University Of Minnesota

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY / ABSTRACT Fetal alcohol spectrum disorders (FASDs) comprise a range of effects from prenatal alcohol exposure (PAE) including neurological abnormalities, cognitive and behavioral impairments, growth retardation, and craniofacial anomalies. Very few treatments have been investigated despite the tremendous public health burden posed by FASD. Cognition is a natural target for intervention because deficits contribute to problems with adaptive functioning, social skills, and independent living. A decade of research suggests that cognitive training has beneficial effects on functional brain networks - mediating cognition in a variety of neurodevelopmental and mental health conditions. We believe it can improve cognitive performance in individuals with FASD. The primary objective of our existing line of neuromodulation research and the proposed RCT is to leverage the ability of tDCS to enhance cortical excitability and neural plasticity in order to maximize the cognitive training benefits in children and adolescents with FASD. Recently we carried out a pilot randomized controlled trial of tDCS-augmented cognitive training to demonstrate the safety and feasibility of this approach in children with FASD (Boroda, Krueger, et al., 2020). We showed that the intervention was safe and well-tolerated in this population. Further, despite applying a relatively low-dose of tDCS and cognitive training, we found that the group receiving active-tDCS demonstrated greater improvements on an attention task than a group receiving sham-tDCS. These promising pilot results merit replication in a larger clinical trial. We propose a fully-powered randomized controlled trial of 60 (at completion) children and adolescents with FASD who will receive cognitive training in combination with tDCS. We hypothesize that, in conjunction with cognitive training, five initial sessions of active tDCS will produce a significantly greater improvement in sustained attention (CPT) than sham tDCS. We also hypothesize that, in conjunction with cognitive training, a total of ten sessions of active tDCS will produce a significantly greater improvement in sustained attention (CPT) than five sessions of active tDCS. We also aim to establish the long-term durability of neurocognitive improvements and associated behavioral changes resulting from tDCS and cognitive training. Finally, we plan to use pre-intervention and post-intervention resting-state fMRI data to show functional network changes related to the intervention and to quantify the relationship between dose and functional network changes. Results of the trial will directly inform future clinical implementation of cognitive training and tDCS as a potential neurodevelopmental intervention.

NIH Research Projects · FY 2026 · 2022-08

Abstract. Human cytomegalovirus (HCMV) is the most common cause of congenital viral infections. HCMV’s ability to infect the placenta plays a central role in its pathogenesis during pregnancy. Placental infection can be sufficient to cause adverse pregnancy outcomes and is likely a prerequisite to congenital cytomegalovirus infection. The placenta is resistant to viral infection in part due to the antiviral activity of trophoblasts. These fetal-derived cells form the physical barrier that separates maternal and fetal circulation and secrete a variety of factors, including type III interferon, exosomes, and antimicrobial peptides, that collectively defend the maternal-fetal interface from infection. However, HCMV can replicate in trophoblasts and may injure the placenta either by directly infecting and killing trophoblasts or by stimulating an injurious maternal or fetal immune response. Two preliminary studies have led us to hypothesize that trophoblast differentiation sensitizes the placenta to infection-associated injury late in gestation. Firstly, we have found that human trophoblast stem cells (TSCs) can be infected by HCMV but are not permissive to replication. Transcriptome profiling revealed that, like other embryonic and multipotent stem cells, TSCs constitutively express a subset of interferon stimulated genes (ISGs). Suspecting that one or more of these factors protect TSCs and their derivatives from HCMV during early differentiation, we will conduct an unbiased CRISPR/Cas9 screen to identify HCMV restriction factors in these cells. Follow up studies will use targeted mutagenesis to study how ISG deletion affects the sensitivity of TSCs, TSC-derived trophoblasts, and trophoblast organoids to infection. Separately, studies in a guinea pig model of congenital cytomegalovirus infection revealed that maternal infection after mid-gestation causes a unique pattern of viral infection in the placenta and a transcriptional response that implicates placental immunopathology as a cause of stillbirth and fetal growth restriction. Infection at an earlier time point had no apparent ill effect on the guinea pig placenta. Thus, we will complete a longitudinal study that examines the effect of maternal cytomegalovirus infection at three different times-- pre/peri-implantation, at the end of the embryonic period, and after mid-gestation--on placental development and function. To complement traditional assays of viral load and placental histopathology, we will use spatial transcriptomics to longitudinally study gene expression in the guinea pig placenta at an unprecedented resolution and elucidate how infection affects the distinct regions of the placenta. Collectively, these studies will reveal how placental susceptibility to viral infection varies across gestation and identify targets for therapeutic interventions that are designed to prevent infection-associated adverse fetal outcomes.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT HIV-1 envelope glycoproteins (Envs) mediate viral entry into host cells and are the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) target highly conserved sites on HIV-1 Envs and neutralize a wide range of diverse strains from different clades. Nevertheless, bnAb immunotherapy aiming to suppress HIV-1 replication sometimes leads to development of bnAb-resistant HIV-1 strains, and HIV-1 strains with pre- existing bnAb resistance can be identified by prescreening before treatment. Thus, understanding the underlying mechanisms of bnAb resistance are critical for the future application of bnAbs for immunotherapy and prevention. Mechanisms that lead to multi-bnAbs resistance and indirect mechanisms that facilitate escape of bnAbs from different groups are of particular public health concern. Our study is structured to provide important insights into bnAb resistance at different levels. In Specific Aim 1 we will study direct resistance mechanisms of rebounded HIV-1 strains that are resistant to multiple bnAbs. We will screen samples from clinical studies of bnAb therapy, identify Envs of HIV-1 strains that exhibit the highest degree of resistance to several bnAbs, study Env sequence, function, glycosylation patterns and determine the structures of resistant Envs at atomic level resolution. Our comprehensive approach will provide unique profiles of selected multi-bnAb resistant Envs that integrate all potential mechanisms contributing to bnAb resistance. In a