity of influenza B candidate vaccine viruses. Briefly, the top 5 candidates from Aim 1 will be used to immunize ferrets. Immunization will be carried out with adjuvanted, secreted IBV HA (sHA) mutants (thus eliminating the contribution of other IBV proteins to immune responses), or with adjuvanted IBV HA presented on nanoparticles composed of a self-assembling phage protein (generated by Dr. R. Kane, Georgia Tech). The sera from vaccinated ferrets will be tested for reactivity with IBV HA antigens, and these data will be integrated into the antigenic map. Using an established phage display approach, Dr. S. Khurana (Federal Drug Administration) will identify the epitopes targeted by the antibodies elicited by our HA mutants. This analysis will allow us to identify antigens that elicit broadly reactive antibodies that target conserved epitopes. Immunology studies will be carried out by Dr. P. Thomas, St. Jude Children’s Research Hospital. On the basis of the data obtained in Aim 2, the top 2 IBV HA immunogens will be used to assess the protective efficacy of influenza B candidate vaccine viruses (Aim 3, R33 phase). Ferrets will be immunized as established in Aim 2 and challenged with IBVs representing both current lineages and an ancestral virus (isolated before the separation of the lineages). Virus titers and immune responses will be compared with those of control animals. We expect that a single immunization with the IBV HA mutants will elicit more broadly protective immunity than a single immunization with wild-type IBV HA. In summary, upon completion of both phases, we expect to have developed a novel strategy for the design of broadly protective IBV vaccines, and to have demonstrated their broadly protective efficacy in ferrets.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Aortic dissection (AD) is a disease characterized by sudden tearing of the inner layers of the aortic wall creating a false lumen (FL) channeling aortic blood flow. The vast majority of acute AD patients survive into the chronic phase, although long-term outcomes are poor with about 50% of patients experiencing aorta-related mortality or requiring surgical repair by 10 years. A major contributor to poor long-term outcomes is growth of the FL. Thoracic endovascular aortic repair (TEVAR) is a minimally invasive surgical therapy which can halt FL growth and reduce AD mortality; however, this treatment comes with cost, risk of procedural complications, and is less effective over time owing to increased tissue stiffness. Accurate prediction of disease trajectory at early phases is limited with current metrics but is highly desirable as this would allow TEVAR to be targeted to high-risk patients in a timely manner, sparing those at lower risk from potentially unnecessary procedures. Current methods for estimating risk in AD are largely based on anatomic metrics (e.g., aortic diameter), which poorly capture functional aspects of AD. To overcome these limitations, we propose to apply advanced imaging techniques, namely 4D Flow magnetic resonance imaging (MRI) and 4D computed tomography angiography (CTA), to characterize and quantify functional processes such as FL pressure and FL wall stiffness, which elude current imaging approches and have been implicated as important factors in predicting long-term behavior of AD. We hypothesize that assessments of these functional metrics will, improve our prediction of false lumen growth rate (FLGR) compared to standard anatomic metrics. We plan to prospectively recruit patients with either uncomplicated type B (n=30) or surgically repaired type A (n=45) aortic dissection to undergo baseline 4D Flow and 4D CTA imaging in the subacute period (1-3 months post-dissection) as well as follow-up studies at 1- and 2-years post-dissection, with the primary outcome being FLGR. To achieve these goals, the Aims of this proposal are: 1) Identify baseline hemodynamic and biomechanical metrics in the subacute period of AD that predict FL growth rate over time. FL pressure will be quantified from 4D Flow MRI using indirect and direct methods based on physics-based image analysis, with regional FL wall stiffness quantified by merging FL pressure with cyclic aortic wall deformation by 4D CT; 2) Determine the trajectories of functional metrics over time that best predict progressive FL growth. Longitudinal changes in pressure and wall stiffness between baseline and 1- and 2-year follow-up scans will be assessed to identify patients who achieve a new equilibrium versus those who continue to progress; 3) Develop a clinical assessment tool to predict risk of progressive FL growth combining functional metrics, anatomic parameters and patient characteristics with a focus on simplicity and accuracy for dissection-type specific prediction of the FLGR at the earliest possible time point. This work seeks to shift the paradigm of AD assessment from pure anatomic characterization by integrating functional imaging biomarkers to provide accurate predictions of disease trajectory and allow for optimal determination of surgical candidacy and timing.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract Bacterial chromosomes are highly structured in order to accommodate their large size in a relatively small cellular package. In E. coli, the chromosome is divided into 31 chromosomal interaction domains (CIDs) and several larger and topologically isolated macrodomains. The structures defining macrodomain boundaries are unknown. One macrodomain of about 1 million bp encompasses the replication terminus and is referred to as the Ter macrodomain. We have discovered two 222 bp and intergenic repeat sequences in the E. coli genome, symmetrically arranged around the replication terminus and just outside what has been defined as the Ter macrodomain. These sequences, now called replication risk sequences or RRS, trigger unusual levels of RecA deposition in local single-stranded gaps. The RRS affect replication and are highly conserved in enterobacteria, including many pathogens. Deletion of one RRS generates a growth defect. It has not been possible to delete both RRS, suggesting that the retention of at least one of them is essential. The RRS represent a new genomic phenomenon and likely represent a chromosomal structural feature involved in genomic replication, condensation, segregation, or all three. We hypothesize that the function of RRS is to relieve topological stress. The RRS may represent the physical reality of the Ter macrodomain boundaries. Given a complete lack of information about RRS, we are proposing a general characterization to understand their effects on replication and transcription. The methods include a variety of standard genetics, microscopy, cell biology, molecular biology, and genomics. Nonstandard methods include a newly devised genomics method that allows the probing of genomic single-stranded DNA called ssGAP-seq and single molecule replication assays carried out in vitro. The goal is basic understanding of the role of RRS in bacterial nucleic acid metabolism to inform future work.