University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2016-07

PROJECT SUMMARY/ABSTRACT The regular beating of the heart requires the orchestrated expression of ion channel ensembles specific for ventricular repolarization, pacemaking, conduction and other functions. Recent progress establishes a new way in which channel expression is coordinated. The central hypothesis of this proposal is that a “microtranslatome” of interacting mRNA species encodes functionally related proteins, such as those producing the ventricular action potential. These ion channels assemble cotranslationally into macromolecular complexes that govern higher-order cardiac excitability. The aims of the proposal are to resolve components of cotranslational complexes mediating cardiac repolarization using single-molecule fluorescence in situ hybridization combined with protein immunofluorescence (smFISH/IF) in both embryonic (iPSC) and adult cardiomyocytes. Hypotheses regarding the composition, stoichiometry and cellular localization of these complexes will be tested. Whether such complexes regulate other cardiac functions, such as pacemaking, will be determined. Mechanisms mediating mRNA association, such as candidate RNA binding proteins (RBPs) identified with affinity-capture mass spectrometry, and direct mRNA interactions, will be resolved. These studies are expected to illuminate new mechanisms by which cardiac excitability is controlled and identify novel targets for disease and therapeutic development.

NIH Research Projects · FY 2025 · 2016-07

Project Summary Approximately 50% of children with cerebral palsy (CP) have the speech disorder dysarthria. Dysarthria is difficult to identify in young children because its characteristics (speech subsystem features) overlap with features of early typical development. Quantitative standards for differentiating typical from atypical speech subsystem development in children do not currently exist. This research seeks to improve early identification and treatment of dysarthria in children with CP by creating clinically accessible, data-based developmental standards that allow us to differentiate between typical and atypical auditory-perceptual speech subsystem features in children. To do this, we will validate an English adaptation of the pediatric version of the well- established Bogenhausen Dysarthria Scales (BoDyS-Kid), including 31 subsystem-based perceptual speech features and 10 domains. The research will capitalize on speech samples from 573 typically developing (TD) children, collected in the first funding cycle; and 74 children with cerebral palsy (699 longitudinal speech samples). We will collect new data from 1300 speech language pathologists (SLPs) who will make auditory- perceptual ratings of children’s speech using the BoDyS-Kid, comprising the first ever large-scale comprehensive study of the development of speech subsystem based auditory-perceptual features in English- speaking children. Specific aims are: 1.) To quantify the maturation of auditory-perceptual speech subsystem features and domains in typically developing children. 2.) To quantify the maturation of auditory-perceptual speech subsystem features and domains in children with cerebral palsy. 3.) To identify and quantify auditory- perceptual speech subsystem features and domains that discriminate among clinical groups and are associated with speech intelligibility for children with CP and TD. Products of this research will be a.) a theoretically grounded and validated clinical tool for auditory-perceptual assessment of speech subsystem features/domains in English speaking children for use by practicing clinical SLPs to support differential diagnosis of speech disorders; b.) a set of normative standards that quantify the course of acquisition and define an expected range of variability by age for auditory-perceptual speech subsystem features/domains; c.) quantitative characterization of longitudinal patterns of subsystem feature/domain acquisition in children with CP (with and without dysarthria), and d.) foundational knowledge of specific features/domains that best differentiate between typical and dysarthric speech development, and a quantitative understanding of the relative contributions of subsystem features/domains and clusters thereof to children’s speech intelligibility. Results will advance the development and validation of evidence-based clinical speech assessment methods for pediatric dysarthria that will provide a clinically accessible explanatory basis for intelligibility deficits across speech subsystems that can be used to direct treatment.

NIH Research Projects · FY 2025 · 2016-07

PROJECT SUMMARY The advent of genome technologies for interrogating gene expression has irreversibly changed the scale at which scientists investigate biological problems. More specifically, large-scale gene expression sequencing technologies have allowed us to glean insights into biology and disease pathogenesis at unprecedented pace. However, RNA levels are only one piece of a highly complex biological puzzle: genes are the starting point of the cellular regulatory process, while metabolites are often the end products and the ultimate biological effector molecules. There are many layers of regulation post-expression that fine-tune the biological system in the path from gene to protein to metabolite. Recognizing the central role of proteins and their post-translational modifications (PTMs) in this process, the National Center for Quantitative Biology of Complex Systems was founded to provide large-scale quantitative data for these molecules. Our Biotechnology Research Resource aims to accelerate the pace, depth, and accuracy of quantifying the proteome, metabolome, and lipidome. Driven by the needs of biomedical researchers, our mission is to develop technologies that provide rapid access to the most comprehensive and accurate reporters of the biological state. Specifically we will (1) extend and ultimately culminate our work to enable comprehensive biomolecule characterization; (2) develop and conclude our work enabling highly multiplexed proteome quantification and (3) break ground on the development of the novel chromatographic and mass spectrometry platform for wholly integrated multi-omic analysis. We shall develop these technologies in the context of several high impact driving biomedical research projects that require the new technological advancements for success and that can serve as technology testbeds. These driving projects comprise two central themes. First, responding to the demands of biomedical researchers to explore the roles of emergent and yet poorly understood biomolecules and their PTMs, we will target projects concerned with new, metabolite-derived post-translational modifications. Second, we have come to appreciate that in addition to protein measurements our collaborators require new technologies to identify and quantify metabolites and lipids to fully understand their biological systems of interest and we will work with a host of projects that allow us to test and refine technologies for the large-scale systems and multi-omics analyses to explore physiology and metabolism. Finally, we propose a multi-faceted approach to dissemination, training, and collaboration, with the ultimate aim of ensuring the sustained impact of our technology. All of our technologies are designed to be sustainable, and we have created both systematic and informal mechanisms to deliver our knowledge and innovations to the scientific community.

