University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Worldwide, over a billion people each year experience high morbidity and/or mortality from the effects of human fungal pathogens. People with AIDS, chemotherapy patients, and transplant recipients are at highest risk of acquiring life-threatening infections, but many fungi also cause disease in apparently healthy individuals. Among these is the spore-forming yeast, Cryptococcus, which is ubiquitous in the environment. Like many pathogenic fungi, Cryptococcus causes disease when it is inhaled into the lung. From the lung Cryptococcus can disseminate to the central nervous system (CNS) and cause fungal meningoencephalitis that is fatal ~25% of the time, even with state-of-the-art treatments. In the United States the case mortality rate overall from invasive fungal diseases is ~50%, indicating the dire need for improved therapeutic strategies. To develop new antifungal therapeutics, we need to identify novel fungal-specific molecules or pathways. Thus, it is imperative that we gain a better understanding of the fundamental biology of pathogenic fungi, especially the development and growth of spores. Our long-term research goal is to understand how infectious spores survive in new environments, including the mammalian lung, and use that information to identify fungal-specific targets for therapeutic interventions. To accomplish this goal, we have developed the Cryptococcus system as a model for the study of infectious spores. The objective of this proposed project is to determine the molecular processes by which infectious spores transition into vegetatively growing yeast (germinate) and how this process influences disease. Our overarching hypothesis is that determining the molecular mechanisms of germination will identify key pathways in spore-mediated infections that can be targeted for inhibition. To test this hypothesis, we will carry out three Specific Aims: 1) Determine the molecular pathways and processes required for spore germination, 2) identify the molecular processes and events that promote germination competence of spores, and 3) determine the effects of spore germination kinetics on host-pathogen interactions and disease progression. We will combine molecular and classical genetics, gene expression analyses, chemical genetics, protein composition analyses, and quantitative germination assays to reveal the developmental and regulatory mechanisms that facilitate spore survival in diverse environments. At the same time, we will use in vitro tissue culture models and a mouse intranasal model of infection to determine how spores infect and escape the mammalian lung. These innovative experiments will result in an in-depth map of spore pathways and insights into how spores invade the host. Understanding pathways and processes associated with spore germination makes significant contributions to the long-term objective of this work to identify new and diverse molecular targets that can be exploited for novel antifungal therapeutics and strategies to prevent and/or treat cryptococcosis and other fatal human fungal diseases.

NIH Research Projects · FY 2025 · 2024-07

This proposal outlines a comprehensive plan for graduate education in chemistry & biology at UW–Madison via the Chemistry-Biology Interface training (CBIT) program. We seek to provide cross-disciplinary research training to our students, so that chemists and biologists not only appreciate, but also use, the tools and techniques developed by each other. These types of fresh approaches can lead to meaningful and truly impactful breakthroughs in science. The overall objective of CBIT is to educate trainees so they understand and can articulate scientific problems that span the chemistry–biology interface, have the technical skills to realize an independent research project at this interface, and can effectively communicate their discoveries to both scientific and general audiences. CBIT’s specific objective is to maximize Ph.D. completion rates within our 5 core departments/programs and measure longitudinal student outcomes (as per degree completion and job placement in the biomedical workforce) that will advance best practices in biomedical graduate training overall. CBIT’s overall & specific objectives are shaped by our mission to cultivate cross-disciplinary scholars capable of strong communication and teamwork. To realize these objectives, we request funds to support the CBIT program at the level of 10 trainees/year, with each trainee funded for 2 years. Key features of our proposed program are underscored below: ● The CBIT program will provide an integrated set of coursework (foundational and area specific, with dedicated courses in ethics and research rigor), research experiences that span the frontiers of the chemical biology field, mentorship training, focused training in communication (with mentors, other scientists, and the public), substantive career development opportunities (internships, annual workshops, and IDPs), and team-based experiences (via courses, research, and outreach). ● We will provide trainees a palette of four professional skill sets aligned with different biomedical careers (academic, industry, government, and legal/non-profit), guided by the needs of current employers within the biomedical workforce (via consultation with our new External Advisory Board), and composed of different activities that are specifically tailored for success in these four career spaces. ● We will evaluate our ability to achieve these results through quantitative assessments of the outcomes of the CBIT program, including gains in science identity, science self-efficacy, and core graduate school competencies such as broad knowledge of a discipline, experimental skills, and critical thinking skills. Our CBIT program fills a unique niche at UW–Madison as the only T32 program centered in the chemical sciences. This interfacial program has significantly impacted student outcomes—notably, of our 57 CBIT Ph.D. graduates since 2008, 55 (96%) are in careers that directly impact human health. We will build on this strong history and continue to innovate in graduate education over the next 5 years.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment for many blood diseases and cancers. However, these procedures still need to be optimized to improve patient outcomes and survival. HSPCs reside in a microenvironment surrounded by niche cells that help regulate their function. Our research proposal seeks to address fundamental questions regarding the cellular interactions between HSPCs and niche cells. During development in the embryo, HSPCs move through different tissues and have changing requirements for contact with the microenvironment. HSPCs first arise in the dorsal aorta, a large vessel in the embryo, and are then released into circulation. Next, HSPCs migrate to the fetal liver where the population of cells expands via symmetric divisions. Finally, HSPCs migrate again to colonize the bone marrow where they will remain throughout adulthood. As HSPCs are migrating between these different hematopoietic tissues, they are also becoming more mature and are programmed towards their adult quiescent state. We use zebrafish and mice as model systems that are highly conserved with humans and have many genetic tools for functional testing and live imaging of cellular behaviors. In zebrafish, we have found a novel role for vascular endothelial growth factor c (vegfc) in the release of HSPCs from the dorsal aorta. Vegfc also regulates a fate decision in pre-HSPCs that determines if they will become a stem cell or a different type of progenitor cell. At later stages, we found that integrin alpha 4 (itga4) in the caudal hematopoietic tissue (CHT), the zebrafish equivalent of the fetal liver, is required for proper epigenetic programming of HSPCs. We hypothesize that dissecting niche-dependent HSPC programming, using mutants with prolonged or shortened retention in developmental niches, will enable us to decode mechanisms governing HSPC fate decisions and track their long-term impact on hematopoiesis. We will address this hypothesis through the following Specific Aims: 1) Determine the role of vegfc in fate determination and release of HSPCs from the hemogenic endothelium of the dorsal aorta; 2) Establish how itga4-dependent contact with the fetal/CHT niche programs HSPCs as they transition from immature to mature states. We will perform in vivo genetic knockdown and small molecule treatments, together with live imaging, to understand the dynamic interactions between HSPCs and niche cells. We will use a multiomics approach to infer and functionally test the gene regulatory networks involved in HSPC programming. Together, our proposed aims will identify functionally critical signals and networks that integrate to program HSPCs. Our goal is for these novel insights and paradigms to improve HSPC-based therapies for patients.

