University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Cell division is a conserved process by which replicated chromosomes are equally partitioned into two daughter cells. Errors in this process often result in gains or losses of chromosomes, known as aneuploidy, which can cause and promote tumors and developmental diseases. During mitotic progression, chromosomes dynamically change their positions in a force-dependent manner via forces generated at kinetochores, macro-molecular protein structures built on centromeric chromatin that serves as platforms for microtubule assembly. While chromosome territories, regions preferentially occupied by specific chromosomes in interphase nuclei, have been established and are known to be involved in gene regulation and genomic protection, the presence and function of chromosome organization in mitosis have not been adequately explored. Our long-term goals are to characterize “mitotic chromosome territories” in mammalian cells and to uncover the function behind spatiotemporal regulation of both chromosome organization and kinetochore dynamics in ensuring faithful chromosome segregation. In this proposal, we will test the hypothesis that there exist chromosome organizations in mitosis as in interphase nuclei using a super-resolution microscopy method we recently developed, which will allow us to identify full sets of individual chromosomes and determine their spatial organization in mammalian cells. If there exist mitotic chromosome territories, we will explore how and when they are established and their evolution throughout mitosis. We also hypothesize that major mitotic defects (unaligned chromosomes, lagging chromosomes, and chromosome bridges) are associated with improper chromosome organization. We will examine this hypothesis by identifying which chromosomes are involved in each defect with increased frequency and determine their positionings. Mitotic cells have two major pathways for correcting mitotic errors, mediated by Aurora A or Aurora B kinases. Both kinases are spatially regulated and phosphorylate a highly conserved microtubule-binding kinetochore protein, Ndc80/Hec1, to destabilize improper microtubule bindings for promotion of error correction and regulation of SAC (spindle assembly checkpoint) activity. Aurora A-mediated error corrections require proximity of erroneous chromosomes to the spindle poles, where Aurora A is concentrated. On the other hand, Aurora B-mediated error corrections depend on dynamic deformations of kinetochores. These suggest that mitotic chromosome positioning, coupled with kinetochore dynamics, orchestrate the cooperation between Aurora A and Aurora B-mediated error correction machineries. We will dissect the contributions of chromosome positioning and kinetochore dynamics towards Aurora A and Aurora B error corrections using force- calibrated microneedles and a semi-automated, quantitative microscopy analysis software that we recently developed called the 3D speckle analyzer (3D-Speckler). Our proposed work will provide new, mechanistic insights into mitotic chromosome organization and its contribution toward ensuring the integrity of chromosome segregation, which will contribute towards developing better therapeutic and detection strategies for cancer and developmental diseases for improved patient outcomes.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Racial biases are both evident and harmful in childhood. White children show especially robust and reliable racial biases by age 5. Many have suggested that parents should play a role in addressing the negative effects of children's racial biases, but research to date has focused primarily on how parents of color can take action in this domain (e.g., to buffer effects of discrimination on their children, who are frequently the targets of discrimination). Here we test whether White parents can be effective as bias interventionists with their White children. The broad, long-term goal of the proposed research is to improve the health and wellbeing of children of color. The key objectives for the proposed work are to test the effects of a novel racial bias intervention program on White parents (Aim 1) and their 5–7-year-old children (Aim 2). Parent-child dyads will be randomly assigned to the intervention condition or to one of two control conditions. Parents in the intervention program will complete a training program informed by theory and previous empirical work. The program will teach parents how to address race with their children, and then parents will be given additional materials and support to practice what they learned in the training program with their children at home. To evaluate the effects of the intervention, participants in all conditions will complete a pre-test and two post-test assessments (one immediate, one delayed). Assessments for parents will focus on their awareness of, and concern about, children's racial biases, as well as parents' motivation and perceived self-efficacy to address race with their children. Assessments for children will focus on their racial attitudes, inclusion decisions, reactions to another person's biased behaviors in the race domain, and beliefs about their parents' racial attitudes. A further goal (Aim 3) is to test for moderation by parent-child relationship quality and the racial diversity of participants' environments. Results from the proposed work will reveal whether, how, and in what contexts White parents can be effective as bias interventionists with their children. By intervening early, we may be able to forestall the entrenchment of racial biases and decrease the likelihood that children of color will experience the harmful effects of racial biases.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT The brain must maintain immunological homeostasis to prevent dysregulation and disease. Coordination of immunity in most tissues involves the drainage of antigens or antigen-presenting cells within conventional lymphatic vessels to the draining lymph nodes. In the lymph node, antigen presented to T cells, typically by dendritic cells (DCs), can initiate an immune response. Control of immune response is unique in the central nervous system as the brain parenchyma lacks conventional infiltrating lymphatic vessels and instead utilizes a combination of intra-tissue glial-dependent clearance pathways and meningeal lymphatics surrounding the brain to drain waste, antigens, fluid, and cells. Recently there has been mounting evidence implicating meningeal lymphatic vessels as passive conductors of drainage in the progression, and resolution of various neuropathologies. We previously discovered that neuroinflammation induces lymphangiogenesis of the meningeal lymphatic vessels at the cribriform plate (cp) (Hsu et al. Nat Comm. 2019). We found that in situ meningeal lymphangiogenesis was driven by VEGF-C producing DCs, and this is unique to the cp, highlighting potentially different roles for dural lymphatics in neuroinflammation depending on their precise location. Here we show single-cell RNA sequencing data revealing that neuroinflammation induces cribriform plate lymphatic endothelial cell (cpLECs) gene expression related to antigen presentation, leukocyte adhesion, and immunoregulation. This indicates that cpLECs are not just passive conductors of drainage, but active contributors to the formation of a neuroimmune regulatory niche. We hypothesize that during neuroinflammation, the cribriform lymphatics represent an immunoregulatory niche in which migratory DCs drained from the brain are retained and communicate with cpLECs to regulate downstream immune response and homeostasis of the central nervous system. The pathways of DCs traffic through the brain to the cribriform lymphatics, the mechanism of their interaction with cpLECs, and the functional consequence of these interactions on both cell types and on the formation of a neuroimmune niche are not known. The long-term objective of this project is to define the pathways and dynamics of interactions between dendritic cells and the cribriform plate lymphatics to understand the regulation of brain homeostasis and disease. The specific objectives of this proposal are to map the timeline, origin, and mechanism of dendritic cell - cribriform lymphatic endothelial cells interaction (DC-cpLEC) in the meningeal lymphatic vessels at the cribriform plate (Aim 1); to define expressional consequences of the interaction between DCs and cpLECs (Aim 2), and to examine the impact of DC-cpLEC interactions on lymphatic functionality and immunity (Aim 3). Pharmacological manipulation of the cross-talk between dendritic cells and cribriform lymphatic endothelial cells in CNS diseases may have potential therapeutic value for diseases related to CNS autoimmunity and homeostasis.

