Washington University

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY This research project will enhance our understanding of the immune responses elicited by the O-antigen of Klebsiella pneumoniae (Kp), an important target in putative vaccine development. The long-term goal of this proposal is to further our knowledge of the innate and adaptive immune responses elicited by two subtypes of the common serogroup O2 of Kp, O2v1 and O2v2. We seek to understand how the addition of the single branched galactose of O2v2 changes its bacterial fitness and host immune susceptibility relative to O2v1. Further, we will determine cross-reactivity between these two subtypes and whether infection or vaccination with one subtype confers cross-protection against the other, information critical to the design of vaccines that can broadly target this increasingly antibiotic-resistant organism. Kp infections, including pneumonia, urinary tract infection, and bloodstream infection, are sharply on the rise among hospitalized patients; CDC has declared that infections with Kp and other carbapenem-resistant Enterobacteriales demand a threat level of urgent. This project builds on the PI's background in bacteriology, murine models of infection, and pathogenesis studies of Gram-negative bacteria, by focusing specifically on the Kp virulence factor O-antigen and the host immunogenic response. We have previously demonstrated that use of bioconjugate vaccines targeting Kp are beneficial in preventing disease and seek to increase our understanding and breadth of coverage of future vaccines. Kp historically has eleven known serogroups of O-antigen. Recently, additional O-antigen subtypes within these serogroups have been identified. Yet, differences in pathogenic fitness, immunogenicity, functional antibody response, and cross-protection between related subtypes are not well understood. We have constructed a mutant of a classical Kp strain expressing O-antigen subtype O2v2 to generate an otherwise isogenic strain expressing O2v1. We will test if these bacteria exhibit similar phenotypic expression of virulence factors by measuring capsule, hypermucoviscosity, fimbriae and biofilm. A well-established murine model of pneumonia will be leveraged to determine pulmonary fitness and delineate host innate immune response to each pathogen. Neutrophil and complement-mediated bacterial killing assays will be employed to further explore bacterial resistance to innate immune attack, which the literature indicates may be enhanced in O2v2 strains. Next, we will perform a range of experiments testing if O2v1 and O2v2 are cross-protective. Using classically immunological techniques including ELISA and flow cytometry, we will determine the IgG subclasses and effector cytokines elicited by respiratory tract infection with these pathogens. Lastly, using novel O2v1 or O2v2 bioconjugate vaccines followed by challenge with O2v1 or O2v2 strains, we will assess cross-protection against weight loss and mortality from pneumonia. We will further characterize antibody functionality using serum bactericidal and opsonophagocytic killing assays. These studies will significantly advance our understanding of immune response to Kp O-antigen and aid in vaccine design to combat this pathogen.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY Preterm infants born between 21 to 30 gestational weeks (GW) have 20-40% chance of developing germinal matrix hemorrhage (GMH), which is a leading cause of neonatal mortality and neurodevelopmental disorders, such as cerebral palsy. Despite decades of research, however, there has been no significant improvement in the prevalence of preterm birth and the mechanism leading to GMH remains unclear. To understand the cause(s) for GMH, we have shown that, during the second trimester, germinal matrix contains enriched populations of Nestin+ radial glia and DCX+ neuroblasts that are fated to become GABAergic interneurons. Furthermore, DCX+ neuroblasts in the germinal matrix are organized as distinct clusters, called DCX-Enriched Nests or DENs, where they expand and migrate to the cerebral cortex and other deep nuclei before becoming mature GABAergic interneurons and integrating into the local neural circuits. To investigate why blood vessels in the germinal matrix are particularly vulnerable to develop GMH, we combined histological and ultrastructural analyses, fluorescence- activated cell sorting (FACS), and single-cell transcriptomics to characterize the properties of nascent blood vessels in the prenatal human brain from 15 to 25 GW. These studies lead to three main conclusions. First, during the second trimester the vascular network in the germinal matrix is much more complex than other brain regions. These nascent blood vessels are tiled by an ensemble of endothelial cells and mural cells, which follow distinct developmental trajectories and use diverse signaling mechanisms to facilitate cell-cell communication and maturation. Second, endothelial cells from younger brain (15-18 GW) exhibit stage-specific transcriptomic and bioenergetic features that are different from those from 20-23 GW. In addition, microglia-vasculature interactions stage-dependently promote angiogenesis in the germinal matrix, but not in the cortical plate. Finally, transcriptomic profiling of CD45+ cells in GMH cases showed that proinflammatory neutrophils and monocytes utilize antibacterial factors and CXCL16-S1PR1 signaling, respectively, to disrupt nascent vasculature in the germinal matrix. Collectively, our results support the overarching hypothesis that proinflammatory neutrophils and monocytes produce cytotoxic factors to disrupt angiogenesis and neurogenesis in the germinal matrix of preterm infants with GMH. To test this hypothesis, we propose to (1) characterize the cytotoxic properties of neutrophil-produced antibacterial factors in disrupting angiogenesis, (2) determine the impacts of CXCL16-S1PR1-mediated signaling in angiogenesis in the germinal matrix, and (3) examine the impacts of GMH on the neurogenesis and migration of GABAergic interneurons. Results from this project will provide important insights into disease mechanism of, and therapeutic targets for, GMH.