University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract: Voice dysfunction impairs the quality of life of affected patients and treating these disorders is associated with substantial and far-ranging social, psychological, and economic costs. Vocal fold (VF) inflammatory lesions cause common voice disorders related to disrupted epithelial homeostasis accompanied by inflammatory infiltrates and changes in the lamina propria. In studies of the epithelium from elsewhere in the body, cell types that originate hyperplasic changes are epithelial stem cells. These cells have the capability to self-renew and give rise to the progeny of differentiated daughter cells which is regulated by the local microenvironment and cell-autonomously via Notch signaling. Inactivation of Notch1 in the presence of inflammatory cytokines can lead to epithelial hyperplasia, which can be modeled using in vitro organoids. The overall objective of this proposal is to provide a comprehensive characterization of VF epithelial stem cells, their requirements for self- renewal and differentiation under physiological conditions and in response to stress factors, namely injury and mechanical load, while also creating VF organoids to elucidate molecular mechanisms that underline aberrant epithelial remodeling as seen in benign inflammatory VF lesions. To achieve our goal we will genetically label epithelial stem cells targeting the Lrig1 gene that has been linked to stemness properties in majority of epithelia and our preliminary data show that Lrig1 is also expressed in human and murine VF epithelial cells. In Aim 1, we will perform transcriptome profiling of murine and human VF Lrig1 cells and measure Lrig1 cell responses to mechanical load during homeostasis. We will delineate the mechanistic role of murine Lrig1 cells in homeostasis, and genetically inactivate Notch1 in murine Lrig1 cells to determine its effect on proliferation and differentiation in vivo. In Aim 2, we will induce VF epithelial injury in a murine model, perform transcriptome profiling of murine Lrig1 cells and measure their responses to mechanical load during epithelial recovery. We will determine the functional role of murine Lrig1 cells and Notch1 signaling in re-epithelization. In Aim 3, we will determine differentiation potential of murine and human VF Lrig1 cells using in vivo subrenal graft assay and in vitro organoids. We will utilize VF organoids to model Notch-mediated epithelial hyperplasia using genetic, pharmacologic approaches, and inflammatory cytokines. We will create a reliable culture system that will improve our understanding of VF epithelial cell biology related to VF inflammatory lesions in the context of personalized medicine.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Epstein-Barr virus is an important cause of human Burkitt lymphomas (BLs), Hodgkin lymphomas (HLs) and diffuse large B cell lymphomas (DLBCLs), particularly in AIDS patients. Although the EBV protein EBNA2 (which mimics Notch signaling) is required for EBV-induced transformation of B cells in vitro, cells that express EBNA2 have the “type III” form of viral latency, which is highly immunogenic. Thus, most EBV-infected human lymphomas, including BLs, HLs and DLBCLs, do not express EBNA2, even in AIDS patients. There is currently no in vivo or in vitro model available to study how EBV infection causes lymphomas in the absence of EBNA2 expression. BLs, which are largely driven by MYC translocations, have stringent type I latency (expressing only a single EBV protein, EBNA1) and currently the major role of EBV in BLs is thought to be prevention of MYC- induced apoptosis by the virally-encoded microRNAs. EBV+ HLs, which have type II latency, express only three viral proteins (EBNA1, LMP1 and LMP2A) and are thought to be driven by the LMP1 (a CD40 mimic) and LMP2A (a BCR mimic) proteins, in conjunction with cellular mutations that activate JAK/STAT signaling and a supportive CD4+ T cell-rich microenvironment. Here we propose to use a novel in vitro cell culture model, in combination with a cord blood-humanized (CBH) mouse model developed by our lab, to examine whether an EBNA2-deleted EBV mutant recently constructed by our lab (ΔEBNA2 EBV) can induce BL-like, HL-like or DLBCL-like lymphomas in vitro or in vivo when specific oncogenic pathways known to be induced in each type of tumor are activated, or tumors suppressor genes (TSGs) associated with these tumors are inactivated. Our promising preliminary results already suggest that over-expressing the MYC oncogene in B cells infected with the EBNA2- deleted EBV allows cells to form BL-like tumors with type I latency in NSG mice. In Aim 1, we will determine if ΔEBNA2 EBV cooperates with MYC, BCL6 and/or cyclin D3 over-expression to induce BL-like and/or germinal center (GC) type DLBCL-like lymphomas in NSG mice. In Aim 2, we will determine if ΔEBNA2 EBV cooperates with JAK/STAT activation or inhibition of plasma cell differentiation to cause HL-like lymphomas in NSG mice. In Aim 3, we will identify novel cellular gene mutations/alterations that cooperate with ΔEBNA2 EBV to induce BL- and/or HL-like or DLBCL-like lymphomas in NSG mice and define the mechanism(s) for this synergy. We hypothesize that EBNA2-deleted EBV will cooperate with cellular alterations commonly found in EBV+ BLs and/or HLs to induce BL-like and/or HL-like lymphomas in vivo (although the cellular mutations required to induce BL-like versus HL-like tumors will be different), and that our approach will also identify novel cellular gene alterations that cooperate with stringent EBV latency to cause AIDS-related lymphomas in humans.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Chronic diseases are a significant problem in aging population causing immense economic burden to healthcare systems around the world. Alzheimer’s disease (AD) is the most common form of dementia, affecting as many as 5.8 million Americans who are aged 65 and older and is the sixth leading cause of death in the United States. As age is the greatest risk factor in developing AD, there is great interest in the possibility of targeting AD through interventions that slow or delay aging. Calorie restriction (CR) can slow or prevent AD in animal models, but reduced calorie diets are notoriously difficult to sustain. In this proposal, I will examine if dietary regimens and drugs that mimic aspects of a traditional CR diet, but which are easier to adhere to, can slow or prevent the development and progression of AD. Studies from the Lamming lab and others have shown that protein restriction (PR) improves many aspects of metabolic health in both humans and mice, and extends mouse lifespan, likely in part through reducing the activity of the amino acid sensitive kinase mechanistic Target Of Rapamycin complex 1 (mTORC1), which is a key regulator of autophagy. In preliminary studies, I have found that PR reduces brain mTORC1 activity and activates autophagy, and slows or prevents cognitive decline and the progression of AD pathology in the 3xTg mouse model of AD. Our lab has shown that restriction of the branched chain amino acids (BCAAs; leucine, isoleucine, valine) recapitulates the metabolic benefits of PR and extends lifespan. Defects in BCAA catabolism, or excess dietary BCAAs, may drive the pathogenesis of AD by increasing BCAA levels in the brain, activating mTORC1, and inhibiting autophagy. However, the role of individual BCAAs in these effects are unknown. I will determine the effects of restricting protein or leucine, the essential amino acid that most strong agonizes mTORC1, on metabolic health, cognition, AD pathology, and autophagy in the APP/PS1 mouse model of AD, which develops cognitive deficits later in life than the 3xTg mice. I will also test if reducing levels of BCAAs via pharmacological stimulation of BCAA catabolism reduces brain levels of BCAAs, inhibiting brain mTORC1 signaling and boost autophagy, recapitulating the beneficial effects of restrictive diets. Finally, utilizing distinct feeding regimens I will determine if prolonged daily fasting, which CR animals are subjected to in laboratory experiments, can recapitulate the beneficial effects of a CR diet on the development and progression of AD. Together, these proposed aims will address long-standing questions about how dietary interventions can affect the development and progression of AD. I will be completing this fellowship under the mentorship of my sponsor, Dr. Dudley Lamming, an expert in aging biology, metabolism, and mTOR signaling, and my co-sponsor, Dr. Luigi Puglielli, a world-leading expert in Alzheimer’s disease. Completing these aims and the accompanying individualized training plan will help me develop the skills necessary to become a successful tenure track faculty member focused on the biology of aging and Alzheimer’s disease.