NIH Research Projects · FY 2026 · 2022-08

Project Summary/Abstract In the United States, 42% of the population is obese, and this obesity predisposes individuals for several diseases including type 2 diabetes which affects 29 million Americans. One established mechanism for the causal relationship between obesity and type 2 diabetes is the development of lipotoxicity, a state where excess lipids build-up to cause increased ectopic lipid droplets, elevated plasma lipids, and subsequently, decreased cellular insulin signaling. These plasma lipids that are increased with lipotoxicity include acylcarnitines, a key intermediate in fatty acid oxidation. Acylcarnitines are an established biomarker of type 2 diabetes, hepatocellular carcinoma, and cardiovascular disease, and are known to cause insulin resistance. Despite the known importance of plasma acylcarnitines in the development of insulin resistance, we do not know how they are imported into cells, how this uptake is regulated, or how imported acylcarnitines are metabolized once they enter the cell from the circulatory system. In this proposal we examine the regulation of acylcarnitine uptake by transporters we have identified through a CRISPR/Cas9 screen. Through targeted knockout of these transporters, we will conduct cell culture experiments to highlight the precise molecular pathways through which acylcarnitines enter the cell and signal for insulin sensitivity. We will also explore tissue specific loss of acylcarnitines in mouse models to determine the contribution of plasma acylcarnitine uptake on whole-body energy expenditure, glucose regulation, and plasma lipid pools. Finally, we will determine how these transporters for plasma acylcarnitines are regulated in cell culture and tissue samples from mice to establish potential therapeutic targets for the treatment of metabolic disease. The proposed work will address long-standing questions on the molecular regulation of plasma acylcarnitine import, metabolism of acylcarnitines after entry into the cell, and the regulation of insulin sensitivity by acylcarnitines. In terms of translatability, this work will generate a mechanistic understanding of how plasma acylcarnitine abundance is controlled and how plasma acylcarnitines impact disease vulnerability to enable the identification of new targets for the pharmacological treatment of obesity and diabetes.