NIH Research Projects · FY 2025 · 2016-07

Project Summary RNA-RNA and RNA-protein interactions lie at the heart of essential steps of the eukaryotic gene expression pathway. Defects in these interactions due to inherited mutations can result in age- dependent degeneration of the retina, motor neurons, and other neural tissues. The proposed studies will result in a better understanding of RNA-based mechanisms of gene expression in both the normal and disease states. In the next five years we will pursue three main goals, using the yeast Saccharomyces cerevisiae as a facile model system. First, we will examine the molecular mechanism of activation of the spliceosome for the first catalytic step of pre-mRNA splicing. This process requires allosteric signal transmission through RNA and protein over distances of 100 angstroms or more and results in large-scale remodeling of the spliceosome to allow progression of the splicing cycle. Second, we will elucidate the basis for regulation of expression of a key enzyme in purine nucleotide metabolism, IMPDH, interrogating both transcriptional and post-transcriptional steps. Inherited alterations in a regulatory domain of IMPDH and in proteins that direct spliceosome activation are associated with autosomal dominant retinitis pigmentosa, which leads to progressive blindness. We will look for commonalities between the two processes that might explain the highly specific pathological consequences of these disease mutations. Third, we will examine the functions of the hnRNP protein Hrp1 in the regulation of elongation and termination by RNA polymerase II. Hrp1 is structurally related to human hnRNP proteins in which inherited substitutions cause neurodegenerative disorders, including ALS and FTLD. Furthermore, Hrp1 exhibits a similar propensity to form intracellular aggregates, which are associated with pathology of the human proteins. A more complete understanding of these fundamental processes should lead to more accurate diagnosis and prognosis of diseases caused by alterations in nuclear RNA-binding proteins, and may ultimately result in new therapeutic approaches. Furthermore, the proposed studies will illuminate basic mechanisms of eukaryotic gene expression that can be exploited for synthetic biology and biotechnology.

NIH Research Projects · FY 2026 · 2016-06

Project Summary/Abstract This research project will investigate the structure and function of RNA-protein (RNP) complexes that regulate gene expression in eukaryotic cells. The research will elucidate how mutations in these systems contribute to disease. A central hypothesis of this research is that protein binding influences RNA folding outcomes that are important for gene expression. This project will reveal how poly(UG) or “pUG” repeat sequences are recognized by proteins, and how these RNAs fold when this process is disrupted by mutations associated with disease. The project focuses on pUG repeat regions of RNA because they are present thousands of times in human RNAs, and cluster near splice sites and regulatory regions. Moreover, we recently discovered that pUG RNAs fold into an unusual quadruplex structure, the pUG fold. The pUG fold directs gene silencing and RNAi amplification in the model organism C. elegans. We hypothesize that a major role of the human protein TDP- 43 is to bind to single stranded pUGs and prevent pUG folding. We further hypothesize that TDP-43 loss of function results in aberrant RNA folding that induces mis-splicing. TDP-43 loss of function and mis-splicing is a hallmark of the diseases ALS, FTD and Alzheimer’s, but the molecular basis for why mis-splicing occurs is not well understood. We will use chemical methods to probe RNA folding and to specifically detect pUG folds in cells with and without TDP-43. The project will further elucidate the three-dimensional structure of TDP-43 bound to RNA. These data will provide a better understanding of the molecular basis for RNA mis-splicing in neurodegenerative disease. We will also investigate how the far upstream binding protein 3 (FUBP3) interacts with pUG sequences, such as in the large non-coding RNA (lncRNA) CMPK2. Because many aspects of the pUG-mediated gene silencing pathway have been conserved throughout evolution but are not well understood, we will also investigate the structure and function of gene silencing machinery. Finally, we will investigate the structure of the U6 snRNP, which is essential for RNA splicing, and will determine how this RNP interacts with U4 RNA. Mutations in the U6 interacting region of U4 RNA have been found to be a major cause of neurodevelopmental disorders. By elucidating the molecular basis of RNP interactions that are essential for gene expression, we will further our understanding of how these molecules can fold into complex three- dimensional structures and how RNP interactions direct distinct biological outcomes in diverse pathways such as gene silencing and alternative splicing.

NIH Research Projects · FY 2026 · 2016-06

ABSTRACT The overall goal of this Maximizing Investigator’s Research award renewal application is to understand the integration of complex signaling networks at both the single cell and multi-cellular level during inflammation and wound healing. Despite progress in understanding the signals that guide wound repair, there remains a significant gap in understanding how different types of cells communicate to integrate a wound healing response. This gap limits our ability to design new therapeutic strategies for a broad range of human disease including diabetes, cancer, cardiovascular disease and autoimmunity. The overall focus of our research is to understand the basic molecular mechanisms that regulate cell migration and how defects in cell migration contribute to human disease in the context of inflammation and tissue damage. The optical transparency and ease of genetic manipulation make zebrafish an ideal model system to dissect multi-cellular and tissue interactions during wound repair. During the prior funding period, we discovered a new pathway involved in neutrophil reverse migration and resolution of inflammation through the paracrine factor Myeloid- derived factor (MYDGF). We have also invested significant effort in developing new tools for real time imaging and manipulating cell dynamics in tissue contexts, metabolism and matrix remodeling during wound healing in both simple and complex wound models. Single cell RNA-seq has identified cell-type specific gene expression changes and using genome editing we are now poised to uncover new signaling mechanisms that regulate neutrophil reverse migration and inflammation resolution. Understanding how wound repair is orchestrated and integrated at both the single cell and multi-cellular level in the different types of tissue damage is the focus of our future research. The overall goal of our work is to identify key signaling networks and guidance cues that mediate cell migration during wound repair, dissect how they are altered in pathological conditions and ultimately may be targeted to understand and treat human disease.

NIH Research Projects · FY 2025 · 2016-05

PROJECT SUMMARY My laboratory innovates mass spectrometry (MS) technology to accelerate the pace, depth, and accuracy of proteome analysis and applies these technologies to globally access proteome structure, function, and regulation. Signature achievements include the development of electron-transfer dissociation (ETD), the GC- Orbitrap mass spectrometer, novel protein quantification technologies, and the targeted technique parallel reaction monitoring – to name a few. Having established our reputation in proteomics instrumentation, our mission came to include the investigation of the metabolome and lipidome. The rationale is simple: fully understanding a biological system requires knowing the interplay between molecular classes. We have demonstrated the capability of these technologies to propel numerous projects in the fields of metabolism, developmental, and systems biology. Building on my program’s expertise in the analysis of disassembled molecular machinery (i.e., shotgun proteomics), we have turned our attention to the machine as a whole, i.e., protein structure. By combining technologies from MS and cryogenic electron microscopy (cryo-EM) our aim is to maximize the information garnered from a single sample to provide insight into protein structure with unprecedented speed and simplicity. That is, we compound the orthogonal information offered by MS measurements of chemical makeup with atomic- level information afforded by cryo-EM imaging. Further, drawing on concepts from astrophysics already resonant with MS, we anticipate that our highly creative method will overcome pervasive bottlenecks in cryo-EM sample preparation that often limit image resolution. To that end, we have adapted a mass spectrometer to purify and land native protein complexes on cryogenically cooled TEM grids, which are then coated with amorphous ice in vacuo. The resulting grid would represent the ideal cryo-EM sample – a high density of particles situated in random orientations in the same focal plane and covered with only a few nanometers of ice. We plan to achieve even greater structural insight through the incorporation of gas-phase dissociation techniques, which partition the macromolecule into its subcomponents. In particular we propose the addition of surface-induced dissociation (SID) and activated-ion ETD (AI-ETD) to the mass spectrometer used for grid preparation. In short, this technology has genuine potential to create a new paradigm of proteome-scale structural biology.