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT The last decade has seen a significant increase in the number of FDA approved treatments for men with metastatic castrate resistant prostate cancer (mCRPC). Even greater improvements in survival were observed for men with metastatic castration sensitive PC (mCSPC) treated with chemotherapy or androgen receptor signaling inhibitors compared to hormone therapy alone (Despite these advances in mCSPC, median OS for men with mCRPC remains less than two years and cross-resistance to therapies within the same class (e.g. Enzalutamide and Abiraterone) occurs in >90% of patients, limiting effective treatments in mCRPC to agents with OS improvements ranging from only 2-4 months. There is a critical need to identify new agents that can eliminate resistant disease5. Understanding the molecular associations driving resistance may identify new therapeutic sensitivities in these aggressive cancers to improve quality and quantity of life for men with mCRPC. Translational research studies focused on understanding the underlying mechanisms driving treatment resistance in CRPC have identified a wide range of genomic, epigenomic and transcriptional alterations. Approximately 15-20% of patients with mCRPC develop lineage plasticity, with small cell neuroendocrine CRPC (SCNPC) representing the most aggressive subtype. However, the field lacks consensus definitions for the diverse lineage plasticity phenotypes observed in mCRPC, and few therapeutic targets have been developed to date. In this study, we propose Trop-2 (Trophoblastic cell-surface antigen) as a high value target for therapy of mCRPC with an antibody-drug conjugate Sacituzumab Govitecan (SG). We hypothesize that ARSI-resistant phenotypes in mCRPC can be identified through integrated solid tumor and liquid biopsy analysis and targeted therapeutically with SG. To test this hypothesis, we propose to study Trop-2 regulation in pre-clinical and clinical specimens and test this agent in a prospective Phase II clinical trial for ARSI-resistant mCRPC. In Aim 1, we will conduct molecular-spatial analysis of Trop-2 expression in mCRPC PDX tissues as well as solid tumor and liquid biopsies from patients with CSPC, CRPC and SCNPC In Aim 2, we will evaluate solid tumor and liquid biopsies collected longitudinally from patients with mCRPC treated with SG in a prospective Phase II trial. Aim 2 will characterize chromatin enhancer profiles in the TACSTD2 gene encoding Trop-2 to identify factors regulating Trop-2 levels in prostate cancer. We will test if Trop-2 levels determine sensitivity to SG therapy, and identify SG-based combination regimens that enhance therapeutic efficacy in vitro and in vivo.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The University of Wisconsin (UW) Prostate Cancer SPORE is a highly collaborative research proposal that links basic scientists with prostate cancer clinicians to advance treatment strategies for prostate cancer patients. The broad objectives of this SPORE are to: 1) Increase multidisciplinary translational research and develop the next generation of prostate cancer researchers, 2) Develop common resources to promote advances, 3) Translate promising new approaches into patients, and 4) Improve overall survival and quality of life for patients with prostate cancer. A crosscutting theme that encompasses this SPORE is understanding tumor resistance in advanced prostate cancer and exploiting this knowledge to improve patient outcomes. The UW Prostate Cancer SPORE has three primary research projects: 1) Tumor Microenvironment Initiators of the Metastatic Cascade in High-Risk Prostate Cancer, 2) Androgen Deprivation as an Immune Modulating Therapy in Combination with Targeted Immunotherapy of Prostate Cancer, and 3) Extending Clinical Benefit by Selective Treatment of Resistant Lesions in mCRPC. The SPORE will support this research with three Cores (Administrative, Integrated Pathology Radiology, and Biostatistics and Bioinformatics). The Career Enhancement Program and Developmental Research Program will engage new and established investigators and further translational goals in a rich multidisciplinary environment. When completed, the research of the UW Prostate Cancer SPORE will advance our treatments and understanding of prostate cancer and undoubtedly beneficially impact patients with this disease.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Bacteriophages (phages) are the most abundant but least understood constituents of microbial communities. This is especially evident in the mammalian gastrointestinal tract, where a diverse community of up to 1012 phages are present per gram of stool. Though mounting evidence suggests the importance of phages in human microbiomes, methods of data generation and analysis routinely used in microbiome science neglect important aspects of phage biology. Furthermore, there is a lack of foundational knowledge and experimental tools for understanding phages and the roles they play in microbiomes. This lack of understanding is especially salient when the burgeoning antibiotic resistance crisis is considered: many important human pathogens are becoming increasingly resistant to our antibiotic arsenal. While a growing number of clinicians and scientists believe that “phage therapy” (the therapeutic application of phages to remove specific bacteria from host- associated microbiomes) will be important in our recovery from the antibiotic resistance crisis, phage therapy is inconsistently effective. This is largely due to an incomplete understanding of how phages impact their target bacteria, their off-target effects on other microbiome members, and their interactions with the mammalian host. Without such knowledge, important facets of phage-centric microbiome community dynamics will remain obscure and hinder robust and reproducible phage therapy applications. The goal of the proposed research program is to build a deep understanding of the roles that phages play in host-associated microbiomes and to eventually exploit this understanding to inform phage-based therapeutic strategies. We will work towards this goal using phage isolates, bacterial culture, bacterial genetics, gnotobiotic mice, and systems biology approaches. Using these tools, the proposed research program will build on my previous work with Bacteroides thetaiotaomicron-infecting phages and will focus on an isolate of the prominent crAss-like phage family, DAC15. We will determine how phenotypic heterogeneity in B. thetaiotaomicron influences resistance to DAC15, the specific interactions between DAC15 and B. thetaiotaomicron that lead to productive infection, and how DAC15 influences microbiome-host interactions. We will additionally build a collection of phages that infect other members of a model human gut microbiome to facilitate similar work with diverse bacteria and phages. Together, this work will be a much-needed foundation to understand the roles and identities of gut- resident phages, will build tools for sustained and powerful phage-centric study of the gut microbiome, and will inform the development of robust and reproducible phage therapy.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Rationale: Gene networks underpin all aspects of bacterial physiology. These networks mitigate antibiotic induced stress in the context of antibiotic resistance, and drive microbe-microbe and microbe-host interactions in the context of the human microbiome. Despite the central role of gene networks in maintaining viability and organizing stress responses, there have been few studies that systematically compare gene networks across bacterial species. Patterns in chemical-gene, gene-gene, and gene-promoter interactions will provide clues to gene functions, pathways, and regulons, broadening our understanding of how the genetic backgrounds of strains alter network connectivity. Objective: Here we propose a cross-species comparison of genetic and regulatory networks in three enteric species relevant to human health: Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae. Comparisons to the well-studied model, E. coli K-12, will drive gene function discovery in E. cloacae and K. pneumoniae, as well as provide a test bed for future cross species comparisons. To facilitate these analyses, we have developed CRISPR-based tools that are easily portable across species and can be used to investigate gene function and regulation at the genome scale. We seek to uncover fundamental mechanisms of homeostasis and stress responses by identifying conserved pathways. Our basic research approach could inform strategies that target weak points in gene networks of bacterial pathogens or could be applied to examine host-modified networks in the context of the human microbiome.