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Children facing adversity are at high risk for poor socio-emotional and cognitive outcomes and later psychopathology. However, there is increasing evidence that these negative developmental trajectories can be significantly improved by enhancing nurturing caregiving early in development. Despite numerous empirically supported early interventions, these programs are not readily accessible to the majority of US children most in need. Based on this, there is an urgent need to make these interventions feasible and readily available to these communities. To do this we propose to test the effectiveness of a brief 6 session parent-child preventive intervention conducted by video conference in the family's home and study two implementation methods within 3 high-risk school districts. The intervention, entitled “THRIVE,” is a previously tested early parent child intervention piloted in a St. Louis county district which proved feasible, acceptable and appeared effective. Caregiver-child dyads aged 4.0-6.11 meeting inclusion criteria will be randomized to a THRIVE condition or an established online parenting education of comparable length. Although the use of schools as a service delivery platform will increase accessibility, this system presents unique and complex challenges for implementation of a mental health prevention. Therefore, we will use a hybrid effectiveness-implementation design to test the effectiveness of THRIVE compared to Parenting Wisely, while also assessing the impact of two forms of implementation (coaching vs. no coaching) on study outcomes. Assessments of key outcome measures, including child behavior, social, and emotional functioning, child psychopathology, parenting stress, optimism, and depressive symptoms, and changes in parenting and the parent-child dyadic relationship (observational and neural using functional near infrared spectroscopy' fNIRS) will be measured at baseline, post-treatment (P1), and 12-weeks post-treatment (P2). We will also test how the preventive intervention mechanistically targets the quality of the caregiver-child relationship by enhancing caregiver responsiveness and sensitivity via baseline, mid-treatment and post-treatment (P1) assessments. This project provides the first test of a brief parent-child early prevention accessed through schools and delivered in home by video conference with minimal therapist training to enhance access to care. It further examines the cost-effectiveness of therapist coaching as a means to improve implementation and clinical outcomes.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT High-throughput functional genomics of variants in genes linked to substance use disorders Understanding of the genetic basis of substance use disorders has advanced significantly in the recent past; the generation of a reference human sequence in the early 2000’s enabled Genome Wide Association Studies (GWAS), which in turn led to the identification of thousands of genetic variants that are statistically significantly associated with substance use and substance use disorders. The number of these clinically relevant variants continues to grow proportionally with the increase in sample sizes for GWAS and genome sequencing studies of substance use disorders. Model organism studies have also led to the identification of genes and pathways that are linked to these phenotypes. However, one persistent bottleneck in this field has been the lack of experimental testing of nearly all (>99%) of these variants in genes and regulatory regions to determine which are functionally significant. Determining the functional effects of genetic variants is crucial to acquiring a molecular understanding of the basis of substance use disorders, and this deeper understanding will enable future development and testing of targeted and rationally designed therapeutic interventions. This forward- thinking application proposes to address this gap by applying highly innovative high-throughput functional genomics approaches, including Massively Parallel Reporter Assays (MPRAs) and Deep Mutational Scan (DMS) techniques, that were recently deployed for the study of rare Mendelian disorders, to the study thousands of both noncoding and coding variants associated with substance use disorders. These approaches have not previously been applied to the genetics of substance use disorders, although they have enabled significant advancements in other fields, such as the genetics of neurodevelopmental and neuropsychiatric disorders. Furthermore, this early-stage investigator’s multidisciplinary training, including MD/PhD degrees, Pediatrics residency, a postdoctoral fellowship, and four years of dedicated K08 Early Career Development work that was focused on acquiring expertise in the use of functional genomics techniques, has established him as a rising leader in the field of human genetics. His prior paradigm-altering and team-based accomplishments include establishment of an exome sequencing testing pipeline for children with autism in 2015, ongoing coordination of the Model Organism Screening Center for the NIH-funded Undiagnosed Diseases Network, and leadership of a $1.5 million institutionally-funded, multi- investigator functional genomics project that resulted in deployment of critical infrastructure and delivered a 10:1 return on investment in subsequent grant funding. Although this talented investigator has not previously focused on substance use disorders, he has established a collaboration with a world expert in this field, Dr. Laura Bierut, who has agreed to advise him in this research program and connect him with other relevant investigators in the field. In sum, this very high impact and extremely innovative proposal from a particularly well qualified early- stage investigator will significantly advance the understanding of the genetics of substance use disorders.