NIH Research Projects · FY 2026 · 2024-07

ABSTRACT The cardiac and pulmonary systems are inherently linked through the pulmonary vascular system which leads to secondary pulmonary disease in cases of cardiac pathology. This is especially the case in congenital heart disease where the pulmonary blood supply is often substantially altered by abnormal outflow tracts and ventricular formation. Cross-sectional imaging has proven to be invaluable for assessing pediatric diseases, including congenital heart disease; however, current cardiopulmonary evaluations typically require multiple exams (SPECT, echo, MRI, and CT) to evaluate the cardiovascular and pulmonary systems. Each exam adds risk to already fragile patients, introduces complex logistics of performing multiple exams, and can delay care of patients who may require urgent management. Often, only a subset of exams are performed, and clinical management is based on incomplete information and disregards the strong potential for cardiopulmonary coupling. In this project, we aim to develop MRI methods that can simultaneously and efficiently evaluate both anatomy and function in pediatric cardiopulmonary diseases. MRI is theoretically well suited for quantitatively imaging both the cardiac and respiratory systems but is traditionally challenged by its slow imaging speed and sensitivity to artifacts. Recently, our group has proposed methods for dramatically more robust lung imaging using the combination of ultrashort echo time MRI with advanced motion corrected reconstruction strategies. In this proposal, we extend these techniques and introduce novel methods to provide improved and comprehensive diagnostics of the entire cardiopulmonary system. First, we introduce a free-running approach to cardiopulmonary imaging to provide anatomical imaging and the quantifications of ventilation, perfusion, cardiac function, and respiratory resolved cardiac flow dynamics. We specifically aim to image continuously with T1 weighted and velocity encoded sequences, and subsequently reconstruct this data with a high-dimensional deep learning approach. The reconstructions use novel motion corrected methods to directly estimate images and apply deep learning in a highly compressed space. Secondly, we aim to develop next-generation motion management using an RF navigator technique, Beat Pilot Tone, that can be applied during any pulse sequence to measure bulk, respiratory and cardiac motion. Beat Pilot Tone provides a basis for motion tracking that enables improved imaging efficiency, a simplified setup without cardiac leads or respiratory belts, and much better measures of bulk motion. These techniques will be evaluated in normal control participants and pediatric subjects with congenital heart disease, each with comparisons to state-of-the-art imaging. The impact of this project is to shift the paradigm for clinical management of cardiopulmonary diseases to a single-scan comprehensive imaging study and supporting an integrated assessment of interaction between the pulmonary and cardiac systems in disease. While this is targeted at pediatric cardiopulmonary diseases, the innovations can be applied broadly to MRI studies throughout age ranges and to other studies that suffer from motion artifacts throughout the body.

NIH Research Projects · FY 2024 · 2024-07

Epilepsy is the third most prevalent neurological disorder after stroke and Alzheimer’s Disease with an incidence of 1 in 26 individuals. It is estimated that 3 million people in the U.S and 65 million worldwide currently live with epilepsy. Approximately 30% of epilepsy patients are drug resistant and this number has remained the same since 1850 despite the marketing of 12 new antiseizure drugs in the past 20 years. A major hurdle to understanding the disease is that seizures are transient and importantly, difficult to predict. This prevents the acquisition of a detailed portrait of molecular, cellular and circuit alterations at the most critical time: just before seizure onset. However, seizures manifest a clear temporal organization: they display circadian and multi day (mulitdien) rhythmicity in patients, regardless of epilepsy type or affected brain region. Combining circadian and multidien rhythms, one can extract high seizure risk (HiSR) and low seizure risk (LoSR) epochs in a patient- specific manner. Importantly, this cyclicity in risk exists in two rat models of epilepsy and canine epilepsy. The existence of HiSR and LoSR times imply that circuit excitability changes in a periodic manner that can be modelled and predicted, and its mechanisms studied. We have developed a machine learning tool that acquires continuous EEG over many weeks, learns the pattern of interictal activity and then predicts when an animal is entering a LoSR or HiSR epoch. The goal of this application is to embark on the first ever global molecular and cellular exploration of HiSR and LoSR epochs. Historically, a common research strategy has been to compare non-epileptic to epileptic brains and, clearly this approach has yielded a wealth of knowledge regarding mechanisms behind seizure genesis and epileptogenesis. The premise of our application is that epileptic and non-epileptic brains are different enough from each other that additional, unique insights will be uncovered by using LoSR epochs as controls for HiSR epochs. Preliminary data from bulk tissue shows a differential expression of nearly 100 hippocampal proteins between HiSR and LoSR times. Together, these findings inform our central hypothesis that large scale hippocampal gene changes, driven by a few Master regulators, contribute to alterations in seizure risk over the multidien cycle. We will perform single cell RNAseq on hippocampi from rats in LoSR and HiSR to generate a high resolution, single cell map of all gene changes that occur as a brain transitions from a low to high seizure risk state. To the best of our knowledge this will be the first ever study comparing molecular and electrophysiological changes at the single cell level in the epileptic brain as it transitions from a low to a high seizure risk state. Using bioinformatic tools developed and published by us, we will identify potential Master Regulators behind these changes in every cell type and subfield of the rat hippocampus. The major deliverable of the proposal will be a detailed cell type specific transcriptome map accompanied by a survey of network properties during HiSR and LoSR epochs