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The prevalence of Nonalcoholic fatty liver disease (NAFLD) is increasing worldwide, affecting a quarter of the global population. NAFLD may progress to its more severe form, Nonalcoholic steatohepatitis (NASH), which will become the number one indication for liver transplant. While there are over 400 ongoing NAFLD-related clinical trials, there are no FDA-approved therapies. There is an immediate need for strategies to counteract NAFLD/NASH development and progression throughout the world. However, little is known about its pathophysiology. Western diet contributes to disease pathogenesis, mediated in part by the gut microbiome. Epidemiological studies indicate dietary cholesterol closely associates with the incidence of late-stage NAFLD. However, the influences of Western dietary components, such as cholesterol, on gut microbiota are largely unknown. There is a considerable gap in knowledge regarding the mechanistic relationships between discrete Western dietary components, gut microbiota, and the development of NAFLD/NASH. Preliminary studies show Western diets containing high levels of cholesterol induce gut microbial imbalances that precede and are a prerequisite for NAFLD/NASH in Specific pathogen-free (SPF) mice, yet germ-free (GF) mice that lack a gut microbiome are protected from disease. Bifidobacteria are key commensal organisms that are beneficial to the host and are commonly downregulated in metabolic disorders such as NAFLD/NASH. However, environmental cues that drive a loss of Bifidobacteria remain elusive. Preliminary studies show they are lost from the gut upon high-cholesterol feeding in a dose-dependent manner and their relative abundance is negatively correlated with liver damage. These data strongly suggest diet drives a loss of Bifidobacteria which compromises the host and contributes to NAFLD/NASH pathogenesis. It is critical to define the underlying mechanisms if microbiome-based therapeutic strategies against NAFLD/NASH are to be developed. The goal of this proposal is to define the role of dietary cholesterol in driving gut microbial imbalances in NAFLD/NASH pathogenesis. I hypothesize dietary cholesterol drives gut Bifidobacteria elimination which promotes a proinflammatory microbial milieu during the pathogenesis of NAFLD/NASH. To test this hypothesis, I will utilize a combination of in vitro, in vivo, and bioinformatics techniques to 1) Determine critical functional elements that impact Bifidobacteria’s capacity to sustain a niche in the presence of high dietary cholesterol alone vs. within a complex gut microbiota community in NAFLD/NASH development and 2) Elucidate the indirect effect of dietary cholesterol mediated through altered bile acid profile on loss of Bifidobacteria from a complex gut microbiota community in NAFLD/NASH development. By exposing me to central aspects of microbiome research, these studies provide the perfect vehicle for my training and will propel me toward achieving my goal of becoming a Principal Investigator studying interactions between diet, gut microbes, and metabolic disease at a R1 institution.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Age-related diseases, including cancer, are the major causes of morbidity and mortality in Western society. While cancer in the genetically heterogeneous human population primarily occurs in the aged, cancer research to-date has primarily utilized young, inbred animals. As the effect of aging and host factors on cancer development and progression has grown increasingly evident, the limitations of this approach have become clear. Understanding how aging impacts cancer development, progression, and the response to interventions will provide mechanistic insights into the prevention and treatment of cancer as individuals grow older, and eventually will permit the development of new pharmacological approaches to this age-associated disease that will enable healthy aging. Here, we build on recent work by our team and others demonstrating that restricting dietary protein, or restriction of specific dietary amino acids, can extend the lifespan and healthspan of mice. We will utilize methionine restriction (MR), a dietary intervention that extends longevity and improves metabolic health in mice, and which in mouse xenograft studies has been shown to slow the progression of certain cancers, including breast cancer, the most common cancer in older women. Limitations of MR research to date include the fact that the effect of MR on healthspan is limited in scope, that the effects of MR have been studied only in a few young mouse strains, and that the cancer studies done to-date have exclusively relied on young hosts. Understanding how MR affects the healthspan, longevity, and natural development of breast cancer during the aging of genetically heterogeneous mice will provide valuable new insights into the potential application of MR-based interventions for the health, longevity and treatment of cancer in the genetically heterogeneous human population. We will use cutting edge techniques to isolate and characterize cancer initiating cells (CICs), examine how changes in levels of methionine and its metabolites affect the epigenome. We will use two breast tumor models to examine how host age impacts CICs, tumor growth and/or metastasis and the response to MR. Finally, we will determine the role of specific molecular sensors of methionine metabolites in the epigenetic and anti-cancer effects of MR during aging. The proposed work will address long- standing questions regarding the molecular mechanisms by which dietary components regulate healthy aging and cancer.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT How do we perceive the three-dimensional (3D) movement of objects in the world when our eyes only sense two-dimensional (2D) projections like a movie on a screen? Accurate and precise perception of 3D object motion is essential to intercept objects (e.g., catch a ball) and evade others (e.g., dodge a passing bicyclist). The goal of this proposal is to elucidate the cortical networks that transform ambiguous 2D retinal signals into high-level 3D object-motion representations. To achieve this goal, we will utilize a synergistic combination of behavioral, electrophysiological, and causal manipulation approaches with macaque monkeys. In Aim 1, we will distinguish 2D retinal motion selectivity from 3D object-motion selectivity at the single neuron level and evaluate functional correlations with behavior. We will test the hypothesis that 3D object-motion representations are created within a cortical network consisting of the middle temporal area (MT), the fundus of the superior temporal sulcus (FST), and the lateral subdivision of the medial superior temporal sulcus (MSTl). The experiments will combine a 3D object-motion discrimination task with simultaneous high-density neuronal recordings from all three areas. Importantly, the stimulus set was rigorously vetted through previous perceptual and computational studies, and maximally discriminates 2D retinal vs. 3D object-motion representations. This work will be the first to assess functional correlations between neuronal activity and the behavioral discrimination of 3D object-motion. To evaluate the cortical network organization of MT, FST, and MSTl, we will compare the areas’ functional properties and measure the Granger causal influences between them using simultaneously recorded local field potentials. In Aim 2, we will apply a complementary approach to assess the causal contributions of each area to 3D motion perception. Specifically, we will use electrical microstimulation (EM) with weak currents to manipulate neuronal activity while the monkeys perform the 3D object-motion discrimination task. These experiments will be the first to use EM to causally probe the relationship between neuronal activity and 3D object-motion perception. Critically, the predicted relationship between neuronal response properties at the site of EM and the induced behavioral biases depends on whether the stimulated neurons are either: (i) selective for 2D retinal motion (with outputs that are used by downstream neurons to compute 3D object-motion, otherwise no effect of EM would be expected) or (ii) selective for 3D object-motion. We will test the predictions locally (i.e., at the level of individual neurons within each area) to assess area-specific functional heterogeneity and globally (i.e., between areas) to assess hierarchical differences across the network. The proposed experiments will together explicate differences in the functional properties of three interconnected cortical areas as well as their causal contributions to 3D motion perception. By elucidating the cortical networks that transform 2D retinal signals into ecologically relevant representations of 3D object-motion, insights from this work will facilitate future studies that explore how neuronal representations of dynamic, object-level information support interactions with the 3D world.