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Chromosomal translocations involving the MLL1 gene often drive infant acute myeloid leukemia (AML). MLL fusion proteins (e.g., MLL-ENL, MLL-AF9, MLL-AF10) activate self-renewal programs in hematopoietic stem and progenitor cells, ultimately leading to transformation. The high frequency of MLL1 rearrangements in infant leukemias suggests that neonatal progenitors are uniquely poised to transform in response to these mutations. Indeed, we have recently shown that MLL-ENL initiates AML more efficiently in neonatal progenitors than in adult progenitors. This raises the question of whether MLL-ENL induces key effectors of transformation more efficiently in neonatal progenitors than in adult progenitors. We identified Ski/Dach Domain Containing 1 (Skida1) as a gene that is highly induced by MLL-ENL in neonatal, but not adult hematopoietic progenitors. SKIDA1 is also highly expressed in human pediatric MLL1 rearranged AML. Expression is largely restricted to leukemias with MLL1 rearrangements. To test whether Skida1 promotes leukemogenesis, we generated a germline loss-of-function mouse allele. Skida1 deletion by itself had negligible effects on normal hematopoiesis, consistent with the lack of expression in normal hematopoietic progenitors. Furthermore, Skida1 deletion did not affect normal HSC function. However, when we induced MLL-ENL expression in Skida1-/- neonates, we observed near complete loss of HSCs and a severe reduction in lineage committed hematopoietic progenitor cells (HPCs). Thus, Skida1-dependence emerges as a consequence of MLL-ENL expression. Next, we generated a conditional loss-of-function mouse allele. Skida1 conditional deletion in the hematopoietic system did not perturb normal hematopoiesis at any age. We are currently crossing the conditional Skida1 mouse to our MLL-ENL mouse model to test whether Skida1 promotes leukemogenesis in the context of MLL-ENL-expressing progenitors. Thus, I hypothesize that Skida1 sustains pre-leukemic HSCs and HPCs and promotes AML during neonatal stages of life. Aim 1 will use a Skida1 conditional knockout mouse to interrogate how Skida1 sustains MLL-ENL-expressing HSCs and HPCs. I will also test whether Skida1 is necessary to maintain fully transformed AML cells. Aim 2 builds upon Aim 1 by adding mechanistic studies to identify Skida1-dependent changes in gene expression and cell fate. I will test whether SKIDA1 binds chromatin to regulate leukemogenesis, and I will identify SKIDA1 binding partners. The aims of this proposal will evaluate SKIDA1 as a potential link between age-specific transcriptional programs and AML initiation, as well as offer a novel therapeutic vulnerability for treating infant leukemia.

NIH Research Projects · FY 2026 · 2023-08

Abstract Mitochondria are complex organelles found in virtually all eukaryotic cells. These organelles orchestrate diverse functions such as energy expenditure, nutrient selection, and ion homeostasis, and do so through the coordination of over 1,000 mitochondria-resident proteins. Most of these mitochondrial proteins are phosphorylated under select physiological conditions, but surprisingly little has been done to characterize these organellar modifications. Motivated by the observation that mitochondria house numerous protein phosphatases, we predicted that regulated protein phosphorylation may play a larger role in organellar homeostasis than is currently appreciated. Indeed, our studies show that the knockout of one mitochondrial phosphatase, Pptc7, leads to stark metabolic dysfunction culminating in fully penetrant perinatal lethality in mice. This surprisingly severe pathophysiology indicates that proper management of protein phosphorylation is requisite for mitochondrial homeostasis. Despite these data, it remains unclear how mitochondrial proteins become phosphorylated and the extent to which individual phosphorylation events contribute to mitochondrial function. We will begin to address these gaps in knowledge by mapping the full breadth of substrates of matrix-localized kinases and by testing the cellular compartment in which mitochondrial-destined proteins become phosphorylated. These studies will begin to address longstanding questions as to the mechanisms enabling mitochondrial protein phosphorylation as well as and the genetic identities of its regulators. To complement this work, we will utilize mechanistic, hypothesis-driven approaches to test the effects of phosphorylation on two proteins, Timm50 and Idh2. These two proteins are reproducibly hyperphosphorylated in Pptc7 KO conditions, suggesting they drive at least a subset of the stark phenotypes associated with the knockout of this phosphatase. Furthermore, these two proteins play key roles in mitochondrial protein import and TCA cycle-mediated metabolism and their regulation would likely have broad influence on mitochondrial function. We will test the effects of Timm50 and Idh2 phosphorylation at the biochemical level (determining how this modification affects protein functions), at the cellular level (determining how modulation of these phosphorylation events affect organellar processes such as protein import and metabolic flux), and at the organismal level (testing how phosphorylation of these proteins may mediate pathophysiology – particularly of phenotypes manifested in Pptc7 KO mice). Collectively, this work will link kinases to mitochondrial function and will establish a workflow to delineate the functions of individual phosphorylation events from the biochemical to the physiological level. As kinases are druggable and have had positive clinical impact in human disease, these studies may uncover novel therapeutic targets through which we can resolve mitochondrial dysfunction found across human pathologies.