NIH Research Projects · FY 2026 · 2024-07

Abstract Numerous animal cell types have extended post-developmental lifespans, necessitating robust mechanisms for cellular maintenance. Cellular maintenance deteriorates in conditions like metabolic and neurodegenerative diseases and upon acute or chronic exposure to stressors such as pollution and radiation. Moreover, decline in cellular maintenance is an evolutionarily conserved pathology of aging. Despite its significance, our understanding of the fundamental mechanisms underpinning cellular maintenance remains deficient. My lab aims to elucidate how post-developmental cells preserve their integrity and function in vivo. We use the nematode C. elegans for its powerful genetic and live fluorescent imaging capabilities, and for its suitability for studying cell biology within the organism’s physiological context. In the next five years, we propose to investigate two key aspects of cellular maintenance: First, within mature cells, proteins and organelles undergo continuous turnover: they are degraded, largely by lysosomes, and replaced to ensure cellular quality. The mechanisms determining lysosomal degradative capacity and controlling the rate at which degradative cargoes are sent to the lysosome during steady-state maintenance are not well understood. Building on research initiated in my independent lab, we will take a three-pronged strategy to address this gap in knowledge, that will: probe the control mechanisms of an established regulator of degradative capacity, discover novel regulators of degradative capacity through an unbiased genetic screen, and develop a new tool to measure homeostatic protein degradation, an indicator of degradative flux, with high spatial resolution in vivo. At present, effective methods to measure this remain elusive; the development of this tool will not only enable the experimental interrogation of lysosomal degradative flux but also facilitate exploration of numerous other cell biology questions. Second, cellular maintenance is not fully cell-intrinsic; rather, it is governed by intricate intercellular signaling that coordinates both maintenance and metabolism across diverse cell types within an animal. The molecular mechanisms underpinning this coordination are incompletely understood. During my postdoctoral research, I developed the dauer – a C. elegans alternate organismal state – as an experimental paradigm to study how cellular maintenance can be dramatically extended via intracellular metabolic signaling. We will expand on this line of research to discover new understandings of the interplay between cellular maintenance, organismal metabolism, and metabolic signaling. Collectively, this research will bridge existing gaps in our understanding of the intracellular and extracellular mechanisms of cell maintenance, laying the foundation for designing interventions against cellular maintenance loss in disease and aging.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY In addition to intellectual disability, individuals with Down syndrome (DS, Trisomy 21, Ts21) have an increased risk for many other health conditions, including Alzheimer’s disease (AD). DS is the leading genetic risk factor for AD, and nearly 90% of individuals with DS develop AD (DS-AD). Beta-amyloid (Aβ) plaques and tau tangles, characteristic AD pathology, begin accumulating in DS brains earlier than in the general population, and most individuals with DS are diagnosed with DS-AD in their early to mid-50s. Basal forebrain cholinergic neurons (BFCNs), involved in arousal, learning, attention, and memory, are a susceptible population prone to degeneration in DS-AD as well as other neurodegenerative diseases, including AD, Parkinson’s disease (PD), and Dementia with Lewy bodies (DLB). Little is known about BFCN development and molecular mechanisms underlying vulnerability. Limited analysis of human DS tissue suggests fewer BFCNs populate the basal forebrain, however, it remains unclear whether the reduction is due to developmental deficits, accelerated degeneration, or a combination of both. The goals of the F99 pre-doctoral proposal are to 1) determine whether fewer BFCNs and AD pathology are present in the early postnatal DS basal forebrain, 2) identify underlying molecular signatures that may contribute to the susceptibility of BFCNs later in life, and 3) model BFCN development in vitro using isogenic of Ts21 and control induced pluripotent stem cells. The goals of the K00 post-doctoral proposal are to 1) analyze tissue across the lifespan at different disease stages to determine the temporal order of AD pathology accumulation in the DS basal forebrain, and 2) sequence basal forebrain tissue from DS-AD, AD, PD, and DLB to determine if shared mechanisms underlie BFCN vulnerability across several neurodegenerative diseases. This F99 proposal will be the first study of human DS post-mortem tissue to reveal early deficits in DS BFCNs, suggesting deficits in DS BDCN development. Elucidating early post-natal deficits in DS BFCNs may inform early interventions and improve BFCN health across the lifespan. The K00 proposal will be the first cross-sectional study of human DS post-mortem tissue to reveal early emerging pathology and molecular mechanisms contributing to the susceptibility of BFCNs in the basal forebrain and elucidate shared mechanisms underlying BFCN vulnerability in several neurodegenerative diseases. Results may provide novel targets for therapeutics impactful for many neurodegenerative diseases. The results from this project will meet the goals of the NIH INCLUDE project by establishing scientific data to improve the health and neurodevelopment of individuals with Down syndrome and have a broader impact on the health of individuals at risk for other neurodegenerative diseases characterized by the loss of BFCNs, including DS-AD, AD, PD, and DLB.

NSF Awards · FY 2024 · 2024-06

This grant provides three years of Community Facility Support for the Wisconsin Secondary Ion Mass Spectrometer National Facility (WiscSIMS Lab). The WiscSIMS Lab performs precise and accurate analysis of isotopes in very small amounts of material to advance earth and environmental science research. These small-scale measurements can reveal hidden information about Earth processes, like the driving forces for volcanic eruptions, the speed of climatic changes, and the formation of critical-element minerals. The principal missions of the WiscSIMS Lab are to drive innovative research and to ensure that all visiting scientists, especially those funded by NSF, can obtain world-class SIMS data. The WiscSIMS Lab has been funded continuously as a National Facility since 2008 and is a leader in training in the next generation of scientists. More than 440 different researchers, including 146 student trainees and 27 postdoctoral trainees, have worked with WiscSIMS since it was established. WiscSIMS provides in situ, high spatial resolution, high-precision isotope analysis to advance geoscience research in paleoclimate, geochemistry, volcanology, geobiology, petrology, sedimentology, and tectonics. The WiscSIMS Lab is built around a large-radius, multicollector CAMECA IMS 1280 ion microprobe, analyzing primarily stable isotopes of Li, C, N, O, Mg, Si, S, Ca, and Fe, but also trace elements, volatiles, and U-Pb isotopes. The WiscSIMS staff has developed state-of-the-art capabilities for precise and accurate in situ analysis of isotope ratios with small to ultra-small spot sizes. With renewed Community Facility Support, WiscSIMS will continue to make at least 50% of its beam time available each year to guest researchers. NSF projects will continue to receive the highest scheduling priority and a reduced fee rate. Community Facility Support funding will allow WiscSIMS to pay the salaries of the expert SIMS operators and support staff, who assist scientists to drive innovative applications of isotope analysis across the breadth of geoscience. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-06

Cyber-Physical Systems (CPS) are typically composed of interconnected hardware and software components, which individually may not be inherently highly reliable or secure. However, several CPS applications demand a high degree of safety, security, and reliability. Thus, the fundamental problem is constructing highly dependable CPS applications from building blocks that are, in themselves, not inherently reliable. There has been enormous progress made in understanding and patching various classes of vulnerabilities in large-scale distributed CPS. However, these efforts at designing and operating resilient CPS have often been stymied by the lack of understanding of the impact of any perturbation to the overall system, under the economic and policy constraints involved in any realistic real-world CPS. We define perturbations as failures due to: (1) unintended errors in hardware/software, (2) security attacks, (3) unexpected interactions among cyber-physical and human elements including natural disasters, and (4) incomplete cooperation among stakeholders. In this project, we address these shortcomings to challenges to create resilient, large-scale CPS through our CHORUS Frontier award. Chorus will develop rigorous, scientific mechanisms to enable CPS resilience against a large universe of perturbations. Our application domain is Connected and Autonomous Transportation Systems (CATS) and thus, the benefits of CHORUS will be demonstrated through improvements in safety and security in this domain. We will achieve goals of CHORUS through three interacting intellectually challenging thrusts in the project. Thrust 1 is on Modeling which will create executable specifications of cyber, physical, and human assets, their interconnections, and the economic and policy constraints. The models will capture various stakeholders in the system and their degree of information sharing and cooperation in defense of the target CPS. Thrust 2 is centered on Proactive planning and deployment. We will develop rigorous game-theoretic formulations to model the spread of perturbations (natural and man-made), their effects, and the appropriate resource allocations that can be applied for resilience at the planning stage, i.e., prior to system deployment. We will also consider which defensive investments are feasible under a rational versus a bounded rational behavioral model of the stakeholders. Thrust 3 focuses on Runtime distributed detection and response. We will determine, at runtime, the security state of the system, through novel uses of existing sensors in the system even though they are imperfect. This will then trigger the response mechanisms, which will be proven to be approximately optimal, through analysis and experimentation. In terms of broader impact, the greatest impact will be that CPS owners will gain a higher degree of trust in the operation of the CPS and policy-makers will understand what level of cooperation among multiple stakeholders in a CPS to incentivize. We will create compelling demonstrations of CHORUS on a connected vehicle testbed distributed between our academic institutions and our industrial partner GM. We will also organize an annual student security competition and develop two MOOCS, both having foundational material on resilient CPS and one focusing more on the CATS application domain. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-06