NIH Research Projects · FY 2024 · 2023-07

The decidua is a specially modified mucosa that harbors a unique composition of leukocytes. Despite the importance of maternal-fetoplacental immune interactions during pregnancy, the gestationally-driven population dynamics of decidual leukocytes has been difficult to ascertain, and the transition from the menstrual cycle to early pregnancy is not readily studied in human pregnancy. Disturbance of the mechanisms that regulate population maintenance and trafficking of decidual leukocytes at the maternal-fetal interface is thought to underlie morbidity and mortality in pregnancy (i.e., preeclampsia, preterm labor, fetal growth restriction) and presents a powerful diagnostic and therapeutic target. Thus, this R21 Exploratory/Developmental Grant application aims to establish a novel pregnant macaque model defining trafficking of peripheral blood cells to the decidua using the novel technique of serial intravascular staining (SIVS) with the following Specific Aims. Specific Aim 1: To determine the trafficking and population dynamics of decidual lymphocytes, including decidual NK cells, in pregnant rhesus monkeys. We will test the hypothesis that trafficking from the peripheral blood and proliferation and apoptosis of tissue-resident decidual lymphocytes drive population dynamics across pregnancy. Further, the distribution of these lymphocytes with respect to the decidual vasculature will provide insight into their function and mechanisms of trafficking. Specific Aim 2. To determine the trafficking of peripheral blood lymphocytes, including NK cells, to the nonpregnant endometrium. We will test the hypothesis that the trafficking of lymphocytes to the developing maternal-fetal interface is initiated in the late luteal phase of the menstrual cycle, independent of the presence of an embryo or developing placenta. Our proposed studies will answer the following fundamental questions: Which lymphocytes actively traffic between systemic vasculature and decidual residency during pregnancy? What is the balance of cell proliferation and cell death of decidua-resident lymphocytes across gestation? What is the distribution of trafficking and resident lymphocytes relative to the vasculature within the decidua? And, is trafficking from the blood to the uterus initiated in the luteal phase in preparation for the establishment of pregnancy? Determining the origin and dynamics of decidual lymphocytes is necessary to advance the hypothesis of their pivotal role in hemochorial placentation into clinically actionable intelligence. The R21 Exploratory/Developmental Grant mechanism supports research projects in their early and conceptual stages. The application of the SIVS paradigm to the pregnant nonhuman primate model could have a major impact on our understanding of the reproductive immunology of the maternal-fetal interface. Furthermore, these methodologies for assessing trafficking of immune cells in vivo in the nonhuman primate model will be powerful tools to apply to other experimental settings, including infectious disease in pregnancy and hematopoietic stem cell and solid organ transplantation.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Heterozygous, pathogenic variants in POU4F1 were recently reported by our group to cause a novel ataxia syndrome (Webb et al., 2021). This neurological disorder is characterized by childhood onset ataxia, intention tremor, and hypotonia, and affected individuals have global developmental delay with mildly impaired intellectual development and speech delay or learning difficulties. POU4F1, also known as BRN3A, encodes a class IV POU-domain containing transcription factor expressed in the developing and adult brain. Little is known about the role POU4F1 plays in neurogenesis, neuronal differentiation, and neuronal survival in human neurons. Therefore, we propose to use iPSC to generate cellular models of the human brain to interrogate the consequences of POU4F1 haploinsufficiency. Our specific aims in this proposal are to 1) determine transcriptional signatures, epigenetic status, and functional abnormalities resulting from POU4F1 haploinsufficiency in iPSC – derived neurons; 2) assess the impact of POU4F1 haploinsufficiency in a iPSC model of the developing brain using cortical organoids; and 3) create a patient registry for POU4F1-related ataxia and better define the clinical features of this newly identified disorder. Taken together, these studies will uncover the pathophysiology, disease mechanism, and clinical spectrum of disease of POU4F1-related ataxia and address fundamental questions of human neurodevelopmental biology.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Cellular responses to available macronutrients and extra-cellular signals rely on the unique epigenetic state of the cell, defined by a layer of biochemical information above the genome that dictates specific gene expression. The epigenome consists of DNA sequence-dependent proteins, non-coding RNAs, DNA methylation and histone post-translation modifications (PTMs) such as lysine acetylation and methylation. The latter two mechanisms are catalyzed by enzymes that must ‘interpret’ incoming signals, ‘read’ the existing epigenetic landscape and ‘respond’ appropriately. Enzymes that modify histones and non-histone proteins such as methyltransferases, demethylases, acetyltransferases and deacetylases use central metabolites (S- adenosyl methionine, SAM; α-ketoglutarate, αKG; acetyl-CoA and nicotinamide adenine dinucleotide, NAD+, respectively) as co-substrates. New evidence suggests that fluctuation in such epi-metabolites caused by diet, environment, microbiota and genetics can drive PTM dynamics, however the relevant mechanisms remain unclear in most cases. Are changes in epi-metabolites sensed by signaling pathways or by substrate-level driven catalysis or both? Also, does local production of epi-metabolites enable/accelerate gene expression mechanisms on chromatin? Non-histone protein acetylation is a major PTM that can regulate many aspects of cellular function and occurs in all cellular compartments. But despite broad knowledge of what gets modified, the most pressing challenge is to understand the how, the why and the when, which constitutes an overarching theme of this proposal. A major portion of the research to understand reversible protein acetylation as a regulatory PTM will involve knowledge of how pathway-specific acetyl-CoA (and other acyl-CoAs) production leads to dynamic acetylation after extra-cellular stimulation. Also, this work will focus on the detailed molecular mechanisms by which nuclear NAD+-dependent deacetylases SIRT6/7 (Sirtuins 6 & 7) are regulated and how these enzymes perform such exquisite deacetylation of nucleosomes. A sub-theme of this proposal that connects these two projects is to understand the fundamental principles that govern PTM enzymes acting on chromatin/nucleosomes. This proposal is uniquely poised to make major advances to these salient questions. To accomplish these goals, the projects synergistically employ in vitro biochemistry/biophysics, complementary genetics and pharmacology, and cell- and animal-based models. Results from these investigations will provide i.) insight into the etiology of diseases resulting from the link between metabolism and the epigenome, ii.) foundations for drug development against the enzymes described here, and iii.) a fundamental understanding of how the cell ‘interprets’ incoming signals in the context of existing epigenetic information and ‘responds’ appropriately.