parallel direction, we will study the ability of rebounded HIV-1 strains to spread through cell-cell transmission, which allows efficient viral replication in the presence of different groups of bnAbs. We will test the hypothesis that bnAb-sensitive HIV-1 strains that replicate despite high levels of bnAb in the serum of participants from the RV397 trial can efficiently spread by cell-cell transmission. Additionally, we will investigate the molecular mechanisms of strains that exhibit increased cell-cell transmission efficiency and bnAb resistance. In Specific Aim 2 we will define optimal bnAb combinations to overcome bnAb resistance and use antibody yeast display technology to bioengineer recombinant bnAbs with improved affinity against bnAb-sensitive and resistant HIV-1 strains. This approach will allow us to confirm mechanisms of HIV-1 resistance to bnAbs and to test the hypothesis that specific changes in bnAbs can improve bnAb breadth and allow targeting of a subset of resistant HIV-1 strains. Overall, our study will provide high-resolution and comprehensive view on multi-bnAb resistant HIV-1 Envs, on alternative pathways of HIV-1 resistance in vivo, and on potential approaches to overcome bnAb resistance. Our results will form a strong basis for the development of new strategies for HIV-1 immunotherapy and prevention efforts.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Adrenoleukodystrophy (ALD) is a progressive X-linked neurological disorder with unpredictable variation in expression. During childhood, about 1 in 3 boys develop rapidly progressing cerebral ALD with brain inflammation and myelin destruction. Although existing treatments stop progression of cerebral ALD when detected at an early stage, no current assessments can identify which boys will develop cerebral ALD or when it will begin. Newborn screening programs that identify ALD at birth offer an opportunity to provide earlier treatment that avoids irreversible neurocognitive decline while new treatments may present safer alternatives to standard stem cell therapy. This K23 Career Development Award aims to provide the PI with the necessary training to become an independent investigator proficient in cross-modal methodologies to measure subtle brain changes in children with ALD and apply those methods in clinical research protocols. With mentorship from a team of experts at the University of Minnesota, the applicant proposes the following training objectives: (1) to obtain knowledge and experience in acquisition and analysis of neuroimaging data; (2) to learn statistical and computational methods for validation of novel neurocognitive tests; and (3) to acquire the expertise to design and execute clinical trials. The overall research objectives are designed to identify early neurocognitive and neuroimaging markers of cerebral demyelination and enhance the capacity of clinical trials of newer therapies to define robust endpoints for boys who receive early treatment. The central hypothesis is that deterioration in white matter fiber integrity and associated loss of interhemispheric function involving the corpus callosum mark the onset of cerebral ALD, and begin prior to the appearance of a lesion on MRI. To test this hypothesis, the PI will test several methods designed to measure changes associated with demyelination. The specific aims will use longitudinal neurocognitive testing (Aim 1) and longitudinal diffusion MRI studies (Aim 2) to identify functional and microstructural markers of cerebral ALD in boys with early stage disease, and test for presence of these markers during the critical pre-emergent stage in at-risk boys. The neurocognitive findings will also be compared to MRI metrics of disease progression. The successful completion of these Aims will identify reliable indicators of disease onset and early progression. The proposed research is significant because detection and quantification of subtle progressive brain changes in boys with ALD will allow earlier and more effective intervention to reduce the neurocognitive morbidity. The training objectives will support the applicant's career goals to lead a long-term program of research to identify sensitive markers of brain dysfunction in neurodevelopmental and neurodegenerative diseases, with the goal of informing timing and efficacy of interventions to achieve better patient outcomes.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT We wish to understand how immunotherapy-induced neurotoxicity occurs. Neurotoxicity is the most pernicious side effect of several immunotherapies for B-cell leukemias and lymphomas, including CAR T-cell therapy. In the latter approach, a sample of the patient’s own T-cells are removed, genetically engineered to recognize B- cell tumors, expanded to large numbers, and then reinfused into the patient. The genetically engineered tumor- recognition component is a chimeric antigen receptor (CAR). CARs reprogram T-cells to recognize and kill tumor cells, regardless of the T-cell’s innate specificity. CAR T-cells specific for the B-cell-associated antigen CD19 can induce durable complete remissions in patients with otherwise terminal B-cell malignancies. Like any therapy, though, it has side-effects. CD19-specific CAR T-cells frequently cause a spectrum of neurological adverse effects (NAE) ranging from disorientation to death. They cannot be prevented or treated adequately because their pathophysiology is poorly understood. To that end, we developed a novel, immune- competent humanized mouse model that replicates the anti-tumor efficacy and toxicities (including NAE) caused clinically by CD19-specific CAR T-cells. In our model, mouse B-cells express a human CD19 transgene (hCD19Tg). Transfer of mouse T-cells – called CART19 cells - that express a hCD19-specific CAR into hCD19Tg mice cause NAEs that are very similar to those experienced clinically. Because our findings mirror clinical reports, we suggest the causes of CART19-induced murine NAE will extrapolate to patients treated with CD19-specific CAR T-cells. Our central hypothesis is that blood brain barrier (BBB) disruption following CART19 infusion permits leukocytes, fluids, and systemic cytokines to enter the central nervous system (CNS). Here these systemic cytokines, and to a greater extent cytokines produced in the CNS by CART19 cells, activate resident microglial cells and extravasated myeloid cells. The differentiation of both into proinflammatory cells ultimately causes NAE. We propose two aims to test these hypotheses. The first aim will reveal what causes BBB dysfunction while the second aim will determine what drives NAE. We will learn how CART19 cells cross the BBB and if their persistent activation in the CNS contributes to or drives NAE. Using genetic, immunological, and pharmacological methods, we also will assess the contributions of resident and extravasated peripheral myeloid cells and specific cytokines to NAE. Finally, we will assess how NAE affects gene and protein expression by brain parenchymal cells using single cell approaches. Our proposed project will significantly impact two areas: 1) basic research in CNS pathobiology as it relates to NAE and 2) translational research as it relates to improving CD19-specific CAR T-cell therapy for human B-cell malignancies.