The 41st International Symposium on Computer Performance, Modeling, Measurements and Evaluation (IFIP WG 7.3 Performance 2023) brings together researchers at the intersection of computer science and applied probability, with the aim to understand and improve the performance of computing and communication systems by means of state-of-the-art quantitative models and solution techniques. Attracting scholars from around the world, this selective, single-track conference series features cutting-edge research on the performance evaluation of computing systems. The conference also includes several half- and full-day workshops for in-depth coverage of topics such as mathematical modelling and performance analysis, learning and optimization in networks, blockchain, cloud computing, and applications in transport, energy and service systems. This student travel grant will fully support approximately 25 students’ attendance (airfare/ground transportation, lodging, and registration) to participate in the conference, potentially share their own research, and interact with leading researchers in the field. Mentoring is a core tenet of the Performance conference, and attendees will have several dedicated opportunities to meet and interact with senior members of the community. In the goal to broaden participation in computing, student attendees will be recruited from under-represented groups both locally and across the country, and there are portions of the funding dedicated to this initiative. Furthermore, this funding support also offers student attendees of Performance 2023 the opportunity to meet one another, fostering a peer group at an early stage that can benefit them throughout their careers. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-06

Abstract: Bacterial pathogens require specific adaptations to survive and thrive in their replicative niche. A subset of important human pathogens, including Shigella Spp. Rickettsia spp and Burkholderia spp., utilize the cytosol of host cells as their primary replication niche. Although the host cell cytosol is an inhospitable environment for bacteria, the stresses encountered in this environment and the mechanisms pathogens utilize to overcome these stresses are not well understood. The goal of this proposal is to utilize the cytosolic pathogen Listeria monocytogenes (Lm) to understand how host cells recognize and respond to bacteria in the cytosol and in turn how cytosolic pathogens have adapted to life in the cytosol. Lm is a Gram positive foodborne pathogen that causes the rare but often fatal disease Listeriosis. To identify mechanisms by which Lm has adapted to survive and thrive in the mammalian cytosol, we have utilized a series of forward genetic screens to identify genes required for Lm cytosolic survival. These screens highlighted robust cell wall stress responses and specific metabolic adaptations as essential for cytosolic survival and virulence. Specifically, we demonstrated that the PASTA kinase PrkA is a master regulator of cell wall stress responses essential for cytosolic survival and virulence in vivo. We defined the phosphoproteome of PrkA and defined the role of one phosphosubstrate, ReoM, in regulating peptidoglycan synthesis in response to cell wall stress during infection. GlmR is another PrkA substrate required for cell wall stress responses, cytosolic survival and virulence in vivo and we defined its role as an accessory uridyltransferase. Finally, an additional gene of unknown function, yvcJ, was identified through our genetic screens and shown to be involved in cell wall stress responses, cytosolic survival and virulence. How the remaining PrkA dependent phosphoproteins coordinate cell wall stress responses, how phosphorylation of GlmR regulates its function, how YvcJ contributes to cell wall stress responses and finally the mechanism by which the host imparts cell wall stress on bacteria in the cytosol are all unknown. These questions are the focus of Aim 1. Our cytosolic survival screens also revealed multiple genes involved in central metabolism, notably the pyruvate dehydrogenase complex and genes required for DHNA biosynthesis. Mutants lacking these pathways secrete lactate instead of acetate as the major metabolic byproduct and interventions that restore acetate production rescue cytosolic survival of DHNA deficient strains. Additionally, we created a series of mutants missing genes essential for Lm glycerol utilization, hexose phosphate utilization, or both. As the only described carbon sources utilized by Lm during infection we were surprised to find minimal defects in ex vivo virulence and only 1-2 log reductions in virulence in mice. How increased concentrations of cytosolic lactate during Lm infection alter host responses, what other carbon sources are used by Lm during infection and how alterations in bacterial carbon utilization impact host responses are currently unknown. These questions are the focus of Aim 2.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract Rapid identification and antimicrobial susceptibility testing (AST) of clinical isolates are crucial to ensure early appropriate treatment and prevent misuse of broad-spectrum antimicrobials. Both phenotypic (i.e., growth, inhibition, or killing event-based) and molecular (i.e., resistant gene or molecule-based) ASTs are routinely used in the hospital clinical microbiology laboratory. The limitations of molecular methods (e.g., qPCR, MALDI-TOF MS) include their narrow scope (i.e., select resistance genes/molecules), or in some cases the inability to predict antimicrobial susceptibility, while phenotypic methods involve time-consuming, multi-step expansion culture resulting in prolonged time to result, which can delay appropriate treatment decisions. This proposal leverages an exclusive liquid repellency (ELR) based under-oil open microfluidic system for a transformative approach to next-generation phenotypic ASTs. With a multidisciplinary team including clinicians, microbiologists/pharmacologists, surface scientists, and bioengineers, we outline three aims for the development of this next-generation, phenotypic AST system. The ELR-based AST system aims to meet the following criteria: i) direct AST using the original clinical isolates (e.g., from blood, sputum, urine, abscess) to eliminate time-consuming expansion culture and passage- associated selection bias ex vivo, ii) comprehensive test coverage including anaerobes, multispecies communities, heteroresistance (i.e., resistant mutants within the wild-type population), and iii) rapid AST with the goal of sample to report in less than 4 hours. In Aim 1 we propose to develop ELR-centrifugation and small-volume ( μ l scale) lossless sample processing for isolation, enrichment, and preparation of sparse (i.e., 1-100 cfu/ml) bacteria from whole blood. The goal is to directly isolate and enrich bacteria from whole blood without expansion culture. We will combine the lossless sample processing enabled by ELR with lysis-centrifugation to maximize the recovery yield of bacteria (> 90%) from whole blood. In Aim 2 we will demonstrate compatibility of ELR AST with intrinsic fluorescence label free imaging modalities and deep learning-based identification. The goal is to develop and apply proof-of- concept advanced label-free, single-cell resolution, live-cell imaging, and deep learning algorithms to integrate bacterial detection, species identification, and antimicrobial screening thereby eliminating isolate passage expansion. In Aim 3 we propose to develop label-free, direct detection of heteroresistance in priority human pathogens in clinical isolates. The goal is to detect heteroresistance [i.e., a small/rare subpopulation (e.g., <1%) of resistant mutants in clinical isolates], which cannot be identified by standard clinical methods. We will utilize sweep distribution to array small numbers of bacteria directly from clinical isolates, thus enabling the detection of these heteroresistant mutants. The outcomes of this project will address many gaps in the current AST landscape and develop a next-generation AST platform with the potential for rapid adoption in many clinical settings.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY / ABSTRACT Stem-loop binding protein (SLBP) is a cellular protein required for the production of replication- dependent histones (RDH). SLBP binds to a conserved stem loop in the 3’ UTR of RDH mRNAs and facilitates their processing in the nucleus, export, and translation in the cytoplasm. To date, no other cellular role for SLBP has been reported. We recently demonstrated that during HCMV infection, RDH mRNA processing and translation is inhibited indicating that SLBP is not performing its only known function. Interestingly, however, we also found that SLBP is required for efficient HCMV productive replication. In the absence of SLBP, similar numbers of progeny virions are produced, but my data suggest a non-virion component of the viral supernatant diminishes infectivity of virus stocks made in the absence of SLBP. Together, these data suggest that HCMV utilizes SLBP for a new, yet uncharacterized function to promote HCMV infectivity. The overarching goal of this proposal is to identify and characterize this novel, pro-viral function of SLBP during HCMV infection. Because the only known function of SLBP requires its ability to bind RNA, I hypothesize that HCMV co-opts the RNA binding function of SLBP to perform a new, pro-viral role required to modulate expression of a secreted factor that affects HCMV infectivity. To test this hypothesis, I will determine the function of SLBP required to promote HCMV infectivity, characterize differences in the secretome during HCMV infection in the presence or absence of SLBP, and determine at which point in a newly infected cell HCMV virus stocks produced in the absence of SLBP are deficient.