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT Sudden cardiac death (SCD) claims >300,000 lives yearly in the US and despite aggressive attempts at phenotype-genotype correlations, ~50% of patients with primary electrical SCD do not meet diagnostic criteria for any SCD syndrome and are labeled Idiopathic Ventricular Fibrillation (IVF). This “catch-all” diagnosis of exclusion encompasses a cohort of individuals that are undefined phenotypically with arrhythmia that is mechanistically unexplored. Moreover, without defined genotype-phenotype characterization, clinical practice guidelines for IVF suggest homogenous treatment for a heterogenous disorder. Recognizing that genetic linkage studies have failed for IVF, we propose a paradigm shift to address these challenging gaps in knowledge. We propose to integrate computational modeling of comprehensive electrophysiologic and multiomic outputs to identify the mechanistic underpinnings of an emerging IVF subphenotype related to Purkinje-triggered VF (PVC- IVF). In collaboration with the Bordeaux group Deep Phenotype efforts, who originally described these emerging subphenotypes of IVF, PVC-IVF patients have been recruited to create induced pluripotent stem cells (iPSCs) from UW and Bordeaux. Our iPSC experimental system has distinct advantages including integration of PVC- IVF iPS-cardiomyocytes (iPS-CMs) with a mixed (ventricular myocyte and Purkinje) cell population with computational myocyte models reflecting region-specific phenotypes. Our group’s design for iPSCs experiments combine innovation of platforms that promote electrical and functional maturity, including incorporation of iPS- cardiac fibroblasts (iPS-CFs), and analysis by computational modeling of iPS-CMs and in silico adult human myocytes and tissue. In our pilot data we demonstrate the effectiveness of using iPSCs to differentiate IVF with experimental evidence and integrate this data into computational modeling approaches to gain mechanistic insight into cellular arrhythmic perturbations. Our overarching goal is to examine the mechanistic underpinning of PVC-IVF and identify specific complementary and synergistic therapeutic targets. In Aim 1 we will integrate experimental functional readouts from our advanced model system designed to recapitulate native cardiac milieu with a combination of micro and nanoscale cues with “bottom-up” computational modeling to unravel the specific cellular perturbations that cause the observed functional PVC-IVF readout. The focus of Aim 2 is to incorporate a broad, unbiased data-driven dataset from multiomic characterization of PVC-IVF patient-specific iPS-CMs into a computational systems pharmacology framework to define synergistic arrhythmogenic pathways and antiarrhythmic polytherapy, which we predict to be safer and more effective than monotherapy approaches. Finally, computational cross-cell translators will predict responses in the adult heart. With completion of our aims, we will define the cellular arrhythmic signature; unravel the down-stream transcriptome, protein expression changes and post-translational modifications; and integrate experimental readouts with computational modeling to create actionable data and find druggable targets for PVC-IVF arrhythmia prevention.

NIH Research Projects · FY 2026 · 2023-06

Frontline treatments for major depressive disorder (MDD), including psycho- and pharmacotherapy, have limited effectiveness, with usual care treatment success at just 29% after 1 year. Remission rates for depression would be enhanced if treatments could be optimized and prescribed to those most likely to benefit. There is a critical need to develop and test novel, efficacious treatments for MDD and simultaneously work to optimize their benefits. Resistance exercise training (RET) is a promising but understudied treatment approach. Our recent meta-analysis found a large antidepressant effect of RET in the few very small trials with clinically depressed samples (d=0.90), highlighting the potential of RET for treating MDD. These trials, while underpowered to determine clinically meaningful effects, showed positive results and provide the foundation for larger mechanistically-informed trials to confirm their promising early effects. Importantly, cerebral blood flow is lower in adults with MDD, linked with a poor treatment response, and RET can improve cerebral blood flow in adults. As such, RET may treat MDD via improving cerebral blood flow. However, the mechanistic pathway linking RET’s antidepressant effects to improved cerebral blood flow in MDD is as-of-yet untested. Further, with advances in machine learning, the identification of modifiable and stable predictors of clinical and mechanistic change as well as adherence can inform future precision medicine initiatives for treating MDD. Thus, a trial to confirm the efficacy of RET for MDD, understand its potential cerebrovascular mechanisms, and uncover the modifiable predictors of its effects is urgently needed. Toward this end, we propose a confirmatory efficacy 1:1 randomized controlled trial (n=200) of 16 weeks of progressive RET or low dose RET (SHAM) in adults with DSM-5 diagnosed MDD. Aim 1 will confirm the efficacy of RET vs SHAM on depressive symptoms at 16 weeks, and evaluate both potentially quicker and enduring effects of RET at 8, 26 and 52 weeks. Aim 2 will determine the effect of RET vs. SHAM on the mechanistic target of cerebral blood velocity and pulsatility and their potential mediation of antidepressant efficacy. Aim 3 will use supervised machine learning tools to predict depression changes, cerebrovascular changes, and participant adherence. Upon completion, this study will build towards our long-term goal of identifying and translating mechanistically-driven behavioral treatments to reduce the global burden of mental illness by determining the extent to which a promising, accessible, translatable RET approach can treat MDD by improving cerebrovascular function. Simultaneously, this project will inform future precision medicine approaches that will target modifiable predictors of treatment response and adherence to behavioral interventions to optimize MDD treatments and individually prescribe them to those most likely to benefit. If RET effectively treats MDD, this trial would lay the foundation to apply RET as a standalone treatment for MDD, and potentially as a standalone or augmentation treatment to reduce the widespread burden of mood disorders and serious mental illnesses.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY The overarching goal of this proposal is to understand, at a mechanistic level, how visual working memory is controlled. It will achieve this by pursuing two Specific Aims: Specific Aim I: To elucidate the mechanisms underlying the control of priority in working memory Specific Aim II: To elucidate the mechanisms underlying the active removal of information from working memory Working memory is understood to be a necessary elemental contributor to many aspects of high-level cognition – including cognitive control, problem solving, and planning – and its impairment is characteristic of many psychopathologies and psychiatric syndromes – including attention deficit hyperactivity disorder (ADHD), major depressive disorder (MDD), and schizophrenia. The research pursuing Specific Aim I entails modeling working memory behavior with recurrent neural networks, to refine algorithm-level models of the operations that contribute to the control of priority working memory, then testing these models in human brains with functional magnetic resonance imaging (fMRI), electroencephalography (EEG), and transcranial magnetic stimulation (TMS). The research pursuing Specific Aim II entails the study of active-removal from working memory with model-based analysis (using a reinforcement learning-based model of cognitive control) and computational simulations of fMRI data, and with repetitive (r)TMS targeting frontal circuits responsible for active-removal versus for the control of priority.