NIH Research Projects · FY 2026 · 2022-08

HIV cure has proven elusive given the persistence of HIV in tissue sanctuaries including the central nervous system (CNS) and lymphoid follicles in people living with HIV (PLWH). Re-emerging data now indicate that myeloid cells in addition to CD4 T cells represent a key population contributing to HIV persistence in tissues and are a major source of viral rebound after antiretroviral therapy (ART) cessation. A better understanding of myeloid reservoirs are needed in order to guide effective cure strategies. Our long-term goals are to 1) understand HIV/SIV reservoirs in myeloid cells in the CNS, and 2) develop a cellular immunotherapy that targets virus-specific chimeric antigen receptor (CAR) T cells to tissue reservoirs of HIV in the CNS and lymphoid tissues to durably suppress HIV replication. Our emerging data suggests that whilst most vRNA+ cells are CD4 T cells during chronic SIV infections, after analytical antiretroviral treatment interruption (ATI), the majority of vRNA+ recrudescing cells may be of myeloid origin in lymphoid tissues. It is unknown what cell types recrudesce HIV post-ATI in the CNS and this will be crucial if we are to have successful remission. In order to achieve sustained HIV remission our group is currently developing a one-time immunotherapeutic for durable remission of HIV in the absence of ART. This treatment is an autologous HIV-specific CAR (specifically CD4-MBL-CAR) T-cell therapy that targets B cell follicles, and may also penetrate the CNS and target cellular viral reservoirs. B cell follicles and the brain are immune protected sites that may permit viral persistence due to low levels of virus-specific CD8 T cells. We will investigate the capacity of CAR-T-cell immunotherapy to clear SIVmac251 in both CD4 T cells and myeloid cells in lymphoid and CNS reservoirs as this will be crucial for an effective HIV cure strategy either alone or in combination with other immuno and non-immunotherapy based approaches in humans. Therefore we hypothesize that myeloid cells in lymphoid and CNS tissue sites represent the predominant cell population responsible for viral recrudescence and using engineered CAR T cells that express CXCR5 will facilitate T-cell egress into both the CNS and lymphoid follicles and lead to durable remission. These pioneering advances will permit the development of animal model systems for cure research that are challenging and impossible to perform in human studies of the CNS for translatability to human clinical studies. Specifically, we propose to 1) To characterize the source of SIVmac251 rebound virus post-antiretroviral therapy treatment interruption (ATI) in the CNS and peripheral tissues, 2) To determine the location, abundance, and persistence of rhesus CAR/CXCR5 T cells in the CNS and their impact on SIVmac251 cellular reservoirs post ART release, and 3) To determine the ability of rhesus CAR/CXCR5 T cells to kill SIV infected myeloid cells in vitro.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT/PROJECT SUMMARY There is a global emphasis and critical need to close the patient-centered outcomes research (PCOR) evidence to practice gap. Forty percent of patients do not receive evidence-based practice, 20% receive unnecessary or potentially harmful care, and sadly, the list continues. We believe interoperable clinical decision support (CDS) is an indispensable solution to help close this gap; however, poor design, lack of interoperability, and implementation barriers hinder broad adoption. At the University of Minnesota, we have extensive experience implementing and scaling user-centered CDS systems, with over 20 use cases scaled each year. Importantly, we have developed and implemented both interoperable and federally-funded CDS systems. Our healthcare system leverages a rigorous approach, SCALED (SCaling AcceptabLE cDs), to guide CDS scaling across the system. But, the current climate of each healthcare system developing “home-grown” CDS for the exact same guidelines is not tenable. Building capabilities to rapidly translate PCOR to the bedside at scale and share interoperable CDS routinely with an updated knowledge base (living evidence synthesis) is necessary. Given this, we partnered with Apervita, developers of a healthcare technology platform for digital quality measurement and decision support, to develop an interoperable clinical practice guideline leveraging CPG-on-FHIR (Fast Healthcare Interoperability Resources) to prevent inpatient COVID-19 venous thromboembolism (VTE). The proposed R18 project will adapt a currently deployed CDS system to also deliver a VTE prevention guideline for adult patients with traumatic brain injury (TBI). We believe this is an ideal PCOR use case given PCORI’s continued effort to combat VTE in trauma and our experience previously implementing this guideline. Our overall goal is to successfully scale, evaluate, and maintain an interoperable TBI CDS across our 4-institution collaborative network. For Aim 1, we will conduct a Hybrid Type 2 randomized stepped wedge effectiveness- implementation trial to scale the CDS across 4 heterogeneous healthcare systems. Trial outcomes will be assessed using RE-AIM. Despite best efforts, it highly likely CDS adoption will vary across each site; Aim 2 will allow us to understand why. In Aim 2, we will evaluate implementation processes across trial sites guided by the EPIS implementation framework (determinant framework) using mixed-methods. Finally, it is critical that PCOR CPGs are maintained as evidence evolves. To date an accepted process for evidence maintenance does not exist. In Aim 3, we will pilot a “Living Guideline” process model for the VTE prevention CDS systems. Ultimately, this project will scale CDS across a diverse collaborative CDS community serving as an important demonstration of this critical healthcare challenge. We will integrate lessons learned for a planned national scaling in collaboration with engagement of U.S. trauma societies. Importantly, we will develop electronic health record (EHR)-specific IT playbooks for integration of interoperable CDS. Finally, we will pilot an approach for the “Living Guideline” and use that to sustain evidenced-based decision logic.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT The discrepancy between health spending and outcomes in the United States compared to other developed countries suggests persistent inefficiency in the nation's health systems. Current provider payment systems often inadvertently penalize, rather than reward, providers who produce better health outcomes at lower cost. One approach to improving efficiency is to give consumers information about higher-quality, lower-cost providers and financial incentives to choose them. Tiered cost-sharing is one such health insurance benefit design that provides information and incentives to patients, thereby rewarding efficient providers with greater patient volume. This study will examine one of the longest-running tiered cost sharing systems in the United States – the State Employee Group Insurance Program Minnesota Advantage health plan (SEGIP). The SEGIP plan covers 130,000 State employees and their dependents across the state, which includes dense urban centers and remote rural areas. Each year SEGIP employees select a primary care “gatekeeper” clinic that is responsible for coordinating the entirety of their care, including referrals to specialists and hospitals, and pharmaceuticals. Clinics are assessed on their risk-adjusted total cost of care and placed into one of four cost-sharing tiers. The cost-sharing differentials are substantial, giving consumers a strong incentive to choose lower-cost clinics. We have addressed some of the initial questions regarding consumer responses to tiering in research funded by the Robert Wood Johnson and Donaghue Foundations. In this proposal, we move to the next step to investigate responses of clinic leaders to inclusion in a tiered cost-sharing system. We will use both quantitative and qualitative methods as part of a triangulation mixed methods design. Our research will assess what actions clinic leaders have taken in response to inclusion in a tiered cost-sharing system, how they think strategically about tiered cost-sharing, and what barriers they face as they try to practice more efficiently.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Physiological and metabolic health relies on the circadian alignment of biological processes with the environment. Since the first documented study of circadian rhythms in the 18th century using a plant system, model organisms have been critical for defining the transcriptional mechanism of the oscillator and revealing the importance of the clock on fitness. Most of this work is based on whole organism or organ level studies leaving many mechanistic questions about how cell specific gene regulation leads to coordination of cellular clocks and a concerted physiological response. We know that gene regulatory networks are effective at modeling gene expression dynamics but resolving cell-type specific networks with time resolution remains a significant challenge. To delineate regulatory connections across cell-types, a single cell view is needed to develop network models that reflect the true cell state rather than the variation among groups of cells or tissues. Only with these cell specific networks can we begin to develop testable hypotheses about what regulatory variation underlies physiological responses. This proposal describes a research strategy that leverages the genetic and molecular power of the plant model system Arabidopsis thaliana and the latest single cell technologies to 1) Identify the cell-types with distinct oscillators and their underlying regulatory networks, 2) Dissect how cell-type specific circadian regulatory networks maintain a synchronized physiological response, and 3) Perturb cell-type specific circadian gene regulatory networks and evaluate their physiological consequences. Over the next five years, our goals are to delineate single cell circadian gene regulatory networks across an entire organ with spatial resolution. Our Arabidopsis model system will be used to dissect how distinct cell type specific circadian regulation controls a physiological response. Through perturbations to tissue specific gene regulatory networks we will gain a better understanding of how cellular gene programming is coordinated. Our long-term goal is to develop Arabidopsis into a model for understanding how cell specific gene regulatory networks influence inter-tissue communication. This research will generate valuable insight into how we interpret the influence of circadian gene regulatory variation on human health and the application of targeted therapies.