NIH Research Projects · FY 2026 · 2024-06

Project Abstract Aphasia is an acquired neurologic language disorder that is among the most challenging long-term disabilities for stroke survivors, often leading to social isolation and reduced quality of life. Recovery from aphasia relies on plasticity in residual brain networks. However, neuroplasticity varies substantially across individuals, making the presence, severity, and phenotype of language impairments challenging to predict. A vital step toward post-stroke precision medicine is identifying neuroplasticity-related biological markers that can improve prognostic models and targeted neurorehabilitation therapies for people with aphasia. The proposed research will test the central hypothesis that individual differences in neuroplasticity, measured through genetic polymorphisms and longitudinal neuroimaging connectivity biomarkers, will account for significant variance in post-stroke aphasia recovery. This 5-year project will include three specific aims. Aim 1 is to index spontaneous recovery by determining relationships between genetic biomarkers of plasticity, longitudinal neural network connectivity, and changes in language during sub-acute to chronic stroke recovery. Aim 2 is to identify genetic and MRI biomarkers predictive of chronic post-stroke aphasia severity and phenotypes. Aim 3 is to characterize genetic and MRI biomarkers associated with verbal learning variability in chronic aphasia. These data will support the development of a larger, multi-site R01 study to examine interactions between multiple biomarkers of neuroplasticity that inform longitudinal aphasia prognostics and treatment efficacy. This career development proposal is designed to provide Haley C. Dresang, Ph.D., a clinical neuroscientist and aphasiologist, with the training required for success as an independent patient-oriented scientist conducting neurotranslational aphasia research. As a junior faculty member at the University of Wisconsin– Madison, Dr. Dresang is in an ideal environment with extensive infrastructure to support early-stage investigator training and research. The proposed career development plan includes both coursework and mentored training in the areas of 1) MRI neuroplasticity biomarkers, 2) genetic polymorphisms associated with neuroplasticity, 3) longitudinal clinical trials design and analysis, and 4) professional skills for an independent translational neuroscientist. To ensure success, she has identified committed, expert mentors in these disciplines and secured protected time for this work. This award addresses a significant clinical dilemma and serious gap in neurobiologically motivated aphasia research, while affording the education and mentored research experience critical for Dr. Dresang to lead an independent aphasia research program.