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract Persistent and symptomatic anxiety during childhood is pathological and is a risk factor for the later development of stress-related psychopathology. Anxious young girls are particularly at risk, as during the transition to adolescence the prevalence of anxiety disorders (ADs) and depression markedly increases in females compared to males. Our work in children demonstrates that persistent and symptomatic anxiety is dimensionally related to altered function of neural circuits identified to be associated with responses to threat. Additionally, anxiety symptoms and levels of distress are highly overlapping between children that do and do not meet DSM-5 criteria for ADs. These findings, along with the risk conferred by early-life anxiety, provide a rationale for studying the broad range of pathological anxiety in preadolescent girls. In addition to daytime worries and fears, sleep-related symptoms (e.g. pre-sleep arousal, poor sleep quality) are common in anxiety, occurring in up to 90% of youth with ADs. It is critical to understand how sleep physiology relates to the pathophysiology of childhood anxiety because sleep is a homeostatic regulator that is involved in learning and memory consolidation, and also influences emotion regulation. Here, we will use a translational approach leveraging our nonhuman primate (NHP) model of pathological anxiety to conduct parallel neuroimaging and EEG sleep studies in preadolescent girls and preadolescent female rhesus monkeys with pathological anxiety. Using multimodal imaging, hdEEG sleep recordings, and home sleep EEG data, studies in preadolescent girls with pathological anxiety will explore hypotheses implicating the basolateral amygdala (BLA) and anterior insula (AI) in mediating altered anxiety- related daytime neural circuit function as well as alterations in REM and regional slow wave sleep. Studies using similar methods will be performed in NHPs that will be aimed at causal mechanisms. By chemogenetically activating BLA or AI neurons prior to sleep, the NHP studies will test the roles of the BLA and AI in mediating the linkage between alterations in sleep and daytime neural circuit function that are associated with pathological anxiety. Understanding daytime neural alterations associated with pathological anxiety in relation to disrupted sleep physiology is highly relevant for elucidating mechanisms underlying childhood pathological anxiety and in conceptualizing new treatment approaches.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Biliary tract disease and inflammation, including pediatric biliary atresia and primary sclerosing cholangitis, are leading causes of liver failure. Chronic biliary inflammation, which can be associated with the presence of gallstones, is a risk factor for development of cholangiocarcinoma, a rare but deadly malignancy. Acute inflammation of the gallbladder, or cholecystitis, accounts for 20% of all biliary tract disease hospitalizations, usually requires surgical intervention and has significant mortality if left untreated. Together, biliary tract disease, including common gallstones, represents an enormous human health burden. Biliary tract inflammation plays clear roles in progression of various common and rare biliary diseases, however our understanding of inflammation and immune processes within the biliary tract are exceedingly limited. The resident immune cell populations in the extrahepatic biliary tree at homeostasis have never been characterized, and whether host or environmental factors can influence the development of the biliary niche has never been explored. It is unknown if resident biliary immune cells could impact development and progression of biliary disease. During my postdoctoral training, I developed a reproducible tissue digest method that yields highly viable immune, epithelial, and stromal cell populations. Single cell RNA sequencing revealed that there is a diversity of resident immune cells present at homeostasis in the mouse gallbladder/extrahepatic bile ducts, including innate lymphoid cells, adaptive lymphocytes, neutrophils, macrophages, and dendritic cells. In the course of studying biliary tuft cells—rare, chemosensory epithelial cells with known immunomodulating properties—I found that the biliary immune niche is altered in the absence of tuft cells, and is also sensitive to the microbiome-status of the host. My data suggest that the microbiome and biliary epithelial cells (including tuft cells) regulate the establishment of the biliary immune niche. In this proposal, I will test the role of the microbiome and microbial metabolites in setting biliary immune “tone,” and will define a role for tuft cells in regulating the biliary immune cell make-up. I will test whether alterations in the biliary immune niche at homeostasis, specifically the presence or absence of neutrophils, can impact the progression of cholesterol gallstone disease. To accomplish these aims, I have formed collaborations with experts in liver/biliary biology and microbiome manipulations, and have established a career development plan that will facilitate increasing computational independence. These studies will reveal new links between host factors and biliary inflammation, with the potential for therapeutic targeting of biliary epithelial cells and the microbiome to impact biliary health and disease.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY – Pathological anxiety commonly emerges during childhood and is a prominent risk factor for the later development of anxiety and depression. To gain insights into mechanisms underlying the risk to develop stress-related psychopathology, we developed a non-human primate (NHP) model, termed anxious temperament (AT). This model allows for mechanism-based studies focused on the well-developed prefrontal cortex (PFC) shared by NHPs and humans. In this regard, we demonstrated involvement of PFC regions such as the dorsolateral PFC (dlPFC) in pathological anxiety, along with the amygdala and other subcortical AT-related regions. Neuroimaging research points to hypoactivation of the dlPFC in anxiety and depression. The dlPFC is involved in emotion regulation, working memory, and cognitive control, and modulates activity of limbic regions, such as the basolateral amygdala (BLA). Importantly, the dlPFC serves as a treatment target for neuromodulation strategies such as repetitive transcranial magnetic stimulation (rTMS) and is thought to be involved in mediating the effects of various cognitive therapies. Because of the evolutionary relatedness between NHPs and humans, especially manifested in PFC development, NHPs are ideally suited for investigations of the role of the PFC in psychopathology. As a translational bridge, our laboratory employs methods that provide an in-depth mechanistic understanding of brain alterations associated with extreme anxiety, including behavioral phenotyping, functional and structural neuroimaging, RNA sequencing and viral vector-mediated gene delivery. The focus of this proposal is to characterize the molecular substrates of the dlPFC in relation to AT, to understand how its top-down regulatory influences impact the BLA, a mediator of AT, and to explore the dlPFC as a treatment target. Our laboratory is uniquely suited for this endeavor as we use an integrative strategy in NHPs with behavioral phenotyping, multimodal imaging, chemogenetics, electron microscopy and single nuclear RNA sequencing (snRNA-Seq).