NIH Research Projects · FY 2025 · 2022-08

Project summary Infectious diarrhea remains a leading cause of morbidity and mortality in children and immunocompromised patients worldwide. However, the lack of neonatal and immunodeficient animal models that closely mirror human infection in these highly susceptible patient groups has limited our understanding of the disease. Therefore, we propose here the use of novel neonatal and immunodeficient mouse models of disease to study the mechanisms of pathogenesis and host immunity during enteric and systemic infection by the pathogens Citrobacter rodentium and Salmonella enterica serovar Typhimurium. Our first hypothesis is that C. rodentium employs different virulence factors to induce lethal systemic infection in neonatal mice compared to those required for initial intestinal colonization. Specific Aim 1 was designed to identify and characterize significant pathogen factors required for the establishment of bacteremia in neonates using genome-wide screens and functional studies in vivo (1A-C). Specific Aim 2 will address our second hypothesis regarding the protective role of IgG against enteric disease both in the neonatal and adult gut, and the existence of compensatory or alternative mechanisms of pathogen eradication in the absence of IgG (2A-C). Findings obtained with C. rodentium will be tested in the human pathogen S. Typhimurium to allow a broader understanding of the general and specific aspects of microbial pathogenesis and host immunity during infection in susceptible individuals (1D & 2D). These studies have high clinical significance as they may provide novel insight into the pathogenesis of enteric and systemic disease both in children and patients with impaired adaptive immunity, which aligns well with the goals of the National Institute of Allergy and Infectious Diseases (NIAID). Additionally, we have also developed a robust and comprehensive career development and training plan that will provide scientific training in alternative models of infection, mucosal and adaptive immunity, pathology and disease progression, high-throughput data analysis, and state-of-the-art technologies to assess cell state and inflammatory environment in the gut. This plan will also develop and strengthen key professional skills essential to successfully lead an independent research program, including training in mentoring and teaching, scientific communication, networking, grant preparation, and laboratory management. A group of highly qualified and renowned scientists will guide and evaluate the applicant’s efforts to achieve his scientific and career goals. Altogether, the research and career development activities outlined in this proposal, along with the outstanding institutional environment at the University of Michigan, will fully prepare the PI to pursue an independent career in health-oriented research, positioning him as a strong junior faculty in the fields of bacterial pathogenesis, host immunity and host-pathogen interactions.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY The colon contains tryptophan derivatives such as indole, which is a microbiota-derived signaling molecule, and the host-derived serotonin neurotransmitter that is primarily synthesized in the GI tract. Indole is also known to be absorbed by host cells and helps strengthen the integrity of the intestinal barrier, being regarded as a beneficial chemical cue within microbial/host interactions. Indole is synthesized by tryptophanase, which is encoded by the tnaA gene. We have shown that the concentration of indole is significantly higher in the lumen of the colon (the compartment where the microbiota resides) compared to colonic tissues (where indole is absorbed by intestinal epithelial cells). Serotonin is synthesized in enterochromaffin cells by the enzyme tryptophan hydroxylase (TpH1). Upon its synthesis, serotonin is released into the lamina propria and is secreted into the lumen. Serotonin signaling in the intestinal mucosa is terminated by removal of serotonin by the serotonin selective reuptake transporter (SERT), which is expressed by epithelial cells. We showed that both serotonin and indole converge to decrease virulence gene expression from enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine pathogen employed as a surrogate animal model for EHEC. We also identified the bacterial receptor for these signals as CpxA. Upon sensing serotonin and/or indole, CpxA functions primarily as a phosphatase, dephosphorylating itself and CpxR, that activates virulence in its phosphorylated state. Through transcriptome studies we also identified the Indole Sequestering Receptor (Isr), which in the absence of indole directly activates virulence expression. However, in the presence of indole, Isr is no longer able to activate transcription of virulence genes. Using TpH1 pharmacological inhibitors (decrease the levels of serotonin in the gut) and SERT knockout mice (have increased levels of luminal serotonin), we showed that the presence of higher levels of serotonin in the intestine of mice decreased virulence in C. rodentium, while decreased levels of serotonin are conducive to increased pathogenesis. Moreover, we synthetically altered the concentration of indole in the GI tract of mice. This allowed us to assess the role of self-produced versus microbiota-produced indole, and show that decreased indole concentrations promote bacterial pathogenesis, while increased levels of indole decreases bacterial virulence gene expression during murine infection. Altogether, both serotonin and indole decrease virulence of C. rodentium during murine infection. Our studies show that fluctuations in the levels of indole and the serotonin neurotransmitter significantly impact disease prognosis. However several questions regarding this exquisite signaling regulation of bacterial virulence remain unanswered. Consequently the specific aims of this grant are: Aim 1. Define the CpxA/CpxR and Isr serotonin/indole signaling cascade. Aim 2. Investigate the intersection of serotonin with endogenous and exogenous indole signaling in bacterial pathogenesis during mammalian infection.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Early life adversity (ELA), such as physical or sexual abuse, witnessing a crime, family conflict, or community violence, can have prolonged effects and significantly increases the risk for later psychopathology. Experiencing such adversity can result in a wide range of psychiatric disorders, which have been found to be more resistant to treatment. Further complicating intervention efforts, some youth exposed to ELA continue to show normal functioning and behavior over time (“adaptive” or “resilient”), while others show increasing psychiatric symptoms and go on to develop psychiatric disorders (“vulnerable”). The factors and neurobiological mechanisms that underlie this variability and that prospectively identify those at greatest risk are not yet well understood. This is a critical knowledge gap in the ability to help youth who have experienced early life adversity. Recent research suggests that youth with ELA have abnormal function and connectivity within and between 3 core brain networks which support emotional reactivity and cognitive-emotional control - the salience network (SN), frontoparietal network (FPN), and default mode network (DMN), and changes in these networks are associated with a wide range of psychopathology. To date, however, no study has systematically examined the relative developmental trajectories of these core networks, how they are altered by ELA exposure, and how such alterations relate to the expression of general psychopathology risk and unique dimensions of internalizing vs. externalizing. The proposed study addresses these gaps of knowledge by examining the association between ELA, FPN, DMN, and SN network development, and both overall psychopathological load as well as specific dimensions of psychopathology using data from a large cohort (n=11,873) of adolescents from the Adolescent Brain Cognitive Development (ABCD) study. Demographic, environmental, and behavioral data are acquired every year and neuroimaging data are acquired every 2 years, starting at age 9-10y. This is a critical developmental period, the entrance into puberty, characterized by both a rapid emergence of psychiatric disorders as well as sex differences in the prevalence of internalizing and externalizing disorders. The function and interaction of SN, FPN, and DMN are examined both at rest and during cognitive load to determine the extent to which ELA-related network alterations are modality specific. Data are analyzed using both model-based and data-driven methods. Finally, this research uses both model- based prediction and machine learning to determine potential early predictors of later psychopathology.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Each year >500,000 Americans diagnosed with cancer have tumors that could be targeted with an FDA- approved drug to treat their disease more effectively. However, there is wide variation in use of these treatments in clinical practice. Rural patients, who are more likely to die from cancer than their non-rural counterparts, are less likely to receive targeted cancer therapies and could benefit greatly from interventions to address this quality gap. This study will test the effectiveness of our Multi-TEAM Systems Framework Precision Oncology Reflex Testing (TEAMSPORT) intervention to standardize delivery of targeted cancer therapy and adapt it for use in rural and non-rural community cancer programs, where the majority of cancer patients receive their care. Our experienced multidisciplinary team of molecular pathologists, medical oncologists, rural implementation scientists, and team system scientists will be augmented by a Community Advisory Board of community-based pathologists, oncologists, cancer program administrators, and cancer network leaders representing approximately 1,000 community oncology programs. Together they will ensure the most cutting-edge, evidence- based precision oncology science will be supported by best practices in effective inter-team coordination and packaged for optimal uptake by rural and non-rural community cancer programs. In this modular R01, responsive to NCI priorities to evaluate geographic disparities in technology dispersion and further team system science, we will conduct an interrupted time series study to test the effectiveness of the TEAMSPORT intervention on guideline-concordant genomic testing and team coordination. We will simultaneously work with community collaborators to adapt the intervention to community settings and test its acceptability, feasibility, appropriateness, and adoptability among a large sample of community cancer programs. This scientific study will result in: 1) An evidence-based toolkit to increase appropriate use of genomic testing and targeted cancer therapy, suitable for rural and non-rural community cancer programs 2) One of the first tests of the effectiveness of a multi-team system intervention, and the impact of relational coordination, a validated measure of team connectedness, on quality of care 3) Effect sizes of a multi-team system intervention for use in future practice-randomized trials 4) Locally-endorsed, best practice protocols for precision oncology testing and reporting 5) A novel audit and feedback platform for institutions to assess adherence to national guideline recommended precision oncology practices Ultimately, this study has potential to lead to greater job satisfaction among cancer care providers; wider and more timely use of genomic testing and appropriate cancer therapies; better cancer outcomes for patients; reduced harms from unnecessary treatment; and a reduction in rural-urban cancer disparities.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Chromosome segregation errors can produce cells with an incorrect number of one or more chromosomes, known as aneuploidy. Aneuploidy is therefore a special class of mutation that can have immediate phenotypic effects. Although aneuploidy is detrimental during mammalian development, it is common in many cancers and a driver in the evolution of drug resistant tumors and fungal pathogens. A major unaddressed question is the degree to which different individuals vary in their ability to tolerate aneuploidy. Understanding how genetic differences influence aneuploidy tolerance has far reaching implications for genetics, human biology, and evolution. But studying this topic mammalian systems is extremely challenging, since it is not possible to systematically manipulate karyotypes in a large number of genetic backgrounds. Here we will address the fundamental question of how genetic variation influences the ability of cells to tolerate chromosome duplications, in the model eukaryote Saccharomyces cerevisiae. Using the power of yeast genetics, we adapted a method to duplicate specific yeast chromosomes in the near absence of selection. We will apply this method to explore the breadth and mechanisms of genetic variation in tolerating chromosome duplications (herein referred to as aneuploidy). Aim 1 will use this approach to duplicate each of the 16 chromosomes in yeast, in dozens of non-laboratory strains across the yeast phylogeny. Results will characterize the range of natural variation in aneuploidy tolerance and will test if this variation occurs sporadically due to rare alleles or persists across many strains within specific lineages. Preliminary results suggest lineage-specific variation in aneuploidy tolerance. Aim 2 will test if variations in aneuploidy sensitivity are due to differences in “generalized” aneuploidy tolerance, in which cells are sensitive regardless of which chromosome is duplicated, versus chromosome-specific sensitivities that are likely driven by the effects of duplicated genes encoded on those chromosomes. We will test how well chromosome-specific sensitivities are explained by an additive gene model that is based on measured fitness costs of the genes’ over-expression, measured here from a gene over-expression library expressed in each strain. Aim 3 will begin to uncover the physiological and genetic mechanisms for variable aneuploidy tolerance. We will first test our hypothesis that genetic variation in aneuploidy tolerance is due to variations in the ability to manage proteostasis stress. We will then use bulk- segregant mapping to study the genetic architecture of that variance and identify casual genes. Yeast is an outstanding model in which to study this fundamental question, since many cellular mechanisms and genetic principles are conserved in other organisms including humans. This project will generate important insights into aneuploidy tolerance that will have broad implications for genetics, human health, and evolution.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Cell type-specific transcriptional networks are gene regulatory networks that dynamically reconfigure to drive precise spatio-temporal expression patterns of genes. These networks are central to cell type specificity and are often disrupted in many diseases. The structure of these networks is defined by a trans component that specifies which regulatory proteins control a gene’s expression and a cis component that species the regulatory regions that can regulate a gene’s expression both proximally and distally. Identifying these regulatory networks has been a significant challenge for mammalian cell types because of the number of potential regulators of a gene and the large number of assays needed to define these networks accurately. Advances in single cell omics technologies, such as single cell RNA-seq (scRNA-seq) and single cell ATAC-seq (scATAC-seq), offer new opportunities to define cell type-specific regulatory networks because of their ability to comprehensively profile the transcriptome and accessibility for thousands of individual cells. However, computational methods for integrating these data to define both cell lineage structure and cell-type specific regulatory networks are limited. Most methods have used only one type of assay focusing either on the cis or trans components and have not modeled temporal or hierarchical relatedness of multi-sample datasets. Finally, performance of computational network inference methods has remained low when compared to experimentally detected networks. To address these challenges, we will develop novel computational methods and powerful resources for mapping gene regulatory network dynamics driving cell type specificity. Our aims are to (a) develop a computational toolkit to integrate scRNA-seq and scATAC-seq datasets to infer both cell type lineage (Aim 1) and cell type-specific transcriptional regulatory networks from scRNA-seq and ATAC-seq data (Aim 2), (b) identify the rewired network components during a dynamic progress such as cellular reprogramming (Aim 2), and (c) develop an active learning based approach to infer causal regulatory networks and apply this framework to refine the regulatory networks for cellular reprogramming (Aim 3). We will apply our tools to public and newly collected datasets as part of this project. Our analysis will reveal cis and trans regulatory network components associated with cell fate specification during a dynamic process such as reprogramming or development. Our active learning approach will use Perturb-Seq to perform regulator perturbations to both validate the predicted networks as well as to establish improved gold standards for a system with high significance for translational and basic research. The tools and datasets generated by this project will be publicly available and will serve as a powerful resource to understand regulatory network dynamics in cell fate specification. Our tools should be broadly applicable to define regulatory network dynamics for diverse biological processes.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Intestinal immune responses are linked to the trillions of microorganisms that colonize the gastrointestinal tract. Thus, inter-individual variations in the gut microbiome could contribute to altered immune responses that impact immune driven diseases such as autoimmunity. Activation of T helper 17 (Th17) cells by members of the gut microbiota can contribute to autoimmunity. Further, evidence is emerging that the diet influences both the immune system and the microbiome. While the pairwise interactions between dietary factors, the microbiota, and immunity have been broadly characterized, the field is just beginning to investigate the mechanistic interplay between diet, microbiome, and immunity and the downstream consequences on autoimmunity. The goals of this work are to investigate microbial mechanisms of Th17 cell activation, their diet-responsiveness, and the functional consequences of these interactions on autoimmune diseases such as inflammatory bowel disease (IBD) and multiple sclerosis (MS). Our preliminary studies reveal mechanistic insights into specific diet- dependent factors that counteract specific pro-inflammatory gut bacterial species. Two prevalent human gut species associated with human autoimmune diseases, Eggerthella lenta and Bifidobacterium adolescentis, induce Th17 cells in the intestine in a diet-dependent manner. Dietary arginine and ketogenic diets (KDs) prevent Th17 induction by E. lenta and B. adolescentis respectively. Further, a specific bacterial gene in E. lenta, cgr2, is sufficient to activate Th17 cells. We aim to determine diet-dependent mechanisms of Th17 activation by E. lenta metabolites and functional consequences IBD and MS mouse models. By combining immunological and microbiome techniques with metabolomics and translational research expertise of our collaborators we aim to identify a small molecule metabolized by E. lenta responsible for Th17 activation and assess the disease relevance of dietary modulation of this metabolism. Secondly, we aim to examine the mechanism and disease relevance of ketone bodies for limiting gut bacterial Th17 induction. A KD-associated gut microbiota reduces intestinal Th17 cells via selective inhibition of bifidobacterial growth by the ketone body β-hydroxybutyrate (βHB). Therefore, we hypothesize that the ketone body βHB selectively inhibits B. adolescentis-mediated Th17 induction resulting in functional consequences for MS disease models. To address this hypothesis and elucidate the mechanism by which βHB impacts the Th17 induction capacity of B. adolescentis, we will use bacterial genetic manipulation and disease models. The proposed aims will leverage the candidate’s expertise in immunology and microbiome studies with new training in metabolomics, bacterial genetics, and translational research studies. UCSF’s institutional focus on the microbiome, metabolomics, immunology and translational research and close collaboration with experts in these areas will provide an ideal environment for the proposed scientific and professional development leading to the creation of an independent research program.