NIH Research Projects · FY 2026 · 2016-05

PROJECT SUMMARY Cell interactions regulate many essential aspects of development and disease. Our research program addresses the role of cell interactions in the formation of organs during development. Using the C. elegans embryo as a model system where genetics, cell biological analysis, and live imaging can be combined, we have developed experimental systems to understand the role of specific cell interactions in organogenesis. The goals of this proposal are to learn how cells form small tubes, how cells bite off pieces of other cells, and how niche cells regulate germ cell mitochondria to influence their inheritance. In one project, we are examining how the smallest biological tubes form. Such cells, like narrow human capillaries, hollow out their cytoplasm to create an intracellular lumen. We are investigating the mechanisms of intracellular lumen formation in the excretory cell. We have developed methods for imaging excretory cell intracellular lumen initiation and branching and have performed genetic screens for mutants with defects in tube formation. Using these approaches, we showed that the lumen forms by plasma membrane invasion into the cytoplasm, and that the basement membrane receptor integrin is needed for the excretory cell to breach a basement membrane and branch. We will extend these findings by determining how integrin regulates basement membrane invasion, and by identifying signals that promote lumen initiation and branching. In a second project, we are investigating how cells bite off pieces of other cells. This process, called trogocytosis, has important roles in immune cell interactions, cancer, and neuronal remodeling, and is employed by some pathogens to kill human cells. Intestinal cells use trogocytosis to bite off large pieces of primordial germ cells in the developing gonad. We have identified roles for the membrane trafficking regulator RAB-35 in both the biting and bitten cells. We will continue these studies by determining how RAB-35 controls membrane scission during trogocytosis and which downstream effectors it regulates in this process. In a third project, we will examine how trogocytosis of primordial germ cells regulates germline mitochondria. We have shown that primordial germ cells lose many mitochondria through trogocytosis, and that there is selection against mutated mitochondrial DNA in primordial germ cells. We will extend these findings by examining the importance of trogocytosis and autophagy in regulating mitochondrial quality. These studies will reveal basic insights into the conserved processes of tube formation, trogocytosis, and mitochondrial regulation using a simple genetic model system, providing a foundation for understanding these events in human development and disease.

NIH Research Projects · FY 2025 · 2016-02

ABSTRACT: Respiratory infections with viruses and fungi constitute major public health problems globally. Except for influenza virus, there are no licensed vaccines against viruses or fungi. It is generally agreed that induction of T-cell memory is critical for defense against viruses and fungi in the respiratory tract. We and others have shown that induction of tissue-resident memory (TRM) CD8 and CD4 T cells and systemic migratory memory CD4 T cells are essential for protection against influenza A virus (IAV) and inhaled fungi, respectively. However, both TRM cells and systemic memory CD4 T cells undergo attrition, leading to short-lived immunity, which is especially true for TC1/TH1 cells. Therefore, induction of durable T-cell immunity poses major challenges for vaccinologists. As compared to TH1 cells, TH17 cells display sought-after attributes of stem-ness, durability and functional plasticity. We propose to tailor combination adjuvants to harness T17 programming and induce durable and protective lung TRM cells and migratory memory CD4 T cells against viruses and fungi. We find that Adjuplex, a nano-emulsion adjuvant, when combined with the TLR4 agonist glucopyranosyl lipid A (GLA), evokes antigen-specific CD8 and CD4 T-cell responses in the lung that are: (i) durable and multifaceted (TC1/TC17/TH1/TH17), and (ii) confer heterosubtypic immunity against IAV that persists >400 days. We further find that combining those adjuvants with fungal CLR ligands Blastomyces endoglucanase 2 (Bl-Eng2; Dectin-2 agonist) and b-glucan particles (Dectin-1 agonist) augments antiviral TC17/TH17/TC1/TH1 and elicits migratory memory T cells that protect against fungal pneumonia. By single-cell RNAseq, we found that our combined adjuvants induce memory antiviral and antifungal CD8 and CD4 T-cell clusters that express ICOS (Inducible T Cell Co-stimulator), the transcription factor c-Maf, and a transcriptome that fosters tissue residency, stem cell-ness and non-pathogenic T17 programming. Cyclic dinucleotides also promote T17 programming in lungs. This, we postulate that programming stem cell-like, functionally plastic, non-pathogenic TC17/TH17 memory cells with our combination adjuvants (that engage TLR-4, Dectin-1/2 and STING pathways) will foster durable protective immunity to viral and fungal pathogens in the lung. Our specific aims will test three hypotheses: Aim 1: Combination adjuvants that evoke TC17/TH17 stem cell-like functionally-plastic TRM or systemic migratory memory will engender durable immunity to respiratory viral and fungal pathogens; Aim 2: Functional plasticity of TC17/TH17 memory is important for protective immunity to viruses and fungi; Aim 3: The ICOS/c-Maf pathway is integral to establishment and/or maintenance of durable vaccine-induced protective immunity to respiratory viral and fungal infections. The proposed work is significant and of high impact because it has the potential to create a tractable adjuvant system/tool kit that will advance the formulation of vaccines: (i) targeted for mucosal or parenteral administration; (ii) designed to induce TRM or systemic T-cell memory; and (iii) tailored to elicit CD8 and CD4 T cells that protect against diverse pathogens such as viruses and fungi.