NIH Research Projects · FY 2025 · 2022-08

ADMINISTRATIVE SUPPLEMENT GRANT SUMMARY The Midwest Murine Tissue Mapping Center (MM-TMC) is focused on identifying, characterizing and spatially mapping senescent cells (SnCs) in 5 different murine tissues – adipose, brain, liver, lung and muscle– from two different mouse strains (C57BL/6J and FVB;C57BL/6J F1 mice) with age. To accomplish this, the MM-TMC proposed in the parent application to use sc/snRNAseq analysis for identification and characterization of SnCs at the transcriptional level and IHC/IF at the protein level and 10X Visium Spatial Gene Expression (SGE) and Nanostring GeoMx Digital Spatial Profiler (DSP) for the initial spatial transcriptomic analysis along with a ~600 antibodies for analysis of the proteome. However, neither of these spatial transcriptomics platforms provides single cell resolution. Low-abundance transcripts (~1.8 transcripts/cell) can be identified when a minimum of ~300 cells are spatially profiled, and high abundance transcripts can be identified in as few as ~50 cells. Thus, we recently expanded our spatial mapping efforts to our new CosMx Spatial Molecular Imager with the currently available CosMx Universal Cell Characterization RNA and Neuroscience panels that allow for the profiling of the expression of 1000 curated RNA targets with subcellular resolution. CosMx, like GeoMx, is compatible with a wide range of tissues, including FFPE. However, even though the 1000-plex CosMx panel can be customized by adding up to 50 RNA targets to the panel, the transcriptional profiling is still incomplete, especially when using the panel for analysis of SnCs in a wide spectrum of tissues including brain, adipose, liver, muscle and lung. Thus, as part of this Administrative Supplement, we propose to apply the CosMx Whole Transcriptome Mouse Panel to enable the detection and quantification of over 18,000 RNA transcripts at single-cell and subcellular resolution. Importantly, the MM-TMC has early access to the Mouse Whole Transcriptome Panel in the late Fall of this year that can be pre-ordered at a reduced cost. This unique early access from Bruker will allow for completion of the proposed whole transcriptome spatial analysis in the 1 year time frame of the supplement. The use of the CosMx Whole Transcriptome Mouse Panel will allow for greater identification and characterization of SnCs as well as their paracrine effects on surrounding cells. The analysis also will allow for determination of changes in cell identities or cell population alterations that correlate with age. Finally, the whole transcriptome analysis will allow for a more detailed characterization of the paracrine signaling in regard to ligands and their receptors between SnC and their SASP with neighboring cells. It is also important to note that the results from the CosMx Whole Transcriptome Mouse Panel also will be compared to other platforms being used by the MM-TMC (e.g., Visium, Visium HD, Xenium, GeoMx, and the 1000 RNA CosMx panel) in regard to not only identifying, characterizing and spatially mapping of SnCs, but also for the characterization of the paracrine effects of SnCs on neighboring cells.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract: TRIM-NHL proteins (named after their TRIpartite Motif and founding members NCL-1, HT2A, LIN-41) are essential for animal development and control of stem cell fate. Their dysfunction causes cancer, infertility, and neurological disorders including congenital hydrocephalus. The recent discovery that multiple TRIM-NHL proteins bind to mRNAs provided a crucial insight into their molecular functions. The long term goal of this research is to discover how TRIM-NHL proteins regulate gene expression to control crucial developmental processes. This proposal focuses on the Drosophila melanogaster TRIM-NHL protein, Brain Tumor (Brat), which binds to specific mRNAs and functions in neurogenesis, oogenesis, and embryogenesis. In stem cells, Brat suppresses stemness and promotes differentiation whereas loss-of-function leads to over-proliferation in the brain and germline. We pursue the central hypothesis that Brat negatively regulates gene expression by causing mRNA degradation and inhibiting translation to control stem cell fate. Our research plan will determine the effect of Brat on translation, mRNA decay, and stem cell fate during neurogenesis and oogenesis. A major strength of our work is that it integrates both molecular regulatory mechanisms of Brat and its biological functions at the organismal level. First, using quantitative assays that specifically measure Brat activity, we will dissect its repressive domains and identify the required pathways and corepressors in cultured cells. Second, we will identify the network of genes that are regulated by Brat. We will map Brat-binding sites across the transcriptome and measure Bratʹs effect on the abundance and translation status of expressed mRNAs. By integrating the resulting data, our data will provide a comprehensive view of the relationship between Brat-RNA occupancy, location of functional binding sites, and impact on mRNA stability and translation efficiency. Third, we use precision genome editing to interrogate the roles of RNA-binding and repression domains of the brat gene during neurogenesis and oogenesis in Drosophila. We also created tissue specific reporter gene assays that specifically measure Bratʹs repressive activity in stem cells. The results of this research will provide a mechanistic understanding of Brat mediated gene regulation and provide a global view of its impact on gene expression. Our results will establish the role of Brat’s mRNA regulatory activities in the control of the stem cell proliferation-differentiation axis. Brat serves as an archetype for the TRIM-NHL family, and our discoveries will also broadly enhance the understanding of TRIM-NHL protein functions in development and disease.

NIH Research Projects · FY 2024 · 2022-08

Project Summary Despite being hailed as the “magic bullet” that would selectively target and cure cancer, only a handful of nanoparticles have been successfully translated to the clinic, and their full potential remains yet to be realized. In fact, nanoparticle accumulation in tumors continues to be dismally low, with less than 1% of the injected dose reaching its target. This is largely attributed to the complexity and heterogeneity of both the biological environment and nanoparticle constructs, making it impossible to deconvolute individual factors contributing to nanoparticle targeting and accumulation in tumors. Therefore, there is a critical need to better understand and define the attributes that define successful nanocarriers. This is particularly urgent in lethal cancers that stand to benefit tremendously from new and targeted therapies, like ovarian cancer, which has a 25% 5-year survival rate and 70% recurrence rate following chemotherapy, often leading to treatment resistant disease. To develop effective drug delivery strategies, it is critically important to understand the characteristics of tumors, nanoparticles, and their interactions, such as by identifying the genetic features associated with high nanoparticle uptake and accumulation. To accomplish this, the work proposed herein features a chemical barcoding approach to enable pooled high throughput analysis of nanoparticles in a pre-clinical context, enabling the identification and correlation of genetic features responsible for successful nanoparticle targeting through a multi-omics approach. Successful development of this barcoding platform will entail 1) rapid integrated in vitro screening of pooled NP formulations, 2) in vivo single system evaluation of nanoparticle accumulation at the tissue and cellular level, and 3) use of pooled barcoded nanoparticles to correlate particle trafficking in patient derived models of ovarian cancer. This strategy will provide a holistic evaluation of nanoparticle structure-function relationships with tumor accumulation and enable the identification of genetic components implicated with meaningful nanoparticle interactions, allowing us to leverage these signatures to develop more effective targeted nanoparticles to specific tumor cell populations. The proposed work will take place at MIT’s Koch Institute for Integrative Cancer Research, a premier institution for cancer research with state-of-the-art facilities, under the mentorship of Prof. Paula Hammond, a renowned chemical engineer and polymer chemist with expertise in the self-assembly of materials and drug delivery. An advisory team has carefully been assembled, consisting of Profs. Stuart Schreiber and Angela Koehler for chemical biology and multi-omics analysis guidance, Prof. Joan Brugge for her cancer biology expertise, and Prof. Nathalie Agar for input on mass spectrometry-based analysis. Combined, this research proposal and mentorship team will lay the scientific groundwork and provide the necessary training for the applicant to reach her ultimate goal of successfully starting her independent academic career at the interface of chemistry, biology, and materials science.