NSF Awards · FY 2024 · 2024-06

Monitoring ground motion caused by human activity (e.g. ships, rail and vehicular traffic, explosions, civil infrastructure), biological activity (e.g. marine and terrestrial animal migrations and behavior), and natural disasters (e.g. earthquakes, tsunamis, lightning) is critical for research, public safety, and security. The key considerations for sensors used in these monitoring applications include precision, response time, accuracy, deployability, and cost. The planning work supported through this project will develop the framework for a future mid-scale research infrastructure proposal that would support the build-out of a national testbed facility focused on fiber-based ground motion and deformation sensing. The foundational idea for this facility is that relatively minor modifications to devices that send and receive data signals on fiber-optic cables deployed in the Internet can enable ground motion sensing capabilities that are not possible with standard technologies. The planning activities for the envisioned Internet Sensor Network (Internet-S) testbed will identify the associated technical requirements and develop a framework for the facility that is feasible in terms of costs and operation. This work will also focus on fleshing out details of the key components of the facility, which include (i) an optics laboratory for controlled, repeatable research with optical components; (ii) a dark fiber network for in situ research of optical sensing devices; and (iii) an optical data analytics and visualization workbench for organizing and interpreting fiber-based sensing data. Working groups comprised of thought leaders from key disciplines including computer science, geoscience, physics, and electrical engineering will be organized to develop the Internet-S testbed design. The planning activities will also include two workshops that will convene a broader group of researchers, equipment vendors, network operators, and government entities to discuss Internet-S and seed future use of the envisioned research testbed infrastructure. The mid-scale research infrastructure envisioned in this work will be used by a diverse community of scholars to develop new Internet-based environmental and infrastructure sensing capabilities that go well beyond current technologies in terms of scale, sensitivity, and manageability -- at low cost. The new sensing systems developed in the testbed will be used in a wide range of applications, including computer networking, optics and geoscience research, smart cities, public safety, and security. This work will also influence the development of new concepts and expertise that will be used in computer networking, geoscience, and data science courses. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Bacterial vaginosis (BV) occurs in nearly one third of women of reproductive age, causing significant problems for sexual and reproductive health. Women with BV are predisposed to pregnancy complications including amnionitis and preterm delivery. Gardnerella spp. are major players in the BV dysbiosis, displacing Lactobacillus spp. and forming a robust biofilm supporting the growth of multiple anaerobes. The biofilm also serves as the infectious form for transmitting bacterial vaginosis between people. Gardnerella make a number of putative virulence factors including multiple pili, lipases, sialidases, and the pore-forming toxin vaginolysin. It has been difficult to study Gardnerella virulence factors due to the inability to make targeted mutations or introduce genetic constructs. We discovered a way to make targeted insertion mutations using suicide plasmids in Gardnerella spp., and we propose to further develop these methods to produce a full set of tools for Gardnerella genetic manipulation. First, we will improve methods for making insertional mutants by determining factors affecting transformation frequency and insert stability, and by developing use of a counter- selectable marker to allow for creation of selectable unmarked mutations such as deletions and point mutations. Furthermore, we will test different methods of insert replacement to optimize creation of unmarked mutations. Second, we will develop methods and constructs for gene expression from the Gardnerella chromosome in order to produce constitutive or regulated complement gene expression, transcriptional fusions to Gardnerella ‘lacZ, or translational fusions to the FLAG epitope. The usefulness of the methods will be tested with mutations in the vaginolysin gene.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT The 75-90% lifetime risk of Alzheimer’s disease (AD) in Down syndrome (DS) is a significant health concern for the DS community. Despite the progress of large international consortiums preparing the field for upcoming clinical trials, there are currently no validated remote assessment options for tracking AD-related cognitive decline for adults with DS. Remote assessment offers numerous benefits to participants and researchers and will support recruitment and retention in AD clinical trials, which involve many study visits to assess cognitive and behavioral changes throughout a pharmaceutical or behavioral intervention. Additionally, remote assessment will enhance the accuracy of longitudinal research studies by allowing more frequent data collection without significantly increasing participant burden. Using remote assessments will open participation to those in rural communities, those with low economic statuses, and racial/ethnic groups who may not have the time or resources for the travel and cost that in-person participation requires. This study will focus on the life stage when AD pathology is beginning (22 – 55 years old) to ensure that those in beginning stages of disease progression are included in the study sample. All cognitive measures selected to transition from in- person to remote administration have been shown to be promising for tracking early AD. The K99 phase of the study will focus on the feasibility and modification of remote administration to ensure families are satisfied with remote assessment procedures. The R00 phase will focus on evaluating the reliability and validity of remote measures by comparing performance to in-person assessments, investigating test-retest performance, and measuring change over 16 months. The R00 phase will also include a blood draw to examine associations between cognitive measures and plasma AD biomarkers. Methodology is innovative in that it involves a new frontier of remote assessment tools and incorporates plasma biomarkers of AD to compare remote and in- person cognitive performance to AD pathology. There are three primary aims of the proposed study (1) Develop and test the feasibility and acceptability of remote assessments to measure AD-related cognitive decline in adults with DS, (2) Evaluate the reliability of each remote AD cognitive assessment and construct validity with in-person cognitive assessments, and (3) Determine sensitivity of remote and in-person AD cognitive assessments to plasma biomarkers of AD pathology. This project’s goals are aligned with the NIH INCLUDE (INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndromE) initiative and will serve to increase measurement options for treatment studies in AD and DS and subsequently improve reach and representation of diverse individuals in clinical trials and DS research.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT Relationships between words organize children’s early vocabularies into interconnected semantic networks. The structure of these semantic networks, specifically how densely connected they are, predict cognitive skills that are central to later academic success, including vocabulary size and word recognition speed. That is, children whose vocabularies include more words that are semantically related to one another have stronger language skills later in life. Thus, to support successful language development outcomes, it is essential to understand (a) how toddlers begin to form semantic associations between individual words and (b) how they use these associations to organize their networks into interconnected clusters of related words. Previous work has explored the possibility that toddlers form semantic associations based on perceptual similarities between the objects that words refer to, as well as similarities in the linguistic structures in which words are used. However, these cues may not always be available and accessible to young word learners. An additional, unexplored possibility is that toddlers take advantage of a different type of cue—the environmental context in which they encounter words and objects, like the kitchen or the bathroom—to form semantic associations between words for objects that appear in the same contexts and to group objects that appear in the same context into semantic categories. Previous research suggests that caregivers systematically talk about semantically-related words in particular contexts (i.e., food-related words in the kitchen and hygiene-related words in the bathroom) and that toddlers remember the contexts in which they encounter words and objects. Despite the reliability and accessibility of environmental context as a cue to lexical associations, its role in semantic network development has not yet been examined. Specific Aim 1 of the proposed project will investigate whether young toddlers form semantic associations between novel words for objects that are encountered in the same environmental context. Specific Aim 2 will examine whether older toddlers can use environmental context to aggregate perceptually dissimilar objects together into broad categories of semantically-related objects. Finally, Specific Aim 3 will examine how individual differences in toddlers’ associations between words and particular environmental contexts are related to differences in the structure of their extant vocabularies. By examining the role of environmental context in semantic network development, the proposed project will incorporate environmental context into word learning theory. Additionally, the proposed work will examine a mechanistic account of semantic network development across toddlerhood, which will inform theories that encompass individual differences in vocabulary acquisition and could serve as a potential target for future language learning interventions. Training Plan. The training plan focuses on acquiring new methodological expertise in diverse experimental paradigms and individual differences measures, gaining theoretical knowledge in semantic development, mentoring junior researchers, completing advanced training in ethical and responsible conduct of research, and honing science communication skills.