NIH Research Projects · FY 2026 · 2022-08

Project Summary/Abstract First language acquisition is a hallmark of typical human development. A substantial body of research suggests that infants’ ability to detect statistical regularities in language input facilitates language learning. However, the impact of this literature has been limited by its failure to connect statistical learning with the burgeoning body of research and theories focused on infants’ real-time language processing. The current application is motivated by the premise that statistical regularities facilitate infants’ attempts to efficiently encode and process language input. To this end, infants generate predictions about likely downstream input. These predictions are often incorrect, rendering prediction errors – a potentially potent source of data for subsequent learning. Input that is probabilistic and/or that has previously led to prediction errors provides information-rich data, guiding infants’ subsequent attention and learning. We hypothesize that statistical regularities are an important source of information influencing this process, along with the other contextual cues available in both the linguistic and nonlinguistic environment. To date, no prior studies have manipulated statistical regularities during infant language processing tasks; research is necessary to adjudicate amongst the possible relationships between statistical regularities in the input and real-time language behaviors during development. In the proposed experiments, we will measure infants’ use of sequential statistical regularities during predictive language processing tasks (Aim 1), assess the impact of statistical regularities on prediction error-based learning (Aim 2), and examine the role of uncertainty due to statistical structure and prediction error in motivating infants’ active exploratory behavior during language learning (Aim 3). The results of the proposed research will promote positive developmental outcomes by expanding our understanding of the relationship between the statistical structure of language input and real-time processes that are unfolding during language development. As in our previous statistical learning research, the outcomes of these studies with typically-developing infants will motivate future investigations that include infants and young children at risk for atypical language development trajectories.