NIH Research Projects · FY 2026 · 2022-08

Project Summary This project aims to solve numerous challenges in aryne difunctionalization en route to highly decorated arene rings, critical components of most pharmaceuticals. Currently, methods to functionalize arenes in multiple positions require iterative coupling reactions. Difunctionalization of arynes provides an attractive strategy to incorporate multiple functional groups into an arene ring. Unfortunately, these reactions are plagued with problems with accessing starting materials due to multistep syntheses, limited reaction scope, and poor site selectivity for the two possible positions of the triple bond in an aryne. Our group aims to overcome all of these challenges by using transition metal catalysis to expand the scope and utility of aryne difunctionalization. We plan to use C1 symmetric ligands in order to control regioselectivity of the functional group addition to the aryne. Additionally, by the introduction of transition metal catalysis with new aryne precursors that have previously never been used in catalysis, we will allow access to aryne rings that were previously inaccessible due to ring strain. The transition metal catalyst will be able to bind to the aryne and relieve ring strain. Finally, a wide variety of new difunctionalization reactions are proposed. These new reactions encompass additions of F, N, and CF3 groups due to their importance in medicinal chemistry. If the goals of this proposal are achieved, we anticipate that this chemistry will provide easy access to a wide variety of highly functionalized arene rings. These will provide important building blocks and opportunities for late stage functionalization of medically relevant molecules.

NIH Research Projects · FY 2025 · 2022-08

PROJECT ABSTRACT Thrombosis is the leading cause of mortality among patients with myeloproliferative neoplasms (MPNs). MPNs are characterized by excessive production of red blood cells, platelets, and/or leukocytes. Thrombosis risk in MPNs is thought to be primarily secondary to excess clonal MPN cells. However, at present, the interaction between the vascular endothelium and clonal MPN cells is poorly characterized. Clonal MPN growth is driven by dysregulated Janus kinase-signal transductor and activator of transcription (JAK-STAT) signaling. The JAK2V617F+ mutation occurs in up to 70% of MPN patients and increases the risk of thrombosis 6-fold. Additionally, MPN patients have a higher risk of VTE in slow-flow splanchnic vasculature. Several in vitro and in vivo studies demonstrate that endothelial cells (EC) with the JAK2V617F+ mutation express pro-adhesive and thrombotic proteins, suggesting that EC signaling may contribute to increased thrombosis. My primary objective is to define how EC activation contributes to MPN thrombosis. My central hypothesis is that within the EC vascular, the JAK2V617F+ mutation evokes a pro-inflammatory and thrombotic cascade. In preliminary studies, I evaluated blood outgrowth endothelial cells (BOEC) isolated from JAK2V617F+ patients. In JAK2V617F+ BOECs and in TNF-α-activated JAK2WT ECs, ruxolitinib and fedratinib (JAK1/2 inhibitors approved for use in MPN) reduced tissue factor (TF) expression and activity. Additionally, Compared to JAK2WT ECs, JAK2V617F+ BOECs express higher levels von Willebrand factor (VWF), and growth arrest specific 6 (Gas6) protein. Gas6 is a vitamin-K dependent protein S homolog, which promotes both TF expression and triggers platelet and monocyte activation after binding to receptors Axl, MERTK, and Tyro3. Interestingly, in preliminary studies, JAK2V617F+ individuals had significantly higher plasma levels of Gas6, Axl, and MERTK than controls. Importantly, recent work has shown that blockade of the Gas6-Axl pathway kills JAK2V617F+ hematopoietic stem cells in vitro and reduces spleen size and prolongs survival in JAK2V617F+ mice. However, these studies did not evaluate whether the Gas6-Axl-MERTK axis contributes to MPN thrombosis. Phenotypic variability limits use of JAK2V617F animal models to assess hemostasis and thrombosis. Therefore, I propose to use endothelialized microfluidics models to assess how JAK2V617F expression increases EC activation. Using an endothelialized microfluidics model, I will culture JAK2V617F+ EC under physiologic shear to assess for changes in pro-coagulant and adhesive function. Furthermore, I will assess pro-adhesive and thrombotic interactions between JAK2V617F+ EC and whole blood. I will also explore how Gas6-Axl-MERTK signaling in JAK2V617F+ ECs increases the pro-coagulant and pro-adhesive environment. Collectively, the proposed research will establish the contribution of shear to JAK2V617F+ EC activation and evaluate Gas6-Axl-MERTK signaling in JAK2V617F+ pro-thrombotic activation.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Limbal stem cell (LSC) deficiency is a blinding disease that accounts for an estimated 15-20% of corneal blindness worldwide. LSC deficiency is caused by excessive loss of LSC, a population of pluripotent cells that regenerate the transparent corneal epithelium. Loss of LSC due to chemical injuries and autoimmune disease results in corneal conjunctivalization, erosions, and melting. Treatment options are limited; however, cultured limbal epithelial cell transplantation (CLET) is a promising emerging therapy. In CLET ex vivo expanded limbal stem/progenitor cells (LSPC) are transplanted onto diseased eyes to replace the native LSC and regenerate the corneal epithelium. Short-term success has been reported with CLET; however, long-term outcomes have been limited by loss of transplanted LSPC and recurrence of LSC deficiency over time. One reason for this is the lack of a supportive niche. Under physiological conditions, the microenvironment of the limbus, known as the limbal niche, sustains the pluripotency and proliferative potential of native LSC. However, in LSC deficiency, the limbal niche is often damaged. Identification of synthetic and biological substrates that can function as niche substitutes to support transplanted LSPC remains an ongoing challenge and an unaddressed barrier to long-term success in regenerative therapies for the ocular surface. Although human amniotic membrane (HAM) is the primary substrate used for CLET, it is limited as a long-term niche substitute by its opacity, rapid degradation, and lack of limbus-specific proteins. In contrast Descemet’s Membrane (DM), is a basement membrane on the posterior surface of the cornea is clear and resistant to collagenase digestion. Furthermore, the anterior fetal banded layer of DM is rich in limbus-specific basement membrane proteins, including collagen IV α1, α2 subtypes, vitronectin, and BM40/SPARC. The goal of this study is to compare the stemness and survival of donor and iPSC-derived LSPC on DM vs HAM. In aim 1, we will perform in vitro phenotypic and functional comparisons of LSPC cultured on DM vs HAM using biomarker expression and an organ culture model of LSC deficiency. In aim 2, we will perform an in vivo comparison of cultured LSPC on DM vs HAM using a mouse model of LSC deficiency. In aim 3, we will compare biomarker expression and capacity to regenerate corneal epithelium in iPSC-derived LSPC cultured on DM vs HAM. This project has the potential to inform our choice of substrate in CLET and improve our therapies for LSC deficiency. The training plan will provide the applicant with technical competencies in the characterization of LSPC, use of animal models of LSC deficiency, and manipulation of iPSC; as well as professional skills in oral and written communication to facilitate development as an independent investigator. Training will take place in University of Minnesota’s (UMN) highly collaborative and well-resourced research environment. The applicant will be mentored by Dr. Deborah Ferrington, a leader in applications of iPSC technology in age-related macular degeneration, Dr. Ali Djalilian, a pioneer in therapeutic interventions for limbal stem cell deficiency, and Dr. James Dutton, director of the UMN Stem Cell Institute, Innovation Facilities.