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Neutrophils are terminally differentiated cells of the innate immune system that are necessary for host defense. Emerging evidence suggests that neutrophils have more heterogeneity and plasticity than previously thought. However, there is a gap in understanding neutrophil developmental heterogeneity and function because they are short-lived ex vivo and are not genetically tractable. Induced pluripotent stem cells (iPSC)-derived human neutrophils (iNeutrophls) offer the opportunity to genetically and developmentally program neutrophils for designed properties that may be used both to understand neutrophil biology and provide an avenue for engineering neutrophils for human treatment. We have engineered GMP-compatible human iNeutrophils that show antimicrobial function in vitro but display significant heterogeneity with distinct subtypes based on preliminary single cell analysis. Our preliminary data also suggest that LPS treatment of progenitor cells “trains” the iNeutrophils for an increased responsiveness to secondary stimuli. Here we propose to use single-cell multi-omics to understand the heterogeneity and function of iNeutrophils and to test the hypothesis that iNeutrophils can be used to understand the metabolic and genomic mechanisms of trained immunity of human neutrophils. Specifically, will use single-cell multi-omics and optical metabolic imaging to identify the transcriptional, epigenetic and metabolic heterogeneity that could inform functions of distinct iNeutrophil subsets and the effects of immune training. We will also develop zebrafish larvae as an in vivo model to screen for iNeutrophil antimicrobial functions in models of bacterial and fungal infection. The proposed work will provide a genetically tractable system to understand the heterogeneity of human neutrophils, and the effect of immune training on neutrophil function that may optimize for antimicrobial effects.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT E-cigarettes, or electronic nicotine delivery systems (ENDS), are the most frequent tobacco product used by adolescents (As, aged 14-17) and young adults (YAs, aged 18-24). While growing evidence suggests ENDS as a possible strategy for smoking cessation among established adult smokers, the majority of A/YAs report never smoking cigarettes. This population needs evidence-based treatment to quit ENDS and avoid life-long nicotine addiction and the potential health harms associated with use. To date, there has been only one randomized controlled trial on e-cigarette cessation which used the texting program, This is Quitting, to help YA ENDS users quit. Our goal is to improve understanding of how to help A/YA ENDS users quit and ultimately increase cessation rates. This career development proposal is designed to increase our understanding of how to: a) promote A/YA engagement with ENDS cessation programs; and b) help A/YA ENDS users successfully quit while providing the training, experience, and data necessary to become an independent physician scientist focused on designing, implementing, and assessing A/YA ENDS cessation interventions. The specific AIMS of this K08 proposal are to: 1) Identify predictors of A/YA ENDS quit attempts (Aim 1a) and cessation success (Aim 1b), in a nationally representative sample, 2) Examine A/YA perspectives on strategies to increase engagement with, This is Quitting, with a focus on how to operationalize financial incentives to promote engagement, and 3) Evaluate the feasibility, acceptability, and preliminary efficacy of two enhancements to This is Quitting (financial incentives and NRT), on engagement and ENDS cessation success in YA users. This proposal is based at the UW-Center for Tobacco Research and Intervention and supported by an expert team of mentors and collaborators who represent leaders in tobacco research, qualitative research, data analysis, and clinical trials. This group, along with my education program, will help guide the achievement of the following training objectives: (1) advance knowledge and skills in experimental design, implementation, and evaluation (2) advance understanding of qualitative methodology (3) Increase knowledge of nicotine addiction treatment via both medication and psychosocial interventions, and (4) gain a better understanding of research ethics, leadership, and skills in manuscript and grant writing. Combined, this research proposal and education plan can improve our understanding of how A/YA ENDS users can quit and prepare me with the knowledge to use those findings to apply for a more definitive treatment study to help A/YA ENDS users quit.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Latinx individuals are 1.5 times more likely to develop Alzheimer's disease and related dementia (ADRD) than non-Latinx White adults. However, compared with non-Latinx Whites, they are less likely to receive a diagnosis, and when they do, they are more likely to be at late stages in the disease process. This is a result of individual and contextual factors that limit access to health care services in older Latinx adults, resulting in worse health outcomes for patients and caregiv- ers. To improve access to care for Latinx individuals, it is necessary to account for Latinx individ- uals' diverse backgrounds and socioeconomic characteristics, and develop innovative interven- tions that are feasible to implement in underserved areas. The proposed K99 phase includes training to fill my gaps in knowledge—the organizational factors affecting access to ADRD healthcare services, the use of behavior change theories and design thinking in health interven- tions, and the conduct of pragmatic trials in ADRD research. The overarching objective of this proposal is to increase access to culturally appropriate ADRD services by performing a compre- hensive assessment of the barriers existing at five locations in the US and engaging a team of stakeholders in a human-centered process to develop a systems-level intervention for increasing access to ADRD services. To achieve these goals, we propose a multiphase mixed methods research process. Aim 1 (K99 Phase) includes the validation of a survey instrument that measures beliefs that affect intentions to seek care for cognitive symptoms. This instrument will be an im- portant tool to describe the individual beliefs that sustain health disparities in subsequent stages. Aim 2 (K99-R00 phase) includes partnering with five (5) community health clinics in different cities around the US to characterize the individual and contextual factors that affect Latinx access to ADRD healthcare services. For this, we will use the BESIC instrument to describe beliefs among Latinx patients, hold five focus groups with Latinx patients with ADRD and their caregivers, and conduct up to 50 interviews with key informant stakeholders. Aim 3 (R00 Phase) will involve part- nering with one community health clinic to co-design a human-centered intervention for promoting Latinx access to healthcare services for ADRD. At the conclusion of this project, we will have a validated survey instrument to assess ADRD beliefs linked to health care seeking behaviors in heterogeneous Latinx populations and a prototype of an intervention to increase access to ADRD services. Future studies will pilot the intervention and subsequently evaluate its effectiveness us- ing an embedded pragmatic trial.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT: Glioblastoma (GBM) has a complex infiltrating tumor microenvironment which extends well beyond the visible enhancing tumor margins and plays a substantial role in GBM recurrence and poor outcomes. Unfortunately, in the absence of a precise spatial map of tumor extent, it is often difficult to differentiate infiltrating tumor from vasogenic edema on clinical MRI during radiation/surgical planning. The untreated infiltrating tumor ultimately contributes to over 90% of GBM recurrences. An equally pressing challenge is the difficulty in distinguishing recurrent tumor from treatment-effects following chemoradiation. Due to the histologically diverse landscape of post-treated lesions, treatment-effects often co-exist with tumor recurrence, and mimic appearance on imaging. In the absence of reliable tools, 15-20% of patients with GBM recurrence are incorrectly diagnosed due to sampling error associated with intracranial biopsy. Thus, developing a non-invasive spatial map of GBM tumor extent that can reliably identify infiltrating/recurrent tumor from confounding pathologies (treatment- effects/edema), will have significant implications in radiation/surgical-planning and post-treatment management. Recently, we developed a Radiomic-Image (Rad-I) map of tumor extent that uses computational features corresponding to the micro-architectural image measurements of disorder in the local intensity gradients (i.e., gradient entropy). The initial version of the Rad-I map has been evaluated to distinguish recurrent tumors versus treatment-effects on post-treatment Gd-T1w MRI with an 85% accuracy on n=75 studies, and to distinguish infiltrating tumor versus vasogenic edema on pre-treatment MRI scans with a 94% accuracy on n=42 studies. In this R01 project, we propose to improve on our initial version of Rad-I map by incorporating (1) additional anatomical (T2w, FLAIR) and functional MR sequences (perfusion) and (2) a novel “lesion complexity” feature, which captures organizational changes in the tissue composition via graph-theoretic approaches on MRI scans. Overcoming limitations pertaining to small cohorts and lack of spatially mapped ex-vivo histology for validation, Rad-I maps will be extensively validated on (1) a large multi-institutional MRI cohort with co-localized histopathology and (2) the PRESERVE clinical trial designed to capture GBM heterogeneity via multiple co- localized tissue samples/lesion. These cohorts will also allow for establishing associations of our new radiomic features with underlying histological/molecular tumor characteristics- a prerequisite for clinical adoption. Lastly, Rad-I maps will be evaluated within a tumor board survey to address the clinically challenging problem of distinguishing recurrent tumors versus treatment effects. Criteria for success for Rad-I maps are that they are at least non-inferior to the accuracy of stereotactic biopsies (85-90%) in identifying tumor niches corresponding to (a) viable/infiltrating tumor vs. edema and (b) recurrent tumor vs. treatment effects. Multi-institutional validation and end-user (tumor board) feedback will further confirm the utility of Rad-I maps as a noninvasive alternative to surgical biopsies; thereby paving the way for radiation/surgical and post-treatment management in GBM tumors.