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is a debilitating neurological disorder, and soon will reach epidemic proportions in the context of an aging population. There is a clear need to identify persons at risk for AD pathology, so that preventative strategies can be developed and implemented. Multiple emerging lines of research demonstrate that sleep disturbance, and particularly excessive daytime sleepiness and obstructive sleep apnea (OSA), both increase the risk for AD pathology and dementia. Excessive daytime sleepiness is an important clinical phenotype of OSA, with individuals with impaired alertness – a function (and salient manifestation) of excessive sleepiness – experiencing more severe sequelae. Preliminary studies from our investigative team demonstrate that diminished daytime alertness measured by the psychomotor vigilance task (PVT) is specifically related to AD pathology and cognition, and that OSA moderates relationships between impaired alertness, AD pathology, and neurocognitive function. This project will advance this vital research area by leveraging emerging Alzheimer's disease biomarkers that can be detected in blood with the wealth of unique sleep data available in the Wisconsin Sleep Cohort (WSC) Study. The WSC has followed participants since the late 1980s, and is the only longitudinal cohort with stored biospecimens, sleep, PVT, and neurocognitive data spanning decades to elucidate the relationships of daytime alertness and OSA with AD pathology and cognitive decline. This investigation will prospectively collect additional blood specimens, PVT, and neurocognitive data in a targeted sample of 450 WSC participants who are now older aged, to address three Specific Aims with testable hypotheses supported by preliminary data. First, it will determine if impaired neurobehavioral alertness is associated with higher levels of pathological phosphorylated tau. Second, it will determine whether impaired neurobehavioral alertness is associated with more severe markers of neurodegeneration. Third, it will determine if impaired neurobehavioral alertness is associated with longitudinal cognitive trajectory. For all Aims, it is hypothesized that OSA moderates relationships between daytime alertness and AD pathology and cognitive decline. Detailed sleep and health history data available in the WSC importantly allows for many key covariates to be included in statistical models used for hypothesis testing. Addressing the Specific Aims of this application will have a sizeable impact on AD and sleep research, by linking a readily obtained objective measure of neurobehavioral alertness to longitudinal risk of AD pathology and cognitive decline. In so doing, this project will ultimately lead to improved preventative and therapeutic strategies that target sleep and alertness as modifiable risk factors for Alzheimer's disease.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT This career development proposal will provide Blair Golden, MD MS, a physician-scientist at The University of Wisconsin-Madison School of Medicine and Public Health, with the training required for success as an independent investigator researching interventions to improve the delivery of patient- and family-centered care for hospitalized adults with Alzheimer's Disease and Alzheimer's Disease Related Dementias (AD/ADRD). Nearly half of all hospitalized patients with AD/ADRD develop delirium superimposed on dementia (DSD), which is associated with significant morbidity and mortality. DSD also leads to adverse impacts for family caregivers, compounding pre-existing caregiver stresses and negative impacts on wellbeing. Despite its substantial burden, DSD remains under-detected and poorly managed in hospitalized patients due in part to under-developed approaches for assessing pre-existing impairment and other individual characteristics that are critical to delivering tailored prevention strategies. Evidence-based programs for delirium have emphasized the role of family engagement in recognition and management, but approaches to proactively engaging family members and patients with AD/ADRD in DSD detection and prevention remain underdeveloped. The overarching goal of this K23 proposal is to establish patient and family caregiver-centered communication and engagement practices capable of proactively addressing information needs and soliciting participation in DSD identification and prevention that are feasible and scalable. Understanding family and patient communication and information needs regarding DSD could not only improve the delivery of patient and family-centered care, but also potentially reduce family and patient suffering and improve DSD identification and prevention. As a junior faculty member at an institution with extensive infrastructure to support early-stage investigators, Dr. Golden is in an optimal environment to complete the proposed research project and pursue advanced training. Her career development plan includes formal coursework, intensive mentorship, and experiential training in 1) mixed methods, survey, and intervention mapping approaches 2) delirium assessment and prevention 3) research engagement and clinical care of patients with AD/ADRD and 4) pragmatic clinical trial design. To ensure success, she has identified expert mentors in these disciplines with outstanding track records in training independent investigators and secured protected time for this work. This award addresses a significant clinical dilemma and serious gap in AD/ADRD and delirium research while affording the education and mentored research experience critical to prepare Dr. Golden to lead an independent research program.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT Cone beam Computed Tomography (CBCT) with a digital flat panel detector (FPD) is the primary imaging workhorse for image-guided interventions. By rotating the tube-detector assembly around the patient, 3D cone beam CT (CBCT) data can be acquired to supplement 2D x-ray imaging and improve disease diagnosis, treatment planning, and treatment execution in medical procedures. Despite its volumetric coverage and high spatial resolution, FPD-based CBCT does not provide the necessary soft-tissue contrast resolution for intraoperative hemorrhage monitoring, gray-white matter differentiation, or the detection of other low-contrast lesions in a variety of imaging tasks. In addition, quantitative imaging capabilities that are much desired by physicians are lacking in the current CBCT technology. The overarching objective of this project is to develop an integrated PCD-FPD CBCT imaging platform by adding an x-ray photon counting detector (PCD) to the current CBCT system, enabling users to freelyswitch between the new PCD-CBCT imaging mode and the existing FPD-based imaging without interference between the two components. This new imaging platform offers the same soft-tissue contrast resolution and z-coverage as those of MDCT, but without requiring additional room space to house both MDCT and CBCT in the same suite. Additionally, the proposed system offers quantitative spectral CT imaging functionality at reduced radiation and contrast dose for image-guided interventions. To develop the integrated PCD-FPD CBCT imaging system and to explore its added value to medical diagnosis, three specific aims are planned. Aim #1 is designed to construct and calibrate a prototype PCD-CBCT system; Aim #2 is dedicated to the development of the novel correction algorithms needed to achieve artifact-free PCD-CBCT images; Aim #3 is dedicated to clinical feasibility studies using in vivo canine subjects and human subjects. Upon the completion of these aims, a prototype for the next-generation CBCT imaging system will have been constructed, calibrated, and evaluated; the system will be capable of consistently producing nearly artifact-free PCD-CBCT images with spectral imaging functionality and MDCT-like low-contrast detectability; the benefits and potential clinical applications of the integrated PCD-FPD CBCT imaging system will have been demonstrated.