NIH Research Projects · FY 2026 · 2022-08

Project Summary/Abstract Neurobehavioral characteristics have the potential to predict initial weight loss and weight loss maintenance in response to various treatment approaches in adolescents with obesity. However, prior research is limited by heavy reliance on cross-sectional analyses and a focus on immersion-type interventions (e.g., inpatient treatment), which are not widely available. Identifying neurobehavioral predictors of response to clinically applicable treatments will stimulate needed progress in the emerging field of precision medicine. Building on a funded, randomized comparative effectiveness trial (R01-DK123273), the current study will prospectively examine neurobehavioral predictors of initial weight loss response and weight loss maintenance for adolescents with obesity participating in intensive lifestyle modification therapy (LMT) or low intensity LMT plus medical management using the glucagon-like peptide-1 receptor agonist (GLP1-RA), semaglutide. In the proposed study we will: 1) identify baseline neurobehavioral predictors of weight loss response in adolescents with obesity undergoing intervention; 2) for participants achieving >5% BMI reduction at 6 months (i.e. initial responders), identify neurobehavioral predictors of weight loss maintenance at 12; and 3) evaluate differences in predictors of response between intensive LMT and GLP-1RA therapy. This prospective, observational study will include 116 adolescents with obesity and will focus on the three main aspects of executive functions - inhibition, cognitive flexibility, and working memory – as well as reward responsivity using gold-standard measures. For each domain, a single factor (i.e., score) will be created from factor analysis and these latent factors will be used as predictors in the statistical models. To our knowledge, this will be the first study to evaluate neurobehavioral predictors of weight loss response to GLP-1RA therapy in adolescents. Moreover, we will be one of few who have prospectively evaluated neurobehavioral predictors of treatment response and weight loss maintenance for adolescents with obesity. Our overarching goal is to move beyond the current “one-size-fits-all” paradigm and understand how to best match adolescents with obesity to personally effective treatment, which will maximize clinical outcomes while minimizing exposure to unnecessary risks.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY Cancer survivors experience age-related diseases at earlier age than cancer-free persons, suggesting that cancer survivors have accelerated aging. Accelerated aging among cancer survivors could be caused by cancer treatment, but it could also be related to immune response and chronic inflammation. Chronic inflammation is also found in individuals who subsequently develop cancer with subsequent risk of cancer development, and it is possible that accelerated aging starts even before cancer diagnosis. To assess biological aging before and after cancer diagnosis, we will use existing proteomic data and construct and validate proteomic aging clocks in the prospective population-based Atherosclerosis Risk in the Community (ARIC) cohort. The proteins have been already measured three times over 20 years using highly sensitive SomaScan assay. The proteomic aging clocks will be externally validated in the Multi-Ethnic Study of Atherosclerosis (MESA). The strengths of proteomic aging clocks are that these clocks are easily measured, accurately predict biological aging, and are associated with age-related diseases and mortality. However, it is unknown whether proteomic aging clocks predict cancer risk or outcomes in cancer survivors, and there is no agreement about the optimal clock in the context of cancer. To overcome this gap in knowledge, we will construct and validate proteomic aging clocks in ARIC participants who are cancer free but may have other conditions, i.e. these clocks will be cancer-specific proteomic aging clocks (so called, CaPACs). In addition, we will examine the previously published and validated proteomic aging clocks. Our central hypothesis is that accelerated aging is associated with (1) increased cancer risk in persons who are cancer-free at baseline and (2) premature mortality and morbidity among cancer survivors at middle and later age. In the first aim, we will examine the associations between age acceleration for all CaPACs and the risk of overall cancer and specific cancer types in ARIC and MESA. The associations will be also stratified by sex and race. In the second aim, we will examine associations between age acceleration for all CaPACs and all-cause mortality, mortality from causes other than their index cancer, and frailty among cancer survivors in ARIC. In addition, we will compare associations with mortality among cancer survivors and those without cancer history. The use of existing data from the ARIC and MESA studies will allow for quick and cost-efficient testing of our hypothesis. The quantification of proteomic aging in cancer is clinically important because the knowledge of biological age will not only inform risk-stratified cancer screening and post-diagnosis cancer surveillance, but also facilitate the development of anti-aging drugs. 1

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY Astrocytes have been shown to play an important role in synaptic transmission. While there is emerging research about the effects of neurotransmitters on astrocytes, there is little known about how other signaling molecules such as hormones impact astrocyte signaling. One important hormone that is well studied with neuronal signaling is estradiol (E2). This has important relevance in motivated behaviors such as drug addiction where astrocyte signaling and estrogen signaling affect drug seeking behavior. However, it is unknown how estrogen specifically affects astrocyte signaling. This proposal will determine how estrogen affects astrocyte signaling and its subsequent regulation of tripartite synaptic transmission. For this proposal, I will focus on astrocytes in the nucleus accumbens (NAc) as this brain region is critical for drug seeking behaviors and displays structural and functional sex differences. I hypothesize that E2 will increase intracellular calcium in astrocytes by binding to estrogen receptors on astrocytes. This receptor binding will lead to gliotransmitter release which will in turn increase excitatory synaptic transmission. To this test hypothesis, I will use two photon microscopy to examine calcium activity in astrocytes. I will first establish that E2 increases calcium activity in NAc astrocytes (Aim 1a). I will then determine the location and ER subtype as well as cell type in which ER is expressed (Aim 1b). For Aim 2, I will test the effects of E2 on excitatory synaptic transmission. First, I will determine where E2 stimulates gliotransmitter release by measuring slow inward currents, a biological assay of astrocytic glutamate release (Aim 2a). In addition, I will monitor excitatory post synaptic currents (EPSCs) and determine the gliotransmitter responsible for changes in EPSCs (Aim 2b). This proposal will fill the knowledge gap of how estrogen affects astrocyte signaling which could lead to further understanding of sex differences in NAc related behaviors. This proposal will also allow me to master many research techniques such as two photon calcium imaging as well as electrophysiology, which will promote my career goals to become an independent academic neuroscience researcher.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Formaldehyde is a naturally occurring metabolite found in all cell types. Although it has been implicated in human disease including dementia and diabetes, it has also shown to have critical roles in beneficial processes such as memory formation and purine biosynthesis. In methylotrophic bacteria, one-carbon metabolites such as methanol can serve as growth substrates in pathways where formaldehyde is an obligate central intermediate. Due to its high chemical reactivity, formaldehyde balance in these organisms is critical; however, their formaldehyde stress response systems have remained elusive. EfgA and TtmR are central players of two distinct systems that modulate formaldehyde resistance and disrupt formaldehyde homeostasis in the methylotroph Methylorubrum (formerly Methylobacterium) extorquens. EfgA is a newly identified conserved formaldehyde sensor that halts growth and translation in response to elevated formaldehyde levels. TtmR is a MarR-family transcription factor that regulates many genes involved in regulation, signaling, and stress response, including efgA. Our work will characterize the EfgA and TtmR homeostasis systems to understand how cells sense and respond to formaldehyde levels to prevent otherwise inevitable cellular damage. Specifically, we will employ unbiased sequencing-based approaches and experimental evolution to home in on the mechanisms of these systems and define their regulation. Formaldehyde-mediated cellular damage is a readout of the status of formaldehyde homeostasis; however, the in vivo reactivity of formaldehyde is poorly understood. Our data suggests that protein damage is the predominant cause of cytotoxicity in M. extorquens. We will use proteomics approaches to define the impact of formaldehyde on the proteome and identify cellular strategies for counteracting formaldehyde-induced protein damage. Through this work, we will leverage a model bacterium that is well adapted to maintain formaldehyde homeostasis to explore the burgeoning field of formaldehyde regulatory biology. The results from this work will define essential cellular processes and has implications for analogous homeostasis systems for toxic metabolites. We envision this work will have substantial impacts on the understanding of how cells sense and regulate formaldehyde levels, how cells navigate and avoid accumulation of toxic metabolites generally, and how metabolite-specific and global systems of stress response intersect to provide balanced cellular metabolism and growth.