NIH Research Projects · FY 2026 · 2015-09

Project Summary We discovered that Nε-lysine acetylation occurs in the lumen of the endoplasmic reticulum (ER) in 2007. From that initial finding, we went on to discover the entire ER acetylation machinery (one membrane transporter, AT-1/SLC33A1, and two acetyltranferases, ATase1 and ATase2) and uncover a novel piece of ER biology. Specifically, we discovered that the ER acetylation machinery regulates proteostasis within the ER and secretory pathway by maintaining the balance between quality control/engagement of the secretory pathway and reticulophagy. By using a combination of biochemistry and high-definition mass spectrometry, we discovered that both the citrate and acetate pathways influence AT-1 activity. Homozygous and heterozygous mutations as well as gene duplication events affecting the ER acetylation machinery and key regulatory elements of the citrate (SLC25A1 and SLC13A5) and acetate (ACSS2 and COASY) pathways are associated with similar forms of epileptic encephalopathies with developmental delay, hereditary sensory and autonomic neuropathies, and autism spectrum disorder with intellectual disability and progeria-like dysmorphism. To dissect the biological functions of the ER acetylation machinery, we have so far generated 19 mouse models. They all mimic rare human diseases associated with dysfunctional ER acetylation and fully validate the essential biological functions currently associated with the ER acetylation machinery. Importantly, the animals display important phenotypic similarities, supporting the conclusion that we have identified a unified metabolic pathway that is at the basis of closely related neurodegenerative and neurodevelopmental diseases. The GENERAL HYPOTHESIS of this research is that AT-1 responds to metabolic signals imparted by the citrate/acetate pathway to regulate dynamics of the secretory pathway. Specific Aim 1 will dissect the molecular mechanisms that regulate engagement and efficiency of the secretory pathway downstream of AT-1. Specific Aim 2 will dissect the mechanisms that regulate the induction and progression of autophagy downstream of AT-1. Specific Aim 3 will determine whether AT-1 acts downstream of the acetate/CoA pathway to regulate protein homeostasis within the secretory pathway. The above Aims include in vitro, ex vivo and in vivo approaches together with novel mouse models and newly developed mass spectrometry-based technology. In conclusion, this proposal is the result of novel discoveries made in our laboratory; it will help us dissect the molecular mechanisms of severe neurodegenerative and neurodevelopmental diseases across lifespan and it will allow us dissect essential molecular and biochemical functions of the ER and Golgi apparatus that will impact other areas of biomedical research.

NIH Research Projects · FY 2025 · 2015-09

This is a continuation application for an additional 5 years of support for a comparative and prospective study of Fragile X Syndrome (FXS), an inherited neurodevelopmental disorder caused by a trinucleotide expansion of CGG repeats in the FMR1 gene on the X chromosome. FXS results in significant health and functional impairments that begin in early childhood and last a lifetime. It is the most common inherited cause of intellectual disability and autism, with substantial family burden and public health impacts. Critically, the great majority of knowledge about the FXS clinical phenotype derives from research on children, leaving adulthood a vast uncharted territory. The purpose of the proposed research is to investigate how the health and behavioral functioning of individuals with FXS change across adulthood and to identify factors that alleviate or worsen health and behavioral functioning during the adult years. It will be the first study to robustly address these questions beyond early adulthood. We address 3 Specific Aims. For Aim 1, we will determine profiles of health conditions and health care utilization for adults with FXS. Using a case-control design, we propose to examine the health of a newly ascertained cohort of 368 adults (age 18 to 80+) who have a code for FXS in their electronic health records (EHRs) – 162 women, 206 men, 77 nonwhite, and 291 white. These adults will be drawn from 11 health care systems comprising the Chicago Area Patient-Centered Outcomes Research Network (CAPriCORN). Using EHRs, we will examine differences between adults with FXS and age- and sex-matched controls who do not have FXS with respect to health and health care utilization. We will probe differences between sub-groups of adults with FXS defined by age (early adulthood, midlife, older adulthood) as well as by sex and race. For Aim 2, using a longitudinal design that will span 18 years, we will define life course trajectories in health and behavioral functioning among adults with FXS and investigate age-related risk. Building on the 5 already- collected repeated measures, we will prospectively gather 3 additional rounds of data, resulting in up to 8 repeated measures over the 18-year period (n=182 dyads of adults with FXS and their premutation carrier mothers). By the end of the proposed study, the adults in our existing longitudinal sample will average 37 years of age, with 59% being age 35 or older (the oldest will be age 72). We will employ an accelerated longitudinal design to elucidate changes in health, executive functioning, communication, behavior problems, and daily living skills across adulthood. We will test for ages of increased risk and the effects of sex and autism on these trajectories. For Aim 3, using the same sample as in Aim 2, we will examine how life course trajectories of adults with FXS are associated with familial and social contextual determinants – family relationships, residential status of the adult with FXS, educational level of parents, family income, type of health insurance, services, and neighborhood SES. By employing multiple innovative methods and varied data sources, the proposed study will extend knowledge about FXS beyond early adulthood and elucidate how FXS changes in midlife and beyond.

NIH Research Projects · FY 2025 · 2015-08

PROJECT SUMMARY/ABSTRACT Cigarette smoking is both common and undertreated among oncology patients. Although smoking is a leading cause of cancer and results in worse cancer prognoses, only half of cancer patients who smoke are offered help in quitting during cancer care, and it is unclear that the help they are offered is effective. This R35 proposal is designed to transform oncology practice so that smoking cessation is an integral part of treatment for all cancer patients who smoke. Electronic health records (EHRs) have tremendous potential to accelerate improvements in clinical care by facilitating such treatment delivery. EHR modifications can also increase discovery by enhancing assessment of smoking treatment reach, costs, equity, and effectiveness both in terms of helping patients quit and improving cancer outcomes. The Principal Investigator has 30 years of experience leading research and policy efforts to promote the treatment of smoking. Over the past 7 years, he has led a team that developed and evaluated new EHR-based tools and workflows that markedly increase the proportion of adult patients who receive evidence-based smoking cessation treatment in primary care. The new R35 proposal will similarly identify intervention strategies that increase smoking treatment engagement and effectiveness when implemented in oncologic care. Over the last 4 years, the Principal Investigator has served as Senior Scientist for NCI’s Moonshot-supported Cancer Center Cessation Initiative (C3I), a nationwide effort of 52 NCI-designated cancer centers to develop and implement programs to aid their patients who smoke. Yet, this incredibly important initiative has not been systematically assessed in terms of which EHR-enabled interventions enhance a smoking program’s reach, patient engagement, implementation, cessation effectiveness, and benefits. This R35 renewal is designed to do just that by systematically assessing best practices via EHR data extraction of intervention delivery, healthcare costs, short-term cancer treatment outcomes overall, and by patient demographics. These EHR data will be supplemented by qualitative analysis of C3I smoking treatment components, delivery, adaptations, and contexts in a mixed-methods approach. Higher- and lower-performing smoking treatment programs will be identified and rigorous methods will then be used to select best practice programs from cessation- and cost-effectiveness perspectives. Further assessment will identify implementation strategies and contextual factors that may contribute to the effectiveness of these programs. Best practices from these programs will be implemented in lower-performing C3I programs, with research team support in adaptation and implementation. Pragmatic implementation guides and strategies will be used to disseminate best practices to cancer care programs nationwide. Thus, the proposed project seeks to extend and adapt transformative EHR-facilitated system changes that enhance smoking treatment in primary care to the high-priority cancer care context. The project will demonstrate the benefits of such system changes to cancer patients in terms of costs, smoking cessation, and cancer recovery.