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract: An antigen test for Neisseria gonorrhoeae (Ng) that is accurate, simple, scalable, non-invasive, rapid, and effective at the point-of-care (POC), would transform public health decisions. Although antigen detection rapid diagnostic tests (Ag-RDT's) for Ng are available, they have poor sensitivity (50% or less) compared to gold standard nucleic acid amplification tests (NAAT). A NAAT based POC test was recently cleared by the US FDA, however its use in resource-poor settings is limited by its high cost. To overcome these barriers for the first time, we will harness ultrabright fluorescent nanoconstructs, called plasmonic-fluors, a recent breakthrough from our labs that enables such antigen testing with sensitivity that matches NAATs. The objective of this proposal is to develop an ultrasensitive plasmon-enhanced lateral flow assay (p-LFA) that detects the lipoprotein H.8 antigen present on Ng surface. The rationale underlying this proposal is that a highly sensitive Ag-RDT that can match the accuracy of NAAT could meet the critical need for diagnosing Ng infection at the POC and in resource-limited settings. We will achieve this by pursuing two specific aims: 1) Develop and optimize the p-LFA for detecting Ng lipoprotein H.8 antigen in urine samples obtained from healthy people. 2) Determine the diagnostic accuracy of the Ng antigen test compared to gold standard NAAT in urine specimens from patients with suspected Ng infection. The proposed approach is innovative in that it harnesses plasmonic-fluor as a fluorescent nanolabel in a LFA as opposed to the conventional gold nanoparticles, which provide only a weak colorimetric signal. With over 1000-fold improvement in sensitivity, this approach has the potential to transform antigen testing. The proposed research is significant as the novel p-LFA will meet the critical needs for widespread Ng testing scalability, speed, and low cost. The expected outcome of this work is a simple, non-invasive, Ag- RDT for Ng infection, implemented as a LFA that can replace NAAT by virtue of its accuracy and low cost in resource-limited settings. This test will have a tremendous positive impact immediately as it will be applicable to large scale Ng testing with an inexpensive test strip and minimal battery-powered portable equipment, without relying on skilled personnel. Furthermore, this test will serve as a prototype for ultrasensitive antigen tests applicable to many other infectious diseases in addition to the possibility of incorporating multiple antigens on a LFA for diagnosing other sexually transmitted infections such as chlamydia and trichomoniasis in addition to Ng.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Children facing adversity are at high risk for poor socio-emotional and cognitive outcomes and later psychopathology. However, there is increasing evidence that these negative developmental trajectories can be significantly improved by enhancing nurturing caregiving early in development. Despite numerous empirically supported early interventions, these programs are not readily accessible to the majority of US children most in need. Based on this, there is an urgent need to make these interventions feasible and readily available to these communities. To do this we propose to test the effectiveness of a brief 6 session parent-child preventive intervention conducted by video conference in the family's home and study two implementation methods within 3 high-risk school districts. The intervention, entitled “THRIVE,” is a previously tested early parent child intervention piloted in a St. Louis county district which proved feasible, acceptable and appeared effective. Caregiver-child dyads aged 4.0-6.11 meeting inclusion criteria will be randomized to a THRIVE condition or an established online parenting education of comparable length. Although the use of schools as a service delivery platform will increase accessibility, this system presents unique and complex challenges for implementation of a mental health prevention. Therefore, we will use a hybrid effectiveness-implementation design to test the effectiveness of THRIVE compared to Parenting Wisely, while also assessing the impact of two forms of implementation (coaching vs. no coaching) on study outcomes. Assessments of key outcome measures, including child behavior, social, and emotional functioning, child psychopathology, parenting stress, optimism, and depressive symptoms, and changes in parenting and the parent-child dyadic relationship (observational and neural using functional near infrared spectroscopy' fNIRS) will be measured at baseline, post-treatment (P1), and 12-weeks post-treatment (P2). We will also test how the preventive intervention mechanistically targets the quality of the caregiver-child relationship by enhancing caregiver responsiveness and sensitivity via baseline, mid-treatment and post-treatment (P1) assessments. This project provides the first test of a brief parent-child early prevention accessed through schools and delivered in home by video conference with minimal therapist training to enhance access to care. It further examines the cost-effectiveness of therapist coaching as a means to improve implementation and clinical outcomes.