NIH Research Projects · FY 2025 · 2022-07

Project Summary and Abstract Parkinson's disease (PD) symptoms of postural instability and gait disorder (PIGD) cause profound disability and are inadequately treated by current therapies. Deep brain stimulation (DBS) of the subthalamic nucleus (STN) and Globus Pallidus (GP) is effective for PIGD, but not nearly as much as for other PD symptoms e.g. bradykinesia and rigidity. Standard DBS optimization relies on assessing changes in symptoms each time stimulation changes, a very time consuming process. PIGD changes slowly in response to changes in stimulation. Hence, DBS is normally optimized to quicker- responding symptoms, which may be why DBS works better for symptoms other than PIGD. The goal of DBS optimization is to affect neural target structures which best ameliorate symptoms. Different target structures may ameliorate different symptoms. While bradykinesia and rigidity are thought to respond to stimulation of the subthalamopallidal projection, PIGD is thought to involve other structures, especially pedunculopontine nucleus (PPN). PPN afferents and efferents, passing through or near STN & GP may therefore be more effective stimulation targets for PIGD. New directional DBS electrodes, coupled with high field strength MRI imaging and patient-specific computational models, now enable more selective targeting of these pathways. We hypothesize that targeting the ascending PPN efferent pathway will increase the effectiveness of DBS on PIGD, whereas targeting the descending pallidofugal pathway, by inhibiting PPN, will decrease its effectiveness for PIGD. We will compare effects on gait, postural instability, bradykinesia, and rigidity, of DBS targeting pedunculopallidal and pallidopeduncular pathways, directly, as well as subthalamopallidal pathway directly, pallidosubthalamic pathway directly, and pallidopeduncular pathway indirectly (by targeting inhibitory afferents to GP pars interna GPi). Preoperative 7 Tesla structural and diffusion-weighted MRI scans and postoperative high-resolution CT scans will determine electrode location and orientation, relative to nuclei and fiber tracts. Patient-specific models from these imaging datasets will estimate percent activation of each fiber tract, and a novel particle swarm optimization will design stimulation settings maximizing the difference in activation between pathways being compared. Aims 1 (STN) & 2 (GP) compare settings in a single- session, within-subjects experimental design. Aim 3 compares settings with much longer wash- in/ out intervals, and will also collect data between laboratory testing sessions using measures of functional status and quality of life. This project will comprehensively investigate how DBS can be tailored on a patient-specific basis to treat disabling symptoms of PIGD.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY Epilepsy, a condition characterized by chronic, spontaneous seizures, is the fourth most common neurological disorder, affecting nearly 1 in 27 individuals in the United States. In addition to the seizures themselves, chronic epilepsy is associated with cognitive deficits, structural changes, and devastating negative outcomes. For as many as half of all epilepsy patients, seizures are not controlled with current treatment options. There is an urgent need to uncover how chronic epilepsy impacts overall function as well as advance new avenues for therapeutic intervention. Despite not being traditionally associated with epilepsy, the cerebellum is emerging as a potentially critical node in the seizure network; cerebellar structural alterations in epilepsy are associated with comorbidities and negative outcomes, and interventions targeting the cerebellum can powerfully attenuate hippocampal seizures in a mouse model of temporal lobe epilepsy. This bi-directional influence of seizures on the cerebellum and vice versa clearly outline the cerebellum area as a potentially key player in epilepsy, whose yet unknown exact role carries major implications for patients. This proposal leverages a novel technique for mesoscale imaging of the cerebellar cortex to probe how diverse seizure networks alter cerebellar activity, as well as determine whether targeting cerebellar outputs can attenuate seizures arising in structures beyond the hippocampus. By uncovering how the cerebellum interacts with different seizure foci, we will not only gain critical insight into the impact of seizures and chronic epilepsy on cerebellar function, but also potentially uncover new targets for therapeutic modulation in the treatment of different epilepsies. My overarching research interest is to understand computations performed by the cerebellar cortex and how they contribute to healthy behavior and disease. My career goals include becoming an independent researcher studying cerebellar network dynamics and interactions with forebrain structures during motor and non-motor behavior, as well as epilepsy and seizures. My previous training background has given me a solid foundation in electrophysiological recordings, behavioral analyses, optogenetic manipulations, and the implementation of rodent models of epilepsy. However, to fully realize my independent research goals, the training outlined in this K99 will equip me with new skills in experimental techniques and analysis strategies to characterize cellular and network dynamics using calcium imaging. In addition to new technical expertise, this K99 will provide me with a broadened scientific network, improved laboratory management skills, and augmented grant writing expertise. Combined with my prior experience, the additional training and expertise provided by this K99 provides key additions that will help me establish a successful, independent laboratory incorporating innovative, cutting edge research techniques to explore cerebellar computations and their specific contributions to healthy behavior and disease.