NIH Research Projects · FY 2025 · 2022-07

SUMMARY: The objective of this study is to determine if the polo-like kinase 4 (PLK4) along with other melanoma driver pathways, is a therapeutically actionable druggable target for melanoma management, and what are the mechanisms and interacting partners of PLK4, during melanocytic transformation and neoplastic progression. Melanoma is a clinically challenging skin cancer, if not diagnosed early. Epidemiological and genomic data suggest that BRAFV600E mutations may be the initiating lesion in melanocytic nevi; however, these mutations alone are not sufficient for malignant transformation. Ultraviolet radiation (UVR) and activation of other oncogenic pathways are known to contribute to the neoplastic progression of melanocytes. In the recent past, the treatment landscape for advanced melanoma management has seen dramatic changes with the approval of new drugs such as BRAF inhibitors as well as immune-checkpoint inhibitors. However, these treatments are linked with acquired resistance occurring in nearly 50% of patients. Therefore, novel mechanism-based therapeutic approaches are needed for effective management of this dreaded neoplasm. Based on limited number of recent studies, PLK4 is being considered as a potential druggable target for certain cancers. PLK4 inhibition has been shown to cause a failure of centriole and centrosome duplication, whereas its overexpression results in excess centriole formation, which are sufficient to drive centrosome amplification (CA) and genome instability that is linked to carcinogenesis. A recent study has suggested a role of PLK4 in epithelial-mesenchymal transition (EMT) via modulating PI3K/AKT pathway. We recently demonstrated that PLK4 is significantly overexpressed in melanoma, and small molecule PLK4 inhibition resulted in a significant anti-proliferative response in multiple melanoma cell lines [Mol Cancer Res, 2018]. Our preliminary data has shown that PLK4 CRISPR K/O A375 melanoma cells show significantly decreased tumor growth in melanoma xenografts suggesting an important role of PLK4 in melanoma. We also found that combined inhibition of PLK4 with BRAF and MEK inhibition exerted synergistic antiproliferative effect in melanoma cells. In this study, we propose to challenge a hypothesis that PLK4 signaling, together with other driver pathways of melanocytic transformation and neoplastic progression, will provide therapeutically-actionable novel co-targeting approaches, for melanoma management. Three aims are proposed to; 1) determine the association between PLK4 and other driver pathways of melanocytic transformation and neoplastic progression ex vivo; 2) determine the functional and mechanistic significance of PLK4 in melanoma progression and metastasis in vivo in a variety of human-relevant genetically engineered mouse models; 3) determine the therapeutic significance of PLK4 inhibition, alone and in combination with other promising target-based anti-melanoma modalities in vivo. We expect that our study will establish the exact role of PLK4 in melanoma, and its diagnostic/prognostic as well as therapeutic significance in this neoplasm.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY Circadian rhythms drastically alter animal behavior through diverse actions on cellular targets throughout the body. Disruptions of sleep-activity cycles account for a wide range of diseases affecting behavior, neurology, metabolism, and muscle physiology. This proposal presents a set of studies designed to understand the mechanisms and interactions of circadian effects on cells and systems spanning the body. Circadian rhythms manifest downstream of pacemakers via signaling molecules that act directly on targets to regulate physiology. Understanding how distinct targets are modified to achieve a constellation of physiological and behavioral rhythms is a major goal in chronobiological research. We study how neurons, muscles, and biomechanics interact in larval zebrafish, a tractable diurnal vertebrate, and our preliminary experiments suggest these animals experience a breadth of circadian changes far more diverse than previously known. Although the zebrafish is an imperfect model of sleep behavior, its clear circadian rhythms combined with its tractability for physiological, behavioral, and genetic approaches make it an ideal system for understanding how circadian rhythms organize and interact across cells and organs. We propose to define how diverse circadian effects on nervous system output and muscle physiology amount to complex behavioral output, by tracking and modeling zebrafish behavior and arousal across the diel cycle. We will manipulate light exposure to disentangle circadian and photic effects on target systems, and we will use computational models to understand how circadian effects interact to shape behavioral output. Furthermore, we will evaluate the breadth of circadian regulation of physiology across cellular targets by performing in vivo electrophysiology and functional imaging. Combining this approach with cellular- resolution transcriptional profiling enables us to define how circadian effector molecules signal divergently across targets. Finally, we will test for interactions across circadian targets by examining rhythms following focal lesions throughout the nervous system. Together these experiments will provide detailed information and models regarding the interaction of circadian effects across cells and systems.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Although the critical roles Hox genes play in establishing skeletal morphology has been known for decades, virtually nothing is understood regarding the molecular mechanisms by which Hox genes function in the skeleton. Utilizing a unique collection of genetic tools that permit live visualization of Hox expression (Hoxa11eGFP), Cre-mediated lineage labeling and/or conditional deletion (HoxCreERT2, Hoxd11LoxP/LoxP) and assessment of Hox11 chromatin binding sites (unpublished, validated Hoxa113XFLAG and Hoxd113XFLAG alleles), the overall objective of this application is to dissect the pathways and targets regulated by Hox transcription factors in skeletal stem/progenitor cells to regulate osteogenic and chondrogenesis differentiation. Previous work has demonstrated that Hox-expressing stem/progenitors are maintained in the skeleton in the absence of Hox function, and osteo- and chondrogenic lineages continue to emerge (Sox9-, Osx-/Runx2-expressing), but differentiation is incomplete. Osteoblasts do not progress to mature stages, and chondrocytes fail to undergo normal apoptosis and replacement by bony matrix in Hox mutants. This differentiation defect can be recapitulated in vitro. Based on previously published work and preliminary data, the central hypothesis is that Hox transcription factors regulate critical downstream events at the top of the hierarchy during osteochondrogenic differentiation from skeletal stem/progenitor cells in parallel with canonical differentiation factors. This project will utilize the Hoxa11eGFP reporter and Hoxa11CreERT2-mediated lineage labeling in the presence and absence of adult conditional deletion of Hoxd11 to probe the single cell trajectories of Hox11- expressing progenitors as they expand and differentiate into cartilage and bone in response to injury (Aim 1). The recapitulation of osteo- and chondrogenic differentiation defects in Hox11 mutants in vitro permits a comparative assessment of differential gene expression during temporally controlled differentiation (Aim 2). Newly generated and validated Hoxa113XFLAG; Hoxd113XFLAG epitope-tagged alleles will be utilized to interrogate the sites of chromatin binding in Hox-expressing progenitors and early differentiating cells (Aim 3). The research proposal is innovative in its use of sophisticated genetic tools generated by the research team, the combined in vivo and in vitro approaches, and critical inclusion of a co-investigator and her team with biostatistics expertise. The proposed research is significant as it addresses the longstanding and highly significant question of the molecular mechanism of Hox function in the skeleton. As Hox expression is only observed in skeletal stem/progenitors and early differentiation markers initiate as cells exit the Hox lineage, dissecting the downstream targets and pathways regulated by Hox that are critical to complete successful osteogenic and chondrogenic differentiation will provide impactful new knowledge of skeletal biology.