NIH Research Projects · FY 2026 · 2023-08

This grant will develop high-performance naturalistic optical functional imaging instrumentation, paradigms, and computational tools for mapping typical and atypical brain development. An exemplar neurodevelopmental disorder, autism spectrum disorder (ASD), affects 1/54 children in the general population. Because early interventions in toddlers with ASD have been proven to result in improved outcomes, innovative methods for early detection of the alterations in brain function underlying ASD prior to manifestation of behavioral symptoms are necessary to advance treatment strategies and improve prognoses. Current brain mapping methods such as functional magnetic resonance imaging (fMRI) offer promising sensitivity to healthy development progression and to atypical ASD brain development, yet pose significant methodological challenges in studies of awake, interacting children due to the loud, claustrophobic environment and the requirement for children to stay still. Further, many imaging paradigms developed for adults are not naturalistic and do not translate well to children Optical neuroimaging, a promising potential surrogate to fMRI, can provide a much more naturalistic imaging experience than MRI. While traditional functional near infrared spectroscopy (fNIRS) systems had poor image quality due to sparse imaging arrays, newer high-density diffuse optical tomography (HD-DOT) systems have improved image quality. However, the large opto-electronic consoles and bulky fiber optics typically used with HD-DOT restrict head motion and require participants to remain stationary to avoid motion induced noise. This grant will develop a unique lightweight HD-DOT system the size of a bike helmet that leverages silicon photomultiplier (SiPM) detection to dramatically improve low light level performance. Naturalistic imaging paradigms aim to recapitulate real-life conditions more closely than traditional reductive protocols (e.g., flashing checkerboard patterns). Ideally, naturalistic paradigms use highly engaging multi-modal content and are particularly well suited for populations (e.g., young children) unable to make overt behavioral responses or perform a repetitive or predictable task. Naturalistic viewing paradigms employing movies or television shows enable repeatability and control over stimulus presentation while preserving greater ecological validity. While feasibility of rudimentary movie regressors have been shown with HD-DOT, the full complexity of movie viewing analyses that has been developed with fMRI has not yet been translated to HD-DOT. To complement movie viewing, we will also advance spontaneous brain activity mapping methods. Functional connectivity analysis of the brain at rest has become a dominant approach to human brain mapping. However, traditional FC analysis rests on bivariate correlation measures that are often susceptible to confounding physiological processes. In contrast, a multi-variate FC (MFC) analysis, developed in this grant for HD-DOT, has the potential to improve the spatial specificity, repeatability, and reliability of the network measures. The movie mapping will complement MFC by providing task localizers, a common feature of modern FC Studies.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Recently, the potential of cellular therapy based upon genetic reprogramming of T cells via CAR-T technology has been explored for diseases of the heart, and the promise embodied in this strategy has captured popular and scientific imagination. However, in current clinical CAR-T cell therapy practice, extracorporeal methods must be applied, which represent a major time and cost barrier restricting wider implementation of this technology. Thus, robust methods to generate CAR-T in situ, within the patients, would greatly facilitate the practical application of this promising approach. For this, we propose to develop a robust gene delivery system consisting of a conjoined “SAd.AAV” nano-vector with multiple T cell-tropic adeno-associated viruses (AAVs) conjugated to a clinically approved, T-cell tropic simian (chimpanzee) adenovirus 36 (SAd) on its capsid's surface. Of note, both capsid-engineered SAd and AAVs are able to selectively target various tissues including T cells with high transduction efficiencies, and we hypothesized that SAd.AAV may therefore provide superior targeting through the combined effects of both engineered viral capsids targeting different receptors on the same tissue or cell type. Importantly, AAVs carrying single-stranded knock-in donor DNA templates have been widely shown to support high editing efficiency of homology directed repair (HDR). Furthermore, the robust but transient expression of gene editor by SAd also provides highly desirable “hit-and-run” gene editing reducing potential adverse effects associated with prolonged editor expression after the on-target editing is achieved. Therefore, by virtue of the advantages of each virus in gene editing, SAd.AAV potentially increases the tissue targeting specificity and efficiency and improves safety of existing CRISPR-Cas gene editing therapies. As a proof of concept, after constructing and characterizing the first T cell-targeted SAd.AAV, we plan to achieve efficient production of the heart disease-specific CAR-T cells in vivo with an SAd.AAV gene editing platform to knock-in a switchable CARi transgene in the T cells in vivo. A proof-of-principle demonstration of in vivo production of gene-edited switchable CARi-T cells will establish a key platform for follow-on studies of CARi-T interventions in murine models of heart diseases. We furthermore anticipate that the SAd.AAV can be rapidly redesigned to target a wide variety of clinically important organs relevant to the NHLBI for gene editing-based therapies.