NIH Research Projects · FY 2026 · 2015-06

ABSTRACT Tuberculosis (TB) remains a major threat to global health despite concerted efforts to bring the pandemic under control. Mycobacterium tuberculosis (M. tb), the causative agent of TB, can survive severe stressors including antibiotics. This is a central challenge to TB control, since it requires lengthy, complex treatments that can fail as a result of drug resistance and for unknown reasons. Here we propose to investigate M. tb biofilms, which provide shelter from antibiotics and immune assault. Our research shows that M. tb can adapt rapidly to selection for biofilm growth with a small number of mutations that have widespread phenotypic effects, but this adaptation can have negative impacts on other phenotypes. We hypothesize that M. tb forms biofilms only in response to specific environmental conditions encountered within hosts with TB. We will test this hypothesis with analyses of natural and experimental M. tb populations contextualized with data from source TB cases, as well as comparative studies of M. tb and less pathogenic mycobacteria.

NIH Research Projects · FY 2024 · 2015-06

Project Summary The overall objective of this proposal is to train graduate students and postdoctoral scientists about the concept of implicit bias, the effects of bias on underrepresented minority students in the biomedical, behavioral and clinical science (BBCS) fields, and the strategies to mitigate the effects of bias on themselves, as well as in their current and future work environments. This training both complements and extends the previously-funded project titled, Breaking the Bias Cycle for Future Scientists: A Workshop to Learn, Experience, and Change (NIH-NIGMS, R25GM114002, 2005-2020). It also leverages a number of other NIH-funded projects to ensure that successful bias-reducing and mentoring models, trainings/workshops, and strategies are applied in different venues and with additional populations (Carnes, 2015, 2017; Kaatz et al., 2017; Sorkness et al., 2017). The specific aims of this project are to: 1) Develop training for graduate students and postdoctoral scientists about implicit bias and its effects to empower them with bias-reducing and resiliency-building strategies that positively influence their current and future work environments; 2) Conduct the training through in-person workshops at universities, national labs, and conferences, as well as through online webinars to deepen participant learning and to provide ongoing support; and 3) Provide Fair Play Workshop Facilitator Training to expert facilitators who have the foundational knowledge, skills, interest and commitment to further disseminate and sustain this initiative. At the completion of the grant period, we expect to have: 1) an enhanced workshop conducted with ~2000 graduate students and postdocs to empower them with bias-reducing and resiliency-building strategies, 2) complementary training offered through webinars to enhance the learning of this subject post-workshop, and 3) approximately 200 trained facilitators who are able to offer workshops immediately after they are trained. The investigators are ideally positioned to carry out the proposed work because they have successfully collaborated on research, development and dissemination of Fair Play and its accompanying materials on which this proposal is based.

NIH Research Projects · FY 2026 · 2014-04

PROJECT SUMMARY/ABSTRACT Type 2 diabetes (T2D) is characterized by the inability of pancreatic islet β-cells to meet insulin demands, often due to genetic and environmental factors that disrupt β-cell function. Most genetic variants associated with T2D affect the differentiation, survival, and nutrient sensing of islet cells, as well as insulin secretion. While hyperinsulinemia can initially compensate for insulin resistance, it may lead to β-cell exhaustion and diabetes over time. Thus, precise regulation of insulin secretion is critical, as β-cells must balance the risks of both hypoglycemia and hyperglycemia. To better understand these regulatory mechanisms, we performed a genetic screen in a genetically diverse outbred mouse population. We identified Zfp148, a transcription factor not previously linked to diabetes-related traits, as a novel negative regulator of insulin secretion. Deletion of Zfp148 in β-cells resulted in improved glucose tolerance and enhanced β-cell nutrient sensing, underscoring its critical role in insulin regulation. We will characterize Zfp148 by identifying its direct transcriptional targets and investigating its potential non-transcriptional regulatory roles. In addition, we identified a small molecule—a known α-adrenergic receptor antagonist—that mimics the gene expression signature of Zfp148 knockout β-cells, providing new insights into downstream pathways. We will explore the mechanism of this compound and its impact on β-cell function. In a discovery aim, we integrate mouse and human islet proteomic datasets to identify protein modules that distinguish diabetic from non-diabetic subjects. Using mouse genetics, we uncovered Cbr3, an NADPH-dependent ortho-quinone reductase, as a driver of one such module. This enzyme may link oxidative stress pathways to β-cell dysfunction. This project combines forward genetics, multi-omics integration, and functional studies to reveal novel regulators of insulin secretion. The results will enhance our understanding of how β-cells adapt to metabolic stress and may lead to therapeutic strategies to improve β-cell function, prevent exhaustion, and mitigate T2D progression.

NIH Research Projects · FY 2024 · 2014-04

ABSTRACT Although many women with estrogen receptor positive (ER+) primary cancers are successfully treated with surgery and adjuvant anti-estrogen therapies, metastatic therapy-resistant ER+ cancers account for the majority of breast cancer related deaths. New understanding of the biology underlying disease processes is required to develop new approaches. In this renewal application, we build on our previous findings demonstrating dynamic reciprocity between estrogen and features of the extracellular matrix (ECM) in the microenvironments of both the primary tumor and the metastatic niche which fuel the pulmonary metastatic burden. We showed that estrogen remodels the ECM architecture of the primary tumor, aligning collagen fibers and increasing synthesis of ECM components associated with more aggressive cancers, and alters ECM components in the lung metastatic niche. Moreover, we showed that this collagen alignment signature in patient tumors correlates with inflammatory markers, including COX-2 and CD163+ macrophages, and poor prognosis. In the current proposal, we hypothesize that estrogen orchestrates synergy among ER+ tumor cells, the extracellular matrix (ECM)/ cancer associated fibroblasts (CAFs), and macrophages/ inflammation, to fuel primary and metastatic tumor aggression. We will utilize our robust immunocompetent in vivo model of metastatic ER+ breast cancer, in vitro systems, PDX models and clinical patient samples to test this hypothesis in the following aims. Aim 1: Identify the steps in metastatic progression of ER+ breast cancer which are altered by estrogen activity in tumor cells as well as other estrogen targets. Aim 2: Determine how estrogen action on CAFs modifies the stromal ECM to mediate tumor progression, determine the receptors that mediate this action, and identify ECM signatures from native stromal matrix from ER+ invasive ductal carcinoma biopsies that instruct estrogen action on tumor cell growth and migration. Aim 3: Determine how estrogen impacts immune metastatic mediators, including macrophage activity and the inflammatory tumor microenvironment. Aim 4: Evaluate the ability of standard of care therapeutic approaches to reverse E2-promoted steps in metastasis and post- treatment estrogen re-exposure to promote growth of residual pulmonary lesions, and interrogate the interrelationships among E2-altered ECM structure, and macrophage activity/inflammation. Our studies will illuminate the role of estrogen in dissemination and pulmonary metastatic colonization, with implications for therapy, metastatic dormancy and recurrence of ER+ breast cancer, and reveal potential sites for intervention.