NIH Research Projects · FY 2024 · 2022-07

Abstract Diabetic foot ulcers (DFU) impact over 2 million Americans annually, result in over 130,000 amputations each year, and are associated with high mortality rates. To date, there has been no single infectious agent of DFU identified as a good marker of healing outcome. This is likely because DFUs are host to a diverse community of microbes (i.e., the wound microbiome). Wound microbiomes analyzed at the community-level are promising predictors of wound healing outcomes. We have shown that wounds persisting beyond 12 weeks exhibit high and persistent proportions of mixed-population, anaerobic bacteria within their microbiomes. Additionally, we found a significant increase of anaerobic transcriptional activity in persistent and amputated wounds, even from species identified as being in low abundance by traditional 16S rRNA based gene sequencing. This suggests microbial transcription, and specifically from anaerobic bacteria, are promising biomarkers of wound healing in diabetic patients. The proposed project will identify microbial biomarkers that can be used as prognostic and monitoring tools for DFU wound healing. We hypothesize using RNA instead of DNA will provide a better snapshot of the wound environment and more sensitive biomarkers: specifically, the proportion and transcriptional activity of anaerobes within the wound microbiome can be used as predictors of wound healing outcomes. Using RNAseq to measure the metatranscriptome of the DFU microbiome, we will leverage advances in machine learning approaches to demonstrate the capacity of the anaerobic component of the wound microbiome to serve as a biomarker for wound healing. We have vigorously evaluated the optimal sample collection techniques and methods of detection, determining a simple swab of the ulcer bed is sufficient to characterize the metatranscriptome and will facilitate clinical implementation. We will develop a multiplexed RT-qPCR assay for the panel of candidate biomarker genes we identify and validate the assay sensitivity, specificity, accuracy and precision. We will work closely with the Diabetic Foot Consortium Steering Committee and Data Coordinating Center to test and validate our biomarkers in a multi-site trial with the DFC. Our goal is to develop a multiplexed biomarker assay integrating the complex interactions occurring within the DFU microbiome and tissue microenvironment. It will use a simple swab of the ulcer bed, rely on objective measurements, and is designed to predict healing trajectories at an early time in the clinical course.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY ABSTRACT While other diabetes complications decreased, amputations (combined major and minor) due to foot ulcers increased 63%, reaching a 20-year peak. More than two million Americans develop a foot ulcer annually, placing them at risk of limb loss. Even worse, rural patients face a 37% greater risk of above-ankle, major amputation compared to urban counterparts, a health disparity identified by our group and others. We urgently need interventions to address this grave rural disparity and escalating amputation rate. Our systematic review of 33 studies spanning four continents reported that urban integrated care models reduce major amputation by approximately 40%. Urban integrated care models work by co-locating multiple specialists in the same clinic and using algorithms to address four physiologic factors: 1) poor glycemic control, 2) vascular disease, 3) mechanical complications, and 4) secondary infection. However, the urban integrated care model has never been adapted to rural, primary care settings. We engineered the first integrated care model for rural patients with diabetic foot ulcers, which is innovative in supporting both rural primary care and care that bridges rural and urban settings. To do so, we partnered with a HRSA-awarded Cooperative of 43 rural healthcare systems with a nationally recognized focus on improving rural diabetes care. Together, we identified the #1 health system barrier to rural, integrated care: poor collaboration across the rural-urban health system divide. Without co-location, rural providers and urban specialists struggle to manage the highest risk patients―those with ischemia and infection. Next, we co- designed an integrated care model to promote cross-setting collaboration without co-location. Our model includes two tools: 1) a care algorithm and 2) a referral checklist. The care algorithm supports rural primary care in providing high quality, local care to most patients. It also addresses obstacles to collaborating with urban specialists by providing a priori agreed upon referral criteria including timeframes, clinical indications, and pre-consultation diagnostics for severe disease. The referral checklist will support rural clinic schedulers, who place referrals to urban specialty clinics, by providing schedulers with a list of documents that should be included, reducing barriers of time-consuming triage and disjointed electronic health records. This early-stage-investigator proposal answers NIDDK’s call for small R01 pilot/feasibility trials in preparation for a statewide trial. We aim to: 1) build recruitment and retention strategies that work across diverse, rural clinics, and 2) evaluate the potential of our integrated care model to reduce major amputations by examining its impact on guideline-concordant care processes, including urban specialty referral. These aims 1) address the top reasons clinical trials fail―poor recruitment and retention, and 2) generate preliminary evidence of efficacy for the statewide trial. Our pilot is the next step towards the first intervention to reduce rural health disparities in major amputations, addressing amputation as a NIDDK priority outcome in a priority, rural population.

NIH Research Projects · FY 2024 · 2022-07

Project Summary/Abstract The gastrointestinal (GI) tract is populated by a dense and diverse microbiota that impacts human health. Although the microbiota composition at the species level for each individual is unique as a fingerprint, its composition at the phyla level is more conserved. The predominant phyla are Bacteroidetes and Firmicutes, followed by Proteobacteria. The gut microbiota has been largely regarded as a resistance barrier towards enteric pathogens. However, the enteric pathogens that cause infectious colitis, enterohemorrhagic E. coli (EHEC) O157:H7 and Citrobacter rodentium (extensively used as a surrogate EHEC model for murine infections, given that EHEC does not infect mice), exploit cues and nutrients made available by members of the microbiota to regulate their virulence program. They sense several metabolites, including sugar sources such as fucose, and organic acids such as succinate to gauge the GI biogeography and precisely regulate their virulence programs. The EHEC Cra/KdpE/FusR signaling cascade plays a crucial role in this regulation. The relationship between EHEC and different members of the microbiota varies. Our studies using a representative member of each of the main phyla, Bacteroides thetatiotaomicron (Bacteroidetes), Enterococcus faecalis (Firmicutes) and commensal E. coli (Proteobacteria) suggest that EHEC virulence expression varies in response to these commensals, as well as to different combinations of them. In this grant proposal we aim to address how different minimal microbiota compositions impact enteric infections. These studies will build from reductionist to holistic approaches to delve into mechanistic aspects of pathogen-microbiota-host interactions. It is notable that these studies will also be relevant to other enteric pathogens, such as Salmonella enterica and Clostridium difficile, among others, which share several of these pathogen-microbiota interaction strategies with EHEC. Hence the specific aims of this proposal are: Specific Aim 1. Investigate the impact of different members of the microbiota in EHEC’s Cra/KdpE/FusR signaling cascade. Specific Aim 2. Investigate the impact of different microbiota compositions on EHEC infection of enteroids. Specific Aim 3. Investigate the impact of different microbiotas in C. rodentium murine infections.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Delirium is a complex neuropsychiatric syndrome characterized by acute and fluctuating changes in cognition and consciousness. Delirium survivors suffer from a cluster of cognitive, physical, and psychological disabilities. These disabilities lead to high healthcare utilization, lower quality of life, and loss of functional independence. Worse, a single episode of delirium increases the risk of Alzheimer disease and related dementias. Based on work from our group and the work of others, over 25% of patients who undergo emergency intra-abdominal surgery suffer from at least one episode of delirium during the index hospitalization, putting them at high risk for cognitive decline. There is a fundamental gap in knowledge regarding the best way to prevent cognitive, psychological and functional decline in patients who undergo emergency general surgery and subsequently develop delirium. The long-term goal of this line of research is to improve the health and quality of care for older emergency general surgery survivors. The objective of this application is to apply the concepts of collaborative care to a high-risk patient population. Indiana University School of Medicine and University of Wisconsin School of Medicine and Public Health researchers have over 20 years of experience developing innovative and effective collaborative care models that integrate with primary care and specialty physicians to address the complex biopsychosocial needs of patients with chronic disease states, such as dementia and depression. Our team has developed a specific collaborative care model called the Emergency General Surgery Delirium Recovery Program. This proposal aims to conduct a randomized controlled trial to evaluate the efficacy of 12-months of collaborative care in improving the cognitive, functional and psychological recovery of emergency intra-abdominal surgery patients who suffer at least one episode of delirium in the post-operative period and are at least 65 years old. The trial has the following specific aims: 1) Evaluate the ability of the Emergency General Surgery Delirium Recovery Model to improve the cognitive recovery of older Emergency General Surgery delirium survivors; and 2) Evaluate the ability of the Emergency General Surgery Delirium Recovery Model to improve the physical recovery of older Emergency General Surgery delirium survivors; and 3) Evaluate the ability of the Emergency General Surgery Delirium Recovery Model to improve the psychological recovery of older Emergency General Surgery delirium survivors. The research proposed in this application is innovative because it represents a new and substantive departure from the status quo. Previous collaborative care models focused on chronic care management and they lack rapid adaptability. We have also adapted the intervention to be completed solely via telehealth. This contribution will be significant as broad application of the Emergency General Surgery Delirium Recovery Program at hospitals across the country could result in better health and improved quality of care for a particularly vulnerable patient population.