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Astroviruses have been recently recognized as an emerging cause of central nervous system infections in humans, with astrovirus VA1 (VA1) as the most frequently identified astrovirus genotype. We previously described the capacity of VA1 to infect primary human astrocytes and induce expression of inflammatory cytokines like CXCL10, which can directly induce neuronal apoptosis. The histology from the VA1 cases of encephalitis identified neuronal injury and apoptosis, but in cell culture, VA1 cannot replicate in primary human neurons. These findings suggest that VA1 could cause neuronal apoptosis through an indirect mechanism, including infection of astrocytes leading to CXCL10 expression and induction of apoptosis. It is also possible that neuronal cell culture does not reflect necessary conditions for VA1 replication and a better model is needed. To further study the neuropathogenicity, we did not detect significant viral replication in brain tissue from mice inoculated with VA1. In contrast, inoculation of human cerebral organoids (hCOs) with VA1 resulted in a >500-fold increase in viral RNA 72 hours post-inoculation. These results demonstrate the capacity of hCOs to support VA1 replication and now we can leverage this system to better understand cell-to-cell interactions that are important during VA1 infection. hCOs consist of multiple cell types from the central nervous system, including mature/immature neurons, astrocytes, and neural progenitor cells. Our protocol can generate hCOs with robust populations of astrocytes and neurons, enabling cross-talk between cell types. In Aim 1, we will study the cellular tropisms of VA1. We developed a novel fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) assays to detect VA1 that can co-localize with host markers of different cellular lineages. Using FISH and IHC, we will define the cell lineages that support infection by VA1. Based on our previous results, we hypothesize astrocytes but not neurons support VA1 replication. Nonetheless, the hCOs will allow us to determine if other cell types, including neurons, support VA1 infection. In Aim 2, we will determine the consequences of VA1 infection. Given that VA1 induces neuronal apoptosis in vivo, we hypothesize that VA1 will also induce apoptosis of neurons in hCOs. We will identify cells that are undergoing apoptosis after infection by staining for activated caspase-3 and by TUNEL. Cells positive for apoptosis will be co-stained with cell lineage markers to identify the cell types that are undergoing apoptosis. We will also perform single-cell RNA-seq to further describe the cellular response to infection. This analysis will determine if VA1 infection induces expression of inflammatory cytokines, like CXCL10, in hCOs. In addition, RNA-seq will identify if other signaling pathways, including cell death pathways, that are activated in VA1 infected and uninfected cells. By dissecting the tropisms and consequences of VA1 infection in hCOs, this is a novel extension of a previously funded K08 award and will establish an essential tool to understand astrovirus induced neuropathology. This application will also enable further research independence of the primary investigator, achieving the goals set forth under PAR-20-291.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract The overall goal of this research and training plan is to define the molecular mechanisms underlying tauopathies in diverse populations. Currently, the contribution of specific MAPT mutations to tau toxicity, the mechanisms by which tauopathies occur, and the contribution of different genetic backgrounds to these mechanisms remain poorly understood. This project aims to define druggable molecular signatures of MAPT mutations using stem cell models from African (Nigeria), Asian (Japan), and South American (Brazil) populations. The investigator, Dr. Miguel Minaya, will gain advanced training in stem cell biology and functional genomics in support of an innovative approach that establishes novel cell models that use human induced pluripotent stem cell (iPSC)-derived neurons, diverse populations, and CRISPR-based screens to study the extent to which MAPT mutations occurring in diverse genetic backgrounds will produce common and/or unique molecular signatures of disease. The mentors who were selected for this training, Drs. Celeste Karch, Carlos Cruchaga, and Martin Kampmann, are internationally recognized experts in the fields of tau biology, human and molecular genetics, stem cell biology, genome editing, and functional genomic screens using CRISPRi. The goal of this proposal is to define druggable molecular signatures of MAPT mutations using stem cell models from diverse populations. The overarching hypothesis of this proposal is that MAPT mutations occurring in diverse genetic backgrounds will produce common molecular signatures of disease. To define these common mechanisms, I will define molecular changes in iPSC-derived neurons from iPSC lines with engineered MAPT mutations and will then functionally annotate those genes using CRISRPi screens. Through this research and mentored training plan, Dr. Minaya will make fundamental contributions to our knowledge of the mechanisms by which disruptions in the MAPT gene are associated with tauopathies and will establish new experimental tools and approaches that will form the foundation for a career as an independent, translational neuroscientist.