NIH Research Projects · FY 2025 · 2024-06

Abstract Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a devastating skin blistering disease for which there currently is no cure. RDEB is caused by mutations in the gene encoding type VII collagen (C7), leading to epidermal fragility, trauma-induced blistering, and long term, hard-to-heal wounds. Fibrosis develops rapidly in RDEB skin and contributes to both chronic wounds, which emerge after cycles of repetitive wounding and scar formation, and squamous cell carcinoma, the single biggest cause of death in this patient group. The molecular pathways disrupted in a broad spectrum of fibrotic disease are also disrupted in RDEB and as such RDEB is a paradigm for understanding the molecular basis of fibrosis. We have shown that RDEB pathogenesis is driven by a radical change in extracellular matrix (ECM) composition and increased TGFβ signaling that is a direct result of C7 loss-of-function in dermal fibroblasts. However, the mechanism of how C7 loss results in extensive fibrosis has until recently remained unclear. Our now published work has identified a direct role for C7 in protein secretion which when defective or absent leads to accumulation of intracellular proteins, and subsequent increases in cellular stress and the initiation of a pro-fibrotic cascade of TGFβ signaling. This paradigm shifting work has shed significant light onto the conundrum in the field presented by RDEB – why does RDEB develop life threatening skin cancers while other forms of Epidermolysis Bullosa (EB), characterized by continued tissue damage and non-healing wounds, do not? In parallel, and using patient cells, we have identified anti-viral drugs, which inhibit the production and release of viral particles (a process known to hijack cellular transport pathways), are normalizing RDEB secretion defects and therefore represent a novel therapeutic approach to treat RDEB by preventing fibrosis. Furthermore, our work comparing RDEB with non-RDEB patient cells has identified increased TGFβ signaling as a potential mechanism of disrupted protein secretion in non-RDEB cells, which is independent of the protein defect in RDEB, and anti-viral drugs may represent a novel anti-fibrotic that could have application to a wider range of diseases, beyond RDEB. Therefore, our proposal seeks to investigate protein secretion defects we have identified in RDEB fibroblasts and determine the mechanism of action of anti- viral drugs in reducing fibrosis. At the same time, we will assess the anti-fibrotic action of anti-viral drugs in a preclinical mouse model of RDEB and if successful future steps (not part of this funding application) will be to initiate a clinical trial of successful candidate drugs. Together, our integrated in vitro and in vivo studies will define and characterize a novel mechanism of progressive fibrosis, and assess preclinical efficacy of new molecular entities that can be translated into the clinic for improved patient treatment.

NIH Research Projects · FY 2025 · 2024-05

Patient genome sequencing has revealed germline genetic variation in DDX41 as one of the most frequent genomic alterations implicated in creating a predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). DDX41 encodes an RNA helicase that regulates RNA splicing, senses double-stranded DNA, operates in the cGAS-Sting pathway and promotes innate immunity. Many questions remain regarding DDX41 mechanisms and how genetic variants impact its functions. To gain fundamental and translational insights, we engineered the genome of HoxB8-immortalized murine hematopoietic progenitor cells, which recapitulate the phenotype of primary progenitors, to yield Ddx41+/- cells. We innovated a rescue system to compare human DDX41 activity with that of clinical variants. Using an unbiased genomic strategy, we identified DDX41-regulated mRNAs and membrane proteins as activity metrics. We will use our foundation and machine learning to construct a matrix that informs the relationship between DDX41 and clinical variants of uncertain significance (VUS) or those deemed pathogenic. Although DDX41 represents one of >60 DEAD box domain (DDX) proteins, unifying principles are not established. Aim 1 will innovate a system to discriminate pathogenic from benign human DDX41 clinical genetic variants. Using a prioritization strategy involving genetic variation attributes, an ensemble of variants was assembled for analysis. We will use our DDX41 activity metrics to create a matrix that establishes the functional signature of any variant. This will enable a classification strategy to predict whether a variant resembles DDX41 (“DDX41-like”) or pathogenic variants (“path-DDX41”). Activity metrics will be extended by quantitative proteomics to identify additional DDX41-regulated proteins and advanced RNA-seq analyses to identify transcript isoforms. Loss-of-function and rescue studies will determine if activity metrics can be extrapolated to primary hematopoietic stem/progenitor cells. Aim 2 will conduct pilot/exploratory studies on a mechanism involving DDX41-dependent alternative splicing at a locus encoding an RNA splicing factor-regulatory kinase. Our results revealed that DDX41, but not a pathogenic variant, promotes intron retention in Clk3 RNA, and DDX41 elevates the CDC-like Kinase-3 (CLK3) protein level in myeloid cells. The CLK3 kinase phosphorylates splicing factor (SR) proteins SRSF1-12, some of which are implicated in MDS and AML. We hypothesize that DDX41-induced intron retention and elevated CLK3 protein have important functional consequences. We will test models to explain the consequences of Clk3 intron retention and CLK3 protein elevation. The studies will establish a foundation to understand the DDX41-CLK3 mechanism, which may have considerable physiological and pathological impact. The rules governing DDX41 function and dysfunction that emerge will advance patient genetic curation and mechanistic logic to inform future lines of biological (e.g., hematology- and immunology-focused) and pathological (e.g., bone marrow failure/MDS) investigations in the contexts of erythroid and myeloid biology and more broadly.

NIH Research Projects · FY 2025 · 2024-05

The proposed studies will uncover components of a core neural system that underlies affiliative, rewarding, non-sexual social communication. In cases of depression, anxiety, and autism spectrum disorders, social interactions that are typically rewarding can become aversive. Deficits can also be context-specific. For example, some autistic individuals are able to make directed requests (e.g., for food) that can be extrinsically reinforced (e.g., by receipt of food) but exhibit profound deficits in affiliative communication (e.g., nonsexual, chitchat) that promotes social bonds and is rewarding but does not result in an immediate, obvious extrinsic reinforcer. Many studies focus on mechanisms underlying goal-directed, extrinsically rewarded behaviors (e.g., food-, mate-, or drug-directed); however, these mechanisms appear to differ from those underlying intrinsically rewarded, affiliative social behaviors, leaving a critical gap in basic knowledge about mechanisms underlying non-sexual, affiliative communication. Studies taking advantage of the unique communication properties of songbirds reveal a previously unappreciated role for the medial preoptic nucleus (mPOA) and in particular mu opioid receptors (MORs) in the mPOA in both facilitating and rewarding a form of non-sexual, affiliative singing behavior. Preliminary RNAseq data reveal for the first time a complex of glutamatergic genes in mPOA to be tightly associated with this type of song. The objective of this application is to integrate findings related to the essential role of glutamate and glutamate-MOR interactions in reward to identify foundational, understudied biological mechanisms that underlie affiliative, rewarding communication. The central hypothesis is that glutamate-related genes and MOR-glutamate interactions in the mPOA play critical roles in affiliative communication and other social interactions. The rationale is the need for basic, mechanistic information on core social circuits that underlie behaviors disrupted by mental illness. Based on past and preliminary data, two specific aims are proposed: 1) Determine selective contributions of glutamate-related genes in mPOA to affiliative song, social motivation, and reward; 2) Explore the role of MOR-glutamate interactions in mPOA in affiliative song and reward. In Aim 1 shRNA will be used to knockdown glutamate-related genes SHANK2, GRM5 or GRIN1 in mPOA or a control region, and affiliative song, other social behaviors, social motivation, and social reward will be tested. In Aim 2 effects of the same manipulations on the ability of pharmacological MOR stimulation to facilitate affiliative song as well as reward will be tested. The approach is innovative because it advances the understanding of intrinsically-rewarded social behavior in songbirds with the goal of identifying fundamental, conserved mechanisms. The proposal is significant because it will elucidate the role of glutamate-related genes and MOR-glutamate interactions in non-sexual, affiliative social behaviors and provide insight into core neural circuits that may be disrupted by mental illness in humans.