NIH Research Projects · FY 2026 · 2022-07

Project Summary: Tissue necrosis is a form of cell death caused by a wide variety of diseases and injuries. Current methods of detecting tissue necrosis to guide surgical decision making are limited. In burn injury, clinical visualization of tissue necrosis is the standard of care; however, it is an imprecise method that can result in delays in care, unnecessary surgery, and removal of viable tissue. There is a critical need to identify novel methods to improve the detection of necrosis in burn injury to aid perioperative clinical decision making. While Indocyanine Green Angiography (ICGA) has been shown to identify burn depth using perfusion as a surrogate marker for necrosis, it has not been widely adopted for clinical decision making. Recently, clinical trials using delayed imaging of high dose ICG (Second Window Indocyanine Green - SWIG) have shown promise in imageguided surgical resection of tumors. We propose that fluorescent imaging with ICGA and SWIG and a novel endogenous fluorophore can be employed to enhance surgical decision-making in burn injury as well as in many disease processes involving necrosis. The knowledge gained from this project will fill the critical need to prevent unnecessary surgery, improve surgical precision, and provide insight into fluorescence localization in inflamed and necrotic tissue. The goal of this project is to characterize the ICGA and SWIG fluorescence in burn inflammation and necrosis on a macroscopic and microscopic level and explore the novel use of protoporphyrin as an endogenous fluorophore. Specific Aim 1 will characterize fluorescent signals from ICGA and SWIG in the healing potential of indeterminate depth burns in humans. Specific Aim 2 will evaluate the diagnostic accuracy of intraoperative fluorescence-guided surgical resection of necrotic tissue in humans. Specific Aim 3 will characterize fluorescence quantification and microenvironmental gene expression in inflamed, necrotic, and healthy tissues and determine substrate localization using cell culture and animal models. To attain our goal, we will use a team science approach including a burn surgeon scientist who has extensive experience in human thermal injury models and clinical expertise in the surgical care of burn patients along with imaging experts who have a track record for developing advanced fluorescence-based technologies for in vivo imaging. These studies will provide foundational data to inform the design of a randomized clinical trial comparing fluorescence-based burn excision to the current standard of care. Quantification of fluorescence and correlation to inflammation and necrosis will support applications of fluorescence-guided surgery in diseases, including cancer, soft tissue infections, and chronic wounds.

NIH Research Projects · FY 2024 · 2022-07

NIA issued RFA-AG-22-011 to test compounds to “prevent, delay, or treat aging-related conditions by modulating fundamental aging-related mechanisms” in humans. RFA-AG-22-011 specified the need for clinical trials to determine the effects on 1) predictors of clinical outcomes, 2) the specificity of molecular target versus off-target effects, and 3) safety. Pharmacological inhibition of mechanistic target of rapamycin (mTOR) has been repeatedly demonstrated to extend lifespan and prevent or delay several age-related diseases in diverse model systems. However, the risk of potentially serious side effects in humans have thus far prevented the long-term use of the mTOR inhibitor rapamycin as a therapy for aging and age-related diseases. Therefore, a critical gap in knowledge is whether rapamycin or rapamycin analogs (rapalogs) can safely improve healthy aging in humans. Our team has demonstrated that inhibition of mTOR complex 1 (mTORC1) is beneficial and extends healthy aging in mice; however, many of the negative side effects of rapamycin result from “off-target” inhibition of a second mTOR complex (mTORC2) in multiple tissues. We and others have systematically identified intermittent dosing schedules and alternative rapalogs that enable more selective mTORC1 inhibition. The objective of this project is to determine if 24 weeks of daily low dose (0.5 mg/day) or weekly intermittent (5 mg/week) treatment with the rapalog everolimus can safely improve physiological and molecular hallmarks of aging in middle-aged to older insulin resistant adults who are at high risk for nearly every age-related condition. Using a double-blinded, randomized, placebo-controlled clinical trial, we will perform a battery of gold standard and innovative techniques to test the hypothesis that daily low dose or weekly everolimus treatment will improve five interrelated domains of physiological aging: metabolic, cardiac, cognitive, physical, and immune function. We will also assess the incidence of adverse events and changes from baseline blood chemistry, hematology, lipids, glucose, insulin, and c-peptide. To comprehensively examine the molecular target specificity and the impact on mechanisms of aging by everolimus, we will evaluate mTORC1 and mTORC2 signaling, assess mitochondrial bioenergetics, and perform a multi-omics approach (epigenomics, transcriptomics, proteomics, lipidomics, and metabolomics) in blood and muscle biopsy samples. We will also explore the role of everolimus on the senescence-associated secretary phenotype, the DNA methylation clock, and proposed biomarkers of aging. To complete this holistic approach, the assembled team of scientists and clinicians are all located at the University of Wisconsin-Madison and will leverage multiple NIH-funded resources to ensure safe, rigorous, and efficient study execution. By completion of this study, we expect to understand if everolimus can safely exploit the potent gero-protective effects of mTORC1 inhibition for the treatment and prevention of age-related diseases in humans.

NIH Research Projects · FY 2025 · 2022-07

Abstract Despite improvements in medical management, hypertension still affects >25% of adults, and 20 to 30% of them are resistant to pharmacological treatment. Similarly, heart failure patients with reduced ejection fraction continue to exhibit dramatically reduced life expectancy, frequent hospitalization, and overall poor quality of life. Chronic electrical stimulation of the baroreflex at the carotid sinus—known as baroreflex activation therapy (BAT)—is FDA-approved to mitigate the marked sympathetic activation associated with both hypertension and heart failure. BAT was demonstrated in multiple controlled clinical trials to produce sustained significant improvements in both hypertension and heart failure outcomes in patients non-responsive to traditional medical management; however, the therapy is limited by side effects. We propose an integrated approach to mitigate side effects and thereby improve the therapeutic efficacy of BAT. We seek to determine the functional neuroanatomy responsible for the side effects of BAT and to use these data to design and test approaches for more effective BAT neural interfaces. These optimized designs will expand the therapeutic window between baroreceptor activation and limiting off-target effects. The outcomes of this project will produce an optimized BAT interface design that could be quickly translated to address a clear clinical need. In addition, we will provide a needed framework for incorporation of local neural and tissue anatomy—which govern therapy-limiting side effects—into the neural interface design process that can be readily applied to myriad neuromodulation therapy targets.