NIH Research Projects · FY 2026 · 2014-02

PROJECT SUMMARY The long-term objective of this proposal is to understand cortical mechanisms underlying loss, recovery, and disorders of consciousness. We have previously characterized neural activity associated with different stages of general anesthesia and sleep. Each of these stages is composed of distinct states of arousal and awareness, such as fluctuations between dreaming and unconsciousness. Finding neural underpinnings of these processes is necessary for understanding the neural basis of consciousness. This proposal takes advantage of the unique opportunity to directly record from the human brain in neurosurgical epilepsy patients. We will use intracranial electroencephalography (iEEG) to identify signatures of unconsciousness and distinguish it from three different states of consciousness: waking consciousness, drowsiness, and dreaming. The scientific premise of this project is that the biomarkers of clinically relevant changes in arousal and awareness share common features, generalizing beyond specific conditions that cause them. Our work to date has identified putative biomarkers that show promise for distinguishing states of arousal and awareness. These biomarkers include region-specific changes in cortical responses to unexpected sounds and speech, and in patterns of cortical connectivity. Using innovative computational approaches applied to data obtained using electrical stimulation and recording of ongoing brain activity at rest, we will track rapid transitions in cortical network configurations. Characterizing these rapid transitions will enable us to identify clinically relevant changes in arousal and awareness. Our multimodal approach combines high resolution iEEG with computational modeling, electrical stimulation tract tracing, conventional scalp-recorded EEG, and magnetic resonance imaging in overlapping sets of human subjects. We will find reliable biomarkers that can identify distinct states of consciousness that occur during induction of and emergence from general anesthesia (Specific Aim 1) and different stages of sleep (Specific Aim 2). We will then leverage these biomarkers to understand mechanisms of delirium, which can occur in neurosurgical patients during recovery from surgery and following seizures (Specific Aim 3). Identifying biomarkers of consciousness has broad clinical relevance to development of novel algorithms for monitoring depth of anesthesia. Knowledge gained from this project will contribute to improved diagnosis, management and prognosis of pathologic states of consciousness including central sleep disorders, delirium, vegetative or minimally conscious states, and coma.

NIH Research Projects · FY 2024 · 2014-01

PROJECT SUMMARY / ABSTRACT There are more than 38 million people living with HIV-1 infection worldwide, and more than 1.1 million in the United States. HIV-1 acute infection and latency reversal are both dependent on viral expression of full-length unspliced viral RNAs (US-vRNAs) that serve dual roles in the cytoplasm as either (1) viral mRNAs encoding Gag and Gag-Pol proteins that drive virus particle assembly or (2) viral RNA genome substrates bound by Gag/Gag- Pol for packaging into virions. This project combines live cell and superresolution imaging, biochemical assays, and other research tools to address key foundational issues in HIV-1 US-vRNA nuclear export and virion assembly relevant to informing the development of new antiviral strategies to target these crucial stages. Aim 1 will define where and when in the nucleus US-vRNAs interface with viral and cellular components of the nuclear export machinery. Our preliminary data have demonstrated surprisingly little overlap between the subcellular trafficking behaviors of retroviral US-vRNAs and the Rev and Rex proteins that mediate their nuclear export through the cellular Exportin-1 (XPO1, also known as CRM1) nuclear export pathway. We will test the hypothesis that key interactions between HIV-1 Rev and US-vRNAs are highly transient, using single molecule detection coupled to an innovative “cell expansion” superresolution light microscopy technique. Our goal is to precisely define the subnuclear sites of Rev-US-vRNA and Rev-XPO1 interaction and study the effects of prescribed perturbations to Rev function on these essential processes. Based on our progress from prior studies of a species-specific block to Rev function in mouse cells, Aim 2 will determine why nuclear Rev-US-vRNA complexes need to recruit multiple XPO1 proteins to achieve efficient nuclear export. We will test the hypothesis that Rev drives XPO1 dimerization in human cells but not mouse cells and attempt to achieve cell-intrinsic resistance to HIV-1 in human CD4+ T cells by inactivation of the putative species-specific XPO1 multimerization domain using precision gene editing. For Aim 3, we will use live cell imaging to test the hypothesis that Rev-dependent nuclear export licenses viral RNAs for enhanced stability and genome packaging in the cytoplasm. These experiments will take advantage of newly described mutant forms of HIV-1 that allow us to, for the first time, study US-vRNAs programmed for either translation or packaging independently. Collectively, these studies will provide new mechanistic insights into the viral and cellular machines that drive HIV-1 genome trafficking, advance the development of new cell-based assays and imaging technologies for studying HIV-host interactions, and test translationally relevant strategies for the targeted inactivation of HIV-1 genomes in vivo.

NIH Research Projects · FY 2024 · 2014-01

Project Summary/Abstract In natural vision, it is rare to encounter an isolated object presented on a blank background. Instead, natural scenes are often complex and contain multiple entities. Image segmentation refers to the process of partitioning visual scenes into distinct objects and surfaces, which includes segmenting a figure from the background (figure- ground segregation) and segmenting multiple objects/surfaces from each other. Segmentation is a fundamental function of vision and is a gateway to perception, recognition and visually guided action. However, the neural underpinning of segmentation remains to be understood. A key question is to understand how the brain represents multiple visual stimuli such that information regarding individual stimuli can be extracted from the activity of populations of neurons. We address this question in the proposed project to elucidate the neural mechanisms underlying segmentation and the principles of coding sensory information in neuronal populations. Visual motion and depth provide potent cues for segmentation. Therefore we focus on understanding how the brain uses motion and depth cues to achieve segmentation. We have made substantial progress in defining how middle-temporal (MT) cortex, an area important for motion and depth processing, represents multiple overlapping visual stimuli. We found that MT neurons show various types of response biases toward one component of multiple stimuli, revealing a set of novel rules by which multiple stimuli interact within neurons’ receptive fields. These physiological findings together with our preliminary data on natural scene statistics led us to hypothesize that the visual system exploits the statistical regularities in natural scenes that differentiate figure from the background and represents multiple visual stimuli efficiently to achieve segmentation. To test this overarching hypothesis, we will integrate the approaches of natural scene statistics, neurophysiology, and theoretical consideration of optimal coding. Specifically, we will characterize natural scene statistics of depth and motion pertinent to image segmentation, elucidate the functional roles of stereoscopic depth in figure-ground segregation, define the rules by which neurons in area MT represent multiple spatially-separated stimuli, which are commonly encountered in natural vision, and determine the signal transformation across multiple brain areas in the dorsal visual pathway to achieve segmentation. Finally, we will use an Information-Maximization approach to determine whether the neural representation of multiple visual stimuli is optimal for segmentation. The proposed study rigorously explores the interaction of multiple stimuli and is expected to provide important insight into how the visual system solves the challenging problem of segmentation in natural vision.