NIH Research Projects · FY 2024 · 2022-07

Senescent cells (SnCs) represent an alternative cellular fate resistant to apoptosis that accompanies and characterizes the aging process. Many of these cells, including immunosenescent cells, manifest a highly pro-inflammatory senescence-associated secretory phenotype that may contribute to a vicious cycle of inflammation following an initial stimulus, such as an acute infection, ultimately leading to organ failure and sepsis. Sepsis represents the leading cause of in-hospital and intensive care unit mortality, and older patients suffer disproportionately poor outcomes, both initially and longer term. Novel senolytic drugs such as fisetin, a flavonoid natural product, effectively reduce senescent cells, inflammation, and organ failure in preclinical models of sepsis. However, dosing, drug target engagement, and biological and clinical efficacy remain unknown in human patients. The overarching goal of this project is to advance the science surrounding the therapeutic potential of senolytics in sepsis. To achieve this goal, we will conduct a multi-center adaptive, dose-finding, placebo-controlled, blinded, randomized control trial with three aims. The first aim is to determine the optimally effective dose of fisetin to reduce SnCs in older admitted patients with an acute infection. We will enroll older patients with acute infection not yet requiring mechanical ventilation or vasopressors and randomize to one of several doses of fisetin or placebo using clinically relevant, bolus dosing and test the short (7 day) and medium term (28 day) effect on peripherally measured SnCs. The second aim will test the effect of treatment on peripherally measured inflammation, with a particular focus on pathways affected by SnCs and relevant to sepsis. Finally, the trial will measure the effect on organ failure at 1 week using validated measures and using a Bayesian paradigm to determine the predictive probability of success of a definitive Phase 3 trial. The anticipated impact of this research is high. This project will promote understanding of the relationship of SnCs to sepsis pathophysiology, determine if fisetin effectively modulates these inflammatory pathways in aging individuals, and establish whether further research investment in a definitive trial is warranted.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Understanding the biological basis of aging-related diseases is becoming paramount as the global population ages and the burden of chronic disease weighs on healthcare systems. Cellular senescence has emerged as a unifying feature of multiple age-associated pathologies, having been identified as a significant player in numerous cancers, neurodegenerative diseases, hepatic fibrosis and steatosis, and age associated muscle dysfunction. Senescence is a state of cell cycle arrest that also features mitochondrial dysfunction, lipid accumulation, and a highly inflammatory senescence-associated secretory phenotype (SASP). This SASP is currently thought to be one of the primary causes of senescence-associated tissue dysfunction. The SASP, consisting of cytokines/chemokines, growth factors, proteases, and lipid-derived eicosanoids, promotes chronic inflammation and dysfunction in neighboring cells. To date, research in this area has mainly focused on detrimental effects of secreted SASP proteins, leaving the lipid/eicosanoid contribution to SASP and senescence largely unstudied. Senescent cells characteristically accumulate lipid droplets (LDs), which have recently been identified as central nodes in the inflammatory response and as sites for eicosanoid production. LD metabolism can either contribute to or protect against inflammation depending on the composition of proteins residing on the LD surface. One such protein, Perilipin 2 (PLIN2), has been shown to play pivotal roles in multiple inflammatory conditions and can regulate the efflux of inflammatory lipids from the LD. My primary objective is to elucidate the mechanisms by which LDs and LD proteins modulate senescence, which could allow for specific targeting of this inflammatory process. My central hypothesis is that PLIN2 plays a key role in the initiation and maintenance of SASP and senescence. We will test this hypothesis by establishing a mechanism by which PLIN2 interacts with eicosanoid synthesizing enzymes in senescence and regulates eicosanoid release from the LD. Then we will show that PLIN2 knockout in senescent cells in aged mice attenuates global inflammation, reduces the SASP in senescent cells, and enhances healthspan of aged mice. No study to date has investigated a role for PLIN2 in senescence. This proposal will lay the groundwork establishing PLIN2 and the LD as central to senescence and SASP, thus unveiling a novel mechanism by which senescence contributes to aging.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY The K23 award will support the career development of candidate, Jiwoo Lee, PhD, RN, LSN, to become an independent researcher with expertise in evaluation of public health nutrition programs that will guide future development of childhood obesity prevention interventions designed to reduce weight-related disparities. Dr. Lee will achieve this goal by conducting an innovative research project and career development activities that are aligned with her career goals. The proposed research will evaluate a summertime, community-based child nutrition assistance program, the Summer Food Service Program (SFSP), funded by the United States Department of Agriculture. This program addresses the nutritional gap of increased food insecurity that low- income children experience during the summer months by providing meals to children at program sites. However, nutritional guidelines of the program are not as comprehensive as other nutrition assistance programs, which may contribute to weight gain among children from low-income households who are disproportionately affected by childhood obesity. Additionally, the program sites vary in operation length and the extent to which there is additional healthy programming beyond the provision of foods. Lastly, characteristics of program participants as well as the program’s impact on child diet quality and weight are not well documented. Without this knowledge, it is challenging to characterize what works and for whom and identify targets for optimizing the SFSP and for future interventions. The proposed research will conduct a prospective observational study to describe program site and participant characteristics at baseline to evaluate the current program implementation (Aim 1) and determine if the site and participant characteristics are associated with child program attendance and changes in food insecurity, diet quality and weight gain (Aims 2 and 3). Two cohorts of elementary school-aged children and their parents (n=210) recruited through SFSP sites in Twin Cities metropolitan area of Minnesota will participate in data collection before and in the final weeks of the program (spring and end of summer, respectively). Data collection components include an online psychosocial survey, three 24-hour dietary recalls, and measurement of height, weight and percent body fat. Additionally, parents will report program usage weekly during the program. A multi-disciplinary team of mentors at the University of Minnesota will guide the proposed project and career development activities. Training areas include: a) health equity approaches in reducing food insecurity and weight gain (Drs. Jayne Fulkerson and Lisa Harnack), b) advanced statistical skills for use in evaluation of the long-term impacts of public health programs (Dr. Weihua Guan), c) implementation science and intervention development (Dr. Nancy Sherwood) and d) collaborations with community partners (Drs. Jayne Fulkerson and Lisa Harnack). The project and training plan will position Dr. Lee to advance her career as an independent investigator while contributing to the mission of the NIH by reducing weight-related health disparities of children living in low-income households.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) has become a widespread global pandemic. While the predominant clinical manifestation of severe COVID-19 is respiratory failure, other organ complications such as cardiac injury are common. Cardiac injury and cardiomyopathy are frequent cardiac manifestations during acute illness. Additionally, some survivors of COVID-19 are experiencing cardiopulmonary symptoms months after the acute illness, referred to as Post- Acute Sequelae of SARS-CoV-2 (PASC) or “Long COVID”. Given the frequency of cardiovascular injury during COVID-19 and the persistence of symptoms for extended periods after the acute illness, there is an urgent need for studies of the late effects of SARS-CoV-2 on the cardiovascular system. We aim to investigate the central hypothesis that immune responses to severe COVID-19 cause acute inflammation and injury that result in clinically relevant myocardial fibrosis and dysfunction over the long-term. Since August 2020, we have been enrolling patients in a COVID-19 Immune Profiling (IP) Study, which includes a protocol to collect blood specimens from patients with COVID-19 at admission, during hospitalization, 1-3 months, and 3-12 months after recovery. We will co-enroll participants from this study and perform cardiac magnetic resonance imaging (CMR) and additional functional cardiopulmonary assessments at 3-12 months and 2-3 years after recovery. Our specific aims include, Aim 1) Identify innate immune profiles during severe COVID-19 that predict long- term cardiac damage. We will focus innate immunity measures in blood specimens collected at admission and early recovery, Aim 2) Establish whether adaptive immune responses contribute to cardiac injury after COVID- 19. We will quantify responses targeting SARS-CoV-2 as well as explore maladaptive responses targeting cardiac proteins. Analysis of blood specimens will be supplemented with exploratory studies of cardiac tissue, and Aim 3) Determine the long-term structural and functional cardiac abnormalities after severe COVID-19. This includes characterization of cardiac fibrosis and dysfunction, cardiopulmonary dysfunction, and clinical symptoms. Comparisons will be made with control participants who had influenza infection 1-2 years prior. Our proposal responds to urgent need for science characterizing long-term cardiac complications following COVID- 19. Our collaborative team has extensive experience spanning cardiology, infectious disease, immunology, and epidemiology, and will be led by a cardiologist with expertise in CMR and inflammatory cardiomyopathies, and an infectious diseases specialist with expertise in cardiovascular complications in the context of chronic viral infections. Successful completion of our work will help understand the long-term cardiovascular effects of severe COVID-19 illness. This knowledge could, in turn, help enhance health, lengthen life, and reduce illness and disability in COVID-19 survivors.