NIH Research Projects · FY 2025 · 2022-07

Project Summary. This proposal details proposed activities by the Midwest Center of Excellence for Vector-Borne Diseases (MCE-VBD) in response to the CDC Funding Opportunity RFA-CK-22-005. The MCE-VBD includes accomplished and enthusiastic partners from academic, public health, and vector control institutions in Wisconsin, Minnesota, Illinois, Michigan, Indiana, and Iowa. Across the region, climate change impacts on vector-borne disease are expected as a result of 1) increased temperatures that can promote vector population growth and affect phenological activity patterns, 2) changes in annual snow cover depth and occurrence which impacts overwintering success of ticks and introduced mosquito species, and 3) extreme weather, with more frequent heavy rainfall and periods of drought affecting mosquito and arbovirus outbreaks, especially in large urban areas. The broad and long-term goal of the Center is to incentivize new and expanded interactions between experts in the region so that responses to endemic and epidemic vector borne disease are improved and accelerated. To achieve this goal, the project is focused on three major objectives: 1) Research to improve prevention of human exposure to vector bites. For this aim, we will evaluate current methods of control for mosquitoes and ticks and will develop new tools to reduce human risks of exposure. Data from research projects will feed back into outreach and education products for use with PH, tax-funded mosquito control districts, private pest control operators, and citizens as partners. 2) Increase the opportunities for training in public health entomology (PHE) for students. This objective will be achieved through graduate student training in PHE and through administration of a Certificate of PHE, with a curriculum including instruction on vector identification, surveillance and control methods, and pesticide application licensure. We will also offer a fellowship program which will provide paid internships in PHE and have established partnerships to enhance recruitment of students from underrepresented backgrounds. 3) Build a community of practice including public health and mosquito control experts at the county and district/municipal level, state public health experts, professional pest control companies, and academic scientists at research institutions. This goal will be achieved by evaluating region-specific public health educational efforts and interacting to establish best practices for VBD management. Successful completion of these objectives will dramatically expand the ability of public health authorities in the Midwest to detect and respond to threats as well as provide a strongly supported evidence-based practice for management of vector-borne disease.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Multiple artificial intelligence (AI) technologies are now commercially available for automated interpretation of screening mammography. These AI technologies hold promise for improving screening performance and outcomes for the 40 million U.S. women who undergo routine breast cancer screening each year. Federal regulatory approval of new AI technologies requires only a demonstration of non-inferior accuracy to existing computer-aided detection systems in small, retrospective reader studies, but their widespread clinical translation is contingent upon more robust population-based evaluation. Specifically, the impact of these AI technologies on actual patient outcomes needs to be assessed, including whether or not they lead to improved detection of clinically meaningful cancers in the general screening population. Robust external validation of AI algorithms for mammography screening has thus far been limited by use of single institution datasets not representative of the entire target population, use of AI algorithms that are not publicly available, comparison to radiologist performance in enriched case sets, limited follow-up time for cancer diagnoses influencing ground truth labels, and evaluation on 2D digital mammography rather than 3D digital breast tomosynthesis (DBT) exams. Our study objective is to conduct a comparative evaluation of four commercially available AI technologies for automated DBT screening interpretation that overcomes all of these limitations and then estimate the long-term benefits, harms, and costs of AI-driven DBT screening at the U.S. population level. Specifically, we will 1) use a centralized honest broker, model-to-data paradigm infrastructure to perform an independent, external validation of four leading commercial AI technologies for DBT screening using prospectively collected data obtained from seven diverse U.S. regional breast imaging registries; 2) stratify AI vs. radiologist performance on detailed woman-, exam-, radiologist-, and tumor-level characteristics to inform targeted algorithm training and refinement efforts to ensure generalizability of the AI algorithms; 3) explore targeted approaches for improving clinical workflow efficiency by using AI to safely triage exams highly likely to be negative; and 4) use a validated breast cancer microsimulation model to determine population-level, long-term health benefits, harms, and costs associated with four commercially available AI technologies for DBT screening both as a standalone screening tool and as a second independent reader to radiologist interpretation. Our proposed study will represent the most objective and rigorous evaluation of deep learning algorithms for DBT screening interpretation in the U.S. to date. Our results will provide urgently needed evidence to inform key stakeholders including women, physicians, payers, industry partners, and policymakers regarding how to maximize the value of AI technologies for DBT screening prior to their widespread clinical translation.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY/ABSTRACT Radiation-induced xerostomia (RIX) is a condition of subjective dry mouth caused by radiation therapy to the head and neck and manifested as hyposalivation and altered sialochemistry. RIX is the most common chronic side effect observed in HNC patients receiving radiation therapy and despite improvements in radiation delivery remains a critical issue for patients. Currently available treatments provide temporary palliation and in some cases (e.g. pilocarpine) can be accompanied by side effects that are as bad or worse than xerostomia. There is a critical need for a treatment that will safely and effectively alleviate RIX without compromising patient quality of life. A small phase I study suggested that MSC therapy could improve the perception of dry mouth and salivary gland function in patients with RIX. However, the mechanisms by which MSCs elicit this effect remain unknown and none of the successful studies investigated to use of cryopreserved MSCs. We aim to fill this important knowledge gap by identifying an optimal source of MSCs for treatment and understanding how MSCs improve salivary function to optimize treatment to maximize patient benefit. This proposal intends to fill this knowledge gap which will potentially lead to the development of novel and effective therapies for RIX. We hypothesize that MSCs provide a reparative effect to the salivary gland through paracrine signaling. We will investigate the short-term and long-term functions of MSCs following injection into the submandibular gland and identify whether there is an ideal potential tissue source for MSCs in terms of cryo-recovery and RIX treatment. We seek to address three questions critical to understand and treat RIX: 1) identify an ideal tissue source for MSC to treat RIX; 2) investigate the effects of MSC treatment on the salivary gland in acute and late-phase radiation damage; and 3) determine if MSC-based products elicit the same effects as MSCs alone. In Aim 1, we will evaluate the ability of MSCs derived from marrow, adipose, and submandibular gland tissue to recover after cryopreservation following IFN-ɣ pre-licensing and evaluate the effects of these MSCs on salivary tissue in vivo and in vitro. We will characterize the secretome of MSCs from different tissue origins alone and investigate bi-directional effects of salivary gland tissue on this secretome using a salivary organoid model. Finally, we will confirm in vivo, differences in reparative effects based on MSC source. In Aim 2, we will define the effects of IFN-ɣ pre-licensed, cryopreserved MSCs in RIX by studying both acute and long-term effects on saliva production and salivary gland architecture in-vivo. We will also investigate the effects of MSC- based products like MSC conditioned media and MSC-derived exosomes on salivary function in vivo and in vitro. Together this work will provide an improved understanding of how MSCs ameliorate radiation damage, support the development of novel therapeutics for the treatment of RIX, and provide an outstanding training opportunity for the PI.