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Infectious diseases are a growing threat to public health owing to increasing antimicrobial resistance (AMR) and stagnation in new antibiotic development. Left unchecked, the annual number of deaths attributable to AMR is estimated to reach 10 million by 2050, exceeding deaths due to cancers and diabetes. Thus, there is an urgent need to develop innovative approaches to tackle this serious global crisis. We aim to develop a new class of dual-stimuli responsive polysaccharide-coated nanoparticles (NP) capable of encapsulating a wide range of FDA-approved antibiotics to effectively treat multidrug-resistant (MDR) bacterial infections. The polysaccharide NP shell ensures good stability and long blood circulation time, thus leading to high NP accumulation in the infected tissues via the enhanced permeation and retention effect. Furthermore, polysaccharides enable the NP to physically bind the pathogens due to multivalent affinity for bacterial lectins. The uniquely engineered NP is activated by high levels of ROS and/or low pH in the inflammatory microenvironment to release both cationic antimicrobial polymers and antibiotics that show a strong synergy to combat MDR pathogens. The cationic polymers can induce pores on the bacterial cell membrane, and significantly diminish the intrinsic resistance of the pathogens by enhancing the transport of antibiotics into the bacteria and allowing them to bypass the efflux pump. The cationic polymers released in the infected tissues can also agglomerate the pathogens and shape a microenvironment entrapping a high level of antimicrobial materials, thus leading to high antimicrobial efficacy. Moreover, the NP can penetrate through bacterial biofilms, and enhance the uptake of antibiotics by macro- phages, thereby effectively eliminating notoriously challenging biofilm and intracellular infections, respectively. Finally, the cationic polymer contains GSH-cleavable bonds in its main chain, which can be readily degraded in the cytosol of mammalian cells, thereby sidestepping the problem of dose-limiting toxicity with other cationic polymers. Following on our successful pilot studies, we will systematically optimize and characterize NPs tailored to treat four different MDR pathogens. In Aim 1, we will determine the optimal polysaccharide NP shell, antibiotics, and NP formulation for each of the four MDR pathogens. In Aim 2, we will study the candidate NPs’ antimicrobial and antibiofilm efficacy, drug resistance development profile, and biocompatibility to gain a fundamental understanding of the design rules for efficacious and safe antimicrobial NP against pathogens of interest. In Aim 3, we will determine the maximum tolerated dose, systemic toxicity, immunological consequences, in vivo biodistribution, pharmacokinetics, and antimicrobial efficacy of the selected NPs in healthy mice and three clinically relevant animal infection models. Altogether, this study will lead to a new class of antimicrobial NPs based on disease-specific stimuli, a unique dual-stimuli responsive and biocompatible cationic polymer we engineered, polysaccharides targeting MDR pathogens, and FDA-approved antibiotics. If successful, it will offer a general, yet effective and safe solution to effectively eliminate the most prevalent MDR pathogens.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY The Wisconsin Registry for Alzheimer’s Prevention (WRAP) is a longitudinal study that follows a risk-enriched cohort from late-midlife into old age and focuses on (1) Defining the preclinical window at the level of the individual including the onset of Alzheimer’s disease (AD) proteinopathy and cognitive decline prior to overt clinical symptoms; (2) gaining a comprehensive picture of the effects of nonmodifiable genetics and modifiable health and lifestyle factors on cognitive and AD biomarker onsets and trajectories; (3) characterizing the presence and impact of other diseases associated with cognitive decline—chiefly vascular disease. WRAP consists of over 1,729 (1386 active) adults who enrolled in midlife (baseline mean age 54 yrs), are followed biannually for an average of 12 years of follow-up so far, and on whom we conduct cognitive, lifestyle, lab, medical and biomarker assessments of AD and related disorders (ADRD). In the prior cycle we: (i) Developed methods for identifying subtle cognitive decline utilizing each participant’s own baseline performance, thereby improving sensitivity to decline while reducing diagnostic bias; (ii) Derived temporal information from amyloid positron emission tomography (PET) and plasma assays showing that amyloid onset age can be estimated and precedes tauopathy, (iii) found that when amyloid and tau proteinopathies are present, cognitive decline accelerates; (iv) showed that lifestyle and health factors affect cognitive decline and likely impact the length of the preclinical window; (v) showed that AD pathology/risk and aspects of vascular changes/risk independently and jointly impact brain health and cognitive decline; (vi) included WRAP data in several multi-cohort collaborations that have advanced the field. With these gains and new supportive preliminary data, WRAP is uniquely positioned to address the following major aims and knowledge gaps in the next cycle: Aim 1 will derive person-level estimates of the preclinical window defined as the interval between onset of AD proteinopathy and the onset of cognitive decline. We will perform 3000 main WRAP study visits from which we will characterize cognitive decline. We will assay 4,400 existing and 2,640 anticipated plasma samples for AD-related biomarkers. Subsets undergo AD (PET and/or CSF) and vascular (MRI and ultrasound) biomarkers. PET—plasma concordances will be established, and analyses using plasma-derived ADRD biomarkers will be conducted on the entire cohort. Aim 2 examines relationships between key genetic and health/lifestyle predictors to cognitive decline in the context of AD biomarkers. Aim 3 examines the inter-relationship between cerebrovascular health spectrum and its associations with cognitive decline relative to AD proteinopathy. Overall, the questions that WRAP is addressing with its longitudinal assessments and advanced temporal modeling are innovative and vital to the field regarding defining preclinical AD with greater precision at the level of the individual, and determining the factors that modify this window.

NIH Research Projects · FY 2025 · 2023-06

Bacterial virulence is closely associated with nutrient acquisition, which is essential for growth and proliferation of pathogens. Metal ions constitute essential nutrients, and the regulation of bacterial metal ion homeostasis within the host environment plays a pivotal role; however, unbound essential metal ions exhibit low bioavailability. For instance, the low solubility of Fe(OH)3 (Ksp = 6.3 x 10-38) at pH 7.4 would result in an insufficient quantity of iron for bacteria to grow, thus bacteria rely on targeting the hosts’ labile iron reserves through synthesis of endogenous, hydrophilic metallophores that are internalized using ATP-dependent bacterial transmembrane shuttles. These metallophores also retain affinity for non-essential xenometal ions with identical charge, comparable ionic radius and chemical hardness to the essential metal ion. For instance, trivalent metal ions with similar ionic radius to high spin Fe3+ (0.78 Å), such as Ga3+ (0.76 Å), Sc3+ (0.87 Å) and In3+ (0.93 Å) are transported to the bacterial peri- and cytoplasm when coordinated by bacterial iron-metallophores such as enterobactin or desferioxamine. These xenometals cannot be utilized for desired biological functions; recent strategies to utilize bacterial metal homeostasis pathways to deliver therapeutics has resulted in renewed interest in xenometals as alternative antibiotics. In bacteria, iron’s cytoplasmic fate and influence on gene and protein regulation is well-understood; however, xenometal homeostasis and utilization, especially in light of differential pH-dependent speciation behavior, remains rudimentary. To this end, we seek to investigate the following questions: (1) Are M3+-metallophore complexes efficiently recognized and transported across bacterial membranes? Size, hardness and Lewis acidity of metal ions influence their coordination complex structure. Substantial divergence from the parent Fe3+ complex results in diminished transport efficiency. We will study xenometal complex speciation under physiological conditions and employ a photoreactive tagging strategy to identify transmembrane shuttle protein interaction. (2) (How) Does M3+ release from metallophores proceed in absence of accessible redox events? Fe3+ is released by reduction to Fe2+ and enzymatic degradation of the metallophore induced by Fe2+-dependent proteins. The xenometals of interest, Ga3+, Sc3+ and In3+, do not have accessible redox events under physiological conditions. We will employ a radiochemical labeling strategy to track their metallophore-mediated uptake and identify metabolites. (3) What is the fate of M3+ xenometals in the cytoplasm and their influence on protein expression? The fate of non-redox active xenometals, once they reach the bacterial cytoplasm, including their effect on the bacterial protein expression is not well understood but hold the key to their growth inhibitory activity. We will combine radiochemical tagging strategies with mass spectrometry isolate and identify xenometal-target proteins. We will assess and quantitate the change in bacterial metabolites following exposure to different xenometal- metallophore complexes, which will inform on altered bacterial metabolism.