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Adult stem cell exhaustion is a hallmark of aging. However, mechanisms of stem cell exhaustion during aging are largely unknown, and there are no therapies that can delay stem cell aging in humans. C. elegans is a premier model organism for studying aging; adult animals are short lived and only contain one stem cell pool, the germline stem cells that generate eggs and sperm. Extensive developmental studies have provided a rich description of the molecular and cellular events that control these stem cells in young animals. My goal is to understand stem cell exhaustion, and my strategy is to exploit the experimental power of C. elegans and the detailed knowledge of stem cell development to elucidate stem cell aging. The somatic distal tip cell (DTC) serves as the stem cell niche by expressing the Notch pathway ligands LAG-2 and APX-1, which bind and activate the GLP-1/Notch receptor in the stem cells. Notch signaling pathways are conserved during evolution and have been repeatedly implicated in regulating stem cells in mammals, suggesting the niche/stem cell system in worms is likely to be broadly relevant. Elucidating the regulatory logic of this system will advance the fields of reproductive aging and stem cell exhaustion. Preliminary results from our lab demonstrate that the number and activity of germline stem cells decline rapidly and progressively with age. Based on these results, I propose two innovative hypotheses. (1) An age-related decline in Notch signaling from the DTC niche causes stem cell exhaustion. (2) Neuronal TGF-β signaling mediates the activity of the DTC niche and contributes to the age- related decline of adult stem cells in the germline. To test these hypotheses, I propose two specific aims. Aim 1: Elucidate mechanisms of Notch pathway regulation during adult stem cell aging in the germline. I will monitor LAG-2 ligand expression in the DTC niche during aging and analyze LAG-2 ligand and Notch receptor function. The results will rigorously test my hypothesis by establishing how LAG-2 ligand expression is regulated during aging and whether LAG-2 ligand and/or GLP-1/Notch receptor are sufficient to sustain stem cell activity during aging. Aim 2: Determine how sensory neurons regulate the DTC niche to mediate germline stem cell aging. I will analyze the DAF-3 binding site in the lag-2 promoter, and the DAF-3 and DAF-5 transcription factors that are the effectors of TGF-β signaling. I will examine multiple levels of organization including protein expression, stem cell dynamics, and progeny production. The results will establish how neuronal signals control the niche and stem cells during aging. The mechanisms of stem cell exhaustion remain mysterious, and these experiments will advance the field by determining the contributions of neurons, the niche, and the stem cells themselves. The results will establish a foundation of knowledge that may stimulate innovative approaches to preserve stem cell function and promote healthy aging in humans.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract The immune system plays an important role during tumor progression, having the ability to recognize and kill tumor cells (T cells and NK cells) [1]. Unfortunately, these immune responses can be bypassed through a variety of mechanisms, including creating an immune suppressive environment that alters the ability of T cells and NK cells to inhibit tumor growth [2]. Major players in suppressing anti-tumor immune responses are immature myeloid populations originating in the bone marrow. Several lines of evidence indicate a correlation between increased numbers of immature myeloid populations in bone marrow, circulation and at tumor site, with disease progression and reduced survival [3, 4]. Altered bone marrow hematopoiesis, with skewing towards myelopoiesis, is indeed observed in cancer patients, regardless of the tumor type and/or dissemination to bone. However, the impact of a primary tumor on bone marrow hematopoietic stem cells (HSC) and the factors involved in changing their frequency and/or functionality remain to be elucidated. Dkk1, a Wnt/β-catenin inhibitor, exerts immune suppressive effects in various cancer types, by either supporting myeloid suppressor populations or inhibiting anti-tumor immune responses [5-7]. However, the mechanisms by which Dkk1 induces the changes in immune populations during tumor progression are not fully understood and evidence of direct effects of Dkk1 on mature immune cells are sparse. Interestingly, Dkk1 is required for hematopoietic stem cell (HSC) regeneration [8], and over expression can promote long-term HSC exhaustion in transplantation models [9]. Currently, the role of Dkk1 on hematopoietic stem and progenitor cells (HSPCs) during cancer progression has not been investigated. My preliminary studies show Dkk1-dependent increases in HSPC frequencies following orthotopic injection of EO771 breast cancer cells, demonstrating systemic effects of a primary tumor on bone marrow hematopoiesis. Based on these observations, this training opportunity will allow me to test the hypothesis that bone derived Dkk1 alters HSPCs frequency and function to increase myelopoiesis and transform the immune landscape during tumor progression. Thus, I propose the following aims: Aim 1: To uncover alterations to HSPCs during breast cancer progression; and Aim 2: To investigate the role of bone derived Dkk1 on HSPCs. These aims will reveal the impact of tumor progression on hematopoiesis and the role of the bone in orchestrating a tumor-conducive, immune suppressive environment. The training received through this proposal will allow me to pursue my interests in bone biology and hematology, strengthen my research skills, and allow me to develop the skill set necessary for an Academic career at the intersection of bone biology, hematology, and immunology.