NIH Research Projects · FY 2026 · 2013-12

PROJECT SUMMARY One major objective facing the HIV research community is to facilitate drug-free viral remission in People Living with HIV (PLWHIV). The host immune responses that can achieve this post-treatment control (PTC) are poorly understood because PTC is rarely observed in PLWHIV and the commonly studied SIV-infected rhesus macaques. CD8+ T cells are essential for control of HIV replication in the presence and absence of ART, but no studies have identified that CD8+ T cells can promote ART- free viral remission or delay the time to viral rebound after ART interruption. We recently identified a Mauritian cynomolgus macaque (MCM) model for sustained control of SIV replication after stopping ART. MCMs who shared a non-protective MHC haplotype called M3 began receiving ART 14 days after SIV infection and stopped receiving ART eight months later. They suppressed SIV replication for more than five months after ART interruption. In vivo depletion of CD8+ cells led to prompt viral rebound, indicating a vital role for CD8+ cells in post-treatment viral control (PTC). Our current proposal aims to unravel the function and type of CD8+ cells responsible for PTC in MCMs. This is the second competitive renewal of an ongoing R01 awarded to our lab. The first grant cycle explored the role of CD8+ T cells targeting invariant SIV epitopes in the control of live attenuated SIV. The second cycle focused on whether the IL-15 superagonist N-803 enhanced CD8+ T cell function to suppress SIV replication. During cycle 2, we serendipitously discovered that MCMs frequently become PTCs when ART is initiated two weeks post-infection, even without additional therapeutic interventions. We are now proposing to use tools developed during both previous grant cycles to test the hypothesis that MHC class I restricted CD8+ T cells are required for PTC through a mechanism that is independent of host MHC class I genetics. We will determine whether the M3 MHC haplotype is necessary for PTC in MCMs. We will use in vivo immune depletion studies and carefully engineered viruses with point mutations in viral peptides restricted by M3 MHC class I alleles to characterize the specific CD8+ population(s) required for PTC. Upon concluding this study, we will know whether PTC is universal among MCMs who begin receiving ART two weeks post-infection and if PTC depends on MHC class I restricted CD8+ T cells. Successful completion of this study will identify whether PTC in MCMs is independent of MHC genotype and if PTC relies upon virus-specific cytotoxic CD8+ T cells. This information could revolutionize our understanding of the specific types and functions of CD8+ cells that mediate PTC.

NIH Research Projects · FY 2026 · 2013-09

Erythropoiesis is a dynamic process governed by quantitative changes in the relative levels of transcription fac- tors (TFs) that control gene regulatory networks (GRNs). Recently, we generated the first high-resolution GRNs of human erythropoiesis that integrate chromatin accessibility, transcripts, and cell surface proteins, all measured simultaneously in the same single cells. While our study provided unprecedented insights into the regulation of erythropoiesis, it lacked TF protein-based measurements and information about 3D genome organization. This significantly limits the understanding of erythropoiesis, ultimately impinging on the capacity to correct erythropoi- esis-associated disorders. Our long-term goal is to obtain a system-level understanding of the GRN that controls human erythropoiesis in health and disease. The objective of this proposal is to use our established GRN model of erythropoiesis as a framework to integrate additional modalities that include TF protein abundances, TF-co- factor interactions and the 3D topology of the genome. Our central hypothesis is that quantitative changes in the protein levels of TFs and chromatin remodeling complexes, along with their dynamic interactions with the ge- nome, are key determinants in establishing gene expression programs that drive erythropoiesis. The rationale is that integrating the dynamic and quantitative nature of the TF proteome with other modalities will enable the development of an expanded and predictive GRN model of erythropoiesis that captures the diverse mechanisms underlying transcriptional regulation. Three specific aims have been designed: 1) Quantification of the TF prote- ome during erythropoiesis; 2) Determine how the interplay between TFs and chromatin remodeling complexes initiate and reinforce cell fate decisions along the erythroid trajectory; and 3) Computational analysis, modeling and in vivo functional validation of the erythropoiesis GRN. For the first aim, the abundances of the TF proteome will be determined during erythropoiesis by applying a hybrid mass spectrometry (MS) approach which combines the throughput of data independent acquisition (DIA)-MS with the sensitivity of parallel reaction monitoring (PRM)-MS in purified erythroid cell populations. For the second aim, we will combine MS proteomic measure- ments with genomic binding assays and 3D genome analyses, in purified erythroid cell populations to understand how TF-cofactor interactions regulate chromatin accessibility and gene expression. Under the third aim, we will integrate joint measurements of TF protein and transcript abundances, along with other transcription-relevant omics data, to build quantitative GRN models of human erythropoiesis which will be validated in vivo by specific factor knockdowns. The approach is innovative in its goal of constructing genome-scale GRNs of erythropoiesis by integrating diverse -omics date including TF protein abundance data, genomic binding and promoter-en- hancer interactions across TEAseq-defined populations spanning the entire erythroid trajectory. The proposed research is significant because it will illuminate complex regulatory processes that control erythropoiesis. Ulti- mately, such knowledge can guide efforts to manipulate cell fate decisions in health and disease.

NIH Research Projects · FY 2025 · 2013-08

PROJECT SUMMARY The University of Wisconsin (UW)-Madison Multidisciplinary Urologic Career Development Program (KURe) K12 began in 2013 to attract and retain aspiring scientists into the benign urologic research field. Our rich training environment includes robust urologic research support including a George M. O’Brien Center in Benign Urology Research; a Summer Program in Undergraduate Urologic Research (SPUUR); and a U24 interactions core focused on Collaborating for the Advancement of Interdisciplinary Research in Benign Urology (CAIRIBU). The K12 scholars and cadre of outstanding mentors also benefit from UW Madison’s NIH-funded Institute for Clinical and Translational Research (ICTR) and an internationally recognized Center for Improvement of Mentored Research Experiences. There has been tangible progress since 2013. In addition to five current scholars receiving training, six scholars have completed K12 training and earned investigator-initiated NIH funding. Five of these scholars are leading independent benign urology focused research programs as tenure track faculty at research-intensive institutions. Building on proven success, the program will continue to recruit and retain promising investigators through formative mentored research experiences and didactic training, as well as new initiatives to enhance professional development, with the goal of expanding the workforce in benign urology research.

NIH Research Projects · FY 2024 · 2013-03

Project Summary/Abstract Myocardial infarction (MI) is the most common cause of heart failure (HF), but the molecular mechanisms underlying cardiac dysfunction in the post-MI myocardium remain unclear. Moreover, current treatments for HF mainly focus on symptom management after maladaptive ventricular remodeling has occurred. However, a comprehensive understanding of molecular changes at the early-phase (adaptive) remodeling could aid in the development of treatments to prevent late-phase (maladaptive) remodeling and HF. Ventricular remodeling is characterized by alterations in the sarcomere composed of myofilaments flanked by Z-discs. The post- translational modifications (PTMs) of the sarcomeric proteins regulate contractility. Moreover, cardiac contractility highly depends on ATP generation and thus impairment in the ATP-generating processes can rapidly lead to contractile dysfunction. The onset of ischemia is known to be associated with dramatic alterations in cardiac metabolism. PTMs of metabolic enzymes play a key role in regulating the metabolic pathways. Therefore, we hypothesize that concerted dysregulations of sarcomeric and metabolic protein modifications contribute to contractile dysfunction at the early-phase post-MI ventricular remodeling. To test our hypothesis, we will employ a novel systems biology approach enabled by multi-omics integrating top-down proteomics and metabolomics with in vivo and ex vivo functional studies to delineate the molecular mechanism underlying early-phase post-MI ventricular remodeling. This novel multi-omics method allows accurate assessment of changes in the proteome and metabolome from the same heart tissue sample to understand the concerted dysregulation of sarcomeric and metabolic PTMs and metabolites. Moreover, we will use a combination of a large animal (swine) MI model and clinical ischemic cardiomyopathy (ICM) tissues to accelerate translation of our findings to aid in diagnostic and therapeutic interventions in humans. Aim 1 will identify concerted changes in the myofilament and Z-disc proteins in post-MI swine myocardium and relate to contractile dysfunction in both sexes. Aim 2 will determine the metabolic alterations in both proteome and metabolome in the post-MI remodeling. Aim 3 will identify sarcomeric and metabolic markers in ICM patient myocardium and assess their functional consequences in consideration of comorbidities and sex differences. This interdisciplinary and translational/clinical application is highly significant with a strong scientific premise and a novel hypothesis of direct clinical relevance. It will provide a comprehensive study to globally characterize a variety of PTMs in metabolic and sarcomeric proteins as well as the interplay between dysregulation between cardiac metabolism and contractile dysfunction. It has direct translational potential leading to an in-depth understanding of the underlying mechanisms at the early-phase post-MI remodeling and discovery of new sarcomeric and metabolic signatures as a panel of markers for diagnosis of ICM at early-stage.

NIH Research Projects · FY 2026 · 2013-02

PROJECT SUMMARY Asthma is an immune-mediated disorder, and early life allergic sensitization (T2 inflammation) and respiratory illnesses are the most common precursors to the development of lifelong asthma. With over 300 million patients affected worldwide, asthma is a significant health care burden and a lifelong illness for most individuals. New information is needed to design preventive treatments, and answers may come from studying children who grow up in farming or traditional agrarian (TA) environments, who have intense and unique microbial exposures and are much less likely to develop allergic diseases and asthma. We hypothesize that TA microbial exposures and colonization promote immune development and airway epithelial cell functions to reduce the risks of respiratory illnesses, allergies and asthma. To test this hypothesis, we started the Wisconsin Infant Study Cohort (WISC) in 2013 and added a cohort of TA families in 2018 to determine mechanisms for protection against allergic diseases and asthma. We find that Wisconsin farm children have distinct gastrointestinal and nasopharyngeal microbiome features and unique gene expression patterns in nasal airway epithelial cells at age 2 years. These findings correspond with lower rates of atopic dermatitis, often a precursor to asthma and respiratory illnesses. The differences in immune and microbial signatures are even more pronounced in TA children. The microbiota in TA children's stool and nasal secretions are enriched for commensals that may promote immune development and reduce rates of allergic diseases and respiratory illnesses. Notably, the bacterial species identified in the TA samples contain genes distinct from those found in samples from the farm and non-farm children. We therefore propose that TA bacteria of the GI tract and airways and their metabolites are mediators of protection from allergic diseases and will be a rich source to determine the mechanistic pathways that promote health. To test our overall hypothesis, we propose three highly interactive projects. Project I will determine how TA children differ in immune development, nasal airway epithelial cell profiles, and clinical outcomes related to respiratory illnesses and allergic diseases. Project II will evaluate how TA children's distinct microbiota and their metabolites relate to their unique patterns of immune development and the resulting clinical outcomes. Project III will use in vitro models of airway epithelial cells to determine the mechanisms of TA microbial metabolites that inhibit viral and bacterial pathogens and modify the immune and inflammatory responses of airway epithelial cells.

NIH Research Projects · FY 2025 · 2012-09

Abstract: DNA replication restart pathways reload cellular DNA replication complexes onto replication forks that have been prematurely terminated, forming an essential link between DNA repair and replication. The proteins that drive these reactions, referred to as the primosome or the Replication Restart Proteins, recognize the structures of abandoned replication forks and reload the DNA replication machinery specifically at these sites. The replication restart process is regulated to ensure loading fidelity and to avoid over-replication that could arise from initiating replication at improper DNA structures. In spite of the broad biological importance of this process, the mechanisms underlying DNA replication restart and its regulation remain poorly understood. Our proposal combines structural, biochemical, and genetic approaches to define the structural and cellular mechanisms of DNA replication restart. Our overall objective is to determine the mechanisms that govern each step of the process, from recognition of diverse structures of abandoned DNA replication forks, to primosome assembly, and finally to reloading the DNA replication machinery. Aim 1 will define the structures of primosome complexes with replication forks, which will provide new insights into structure-specific recognition of DNA replication forks and the mechanisms that regulate DNA replication restart. Structural advances are coupled to genetic studies that will define the cellular mechanisms of action of primosome proteins in bacteria. Aim 2 blends structural and genetic studies to focus on maturation of primosome complexes and their activities in reloading the replicative helicase back onto replication forks. Completion of our specific aims will define the molecular and cellular mechanisms that mediate and regulate bacterial DNA replication restart.