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Phage therapy, the practice of treating bacterial infections with bacteria-targeting viruses, bacteriophage (or phage), is a promising and urgently needed alternative to antibiotics. A major challenge holding this approach back from widespread adoption is that phage treatments need to be customized for the infecting strains in each patient, a slow and labor-intensive process. This requirement arises from the exquisitely narrow host-range that many phages display, even among closely related bacterial strains. A major factor driving phage host range is the immense collection of bacterial anti-phage immune mechanisms that are unevenly distributed across bacterial strains. However, the underlying molecular arms race between bacteria and phage has given rise to an equally impressive set of corresponding phage counter-defense pathways, and thus collectively phage have already evolved mechanisms by which to overcome most bacterial defenses. Similar to their bacterial counterparts, each phage strain encodes only a miniscule fraction of existing counter-defenses, thus explaining the narrow host-range of individual phages. Developing a phage treatment that could amass these naturally occurring phage solutions into a “super phage cocktail” would enable production of an off-the-shelf phage treatment with a greatly expanded species range and the ability to forestall bacterial resistance. Here, I propose developing a pipeline leveraging existing phage counter-defense mechanisms to create a powerful proof-of-principle phage cocktail for the opportunistic pathogen, Pseudomonas aeruginosa. To realize this vision, I will take an experimental genomic approach to map the immune system of clinically relevant P. aeruginosa isolates and thereby determine which bacterial defenses the phage will encounter during infections. I will then develop a powerful, high throughput screen to identify existing phage counter-defense mechanisms that can overcome these bacterial defenses. Finally, I will create a super phage cocktail encoding an extensive collection of counter-defense gene cassettes with the ability to infect a broad set of P. aeruginosa strains. These studies seek to leverage the existing biology underlying the bacterial-phage molecular arms race to overcome a major hurdle in the development of phage therapy. This work will provide unprecedented insight into the breadth and diversity of both bacterial immunity and phage counter-defenses and uncover a multitude of novel biological mechanisms to be characterized in future studies. The engineered phage cocktail also constitutes an innovative experimental system that can be used to answer fundamental questions about viral population diversity and evolution. This initial study will serve as the blue print for development of phage therapy for other multi-drug resistant opportunistic pathogens.

NIH Research Projects · FY 2025 · 2023-07

Many infectious diseases that threaten humans originated among wildlife, yet we know relatively little about the real-world ecological conditions that enable spillover events. Despite its importance, identifying novel viral pathogens and characterizing their transmission dynamics remains difficult because it requires advanced genetic sequencing technologies, sampling wildlife likely to harbor pathogens of concern to humans, and sophisticated modeling techniques. We will study red colobus monkeys in Kibale National Park, Uganda, other nonhuman primates, and people who neighbor these wildlife populations to quantify transmission dynamics within and between species. Our team will collect behavioral ecology data on red colobus monkeys living in areas of the forest with different degrees of anthropogenic disturbance and conduct interviews with people living along the boundary of the park with varying exposure risks for zoonotic diseases. We will conduct repeat sampling of people and individually identifiable red colobus monkeys to analyze the gut virome, assess infection with gastrointestinal parasites known to infect both red colobus and people, discover previously undocumented viral diversity, detect the presence of novel pathogens of concern to humans, red colobus monkeys, and other primates (e.g. SARS-CoV-2), and track the evolutionary spread of detected pathogens. To model how red colobus-associated viruses spread, we will develop new phylodynamic models that allow longitudinal ecological and biogeographical data to structure time-heterogenous epidemiological event rates. We will also create, test, and distribute new software for simulation, Bayesian inference, and deep learning-based inference to model how infectious diseases spread in a wide variety of ecosystem-level transmission scenarios. Our proposed project will benefit public health and wildlife conservation and expand STEM training in the USA and Uganda. Working with Ugandan communities, we will co-create solutions to address risks for zoonotic disease transmission and test mitigation strategies to reduce transmission pathways.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Aneurysmal subarachnoid hemorrhage (SAH) is a highly morbid condition, in large part due to secondary brain injury from Early Brain Injury (EBI) and Delayed Cerebral Ischemia (DCI). EBI occurs 1-3 days after ictus and is characterized by blood brain barrier breakdown, neuroinflammation, and neuronal cell death. DCI occurs 4-12 days after ictus and results from a combination of large artery vasospasm and microcirculatory deficits. Given that EBI and DCI are caused by wide-ranging neurovascular deficits, we believe that effective SAH therapy will require a multiplicity of protective effects to maximize the chance of efficacy. We therefore applied a powerful protection strategy with known pleiotropic effects – Conditioning-based therapy – to experimental models of SAH. Conditioning is a concept whereby the brain's inherent resistance to injury can be enhanced by exposure to non-harmful stress stimuli. Previously, we showed that hypoxic conditioning initiated before SAH (Hypoxic Preconditioning) provides robust protection against DCI in an eNOS-dependent manner. Recently, we extended upon these results in three important ways: 1) We showed that hypoxic conditioning initiated 3h after SAH (Hypoxic Post-Conditioning; HPostC) also produces robust neurovascular protection; 2) We showed that the NAD+-dependent deacetylase, Sirtuin 1 (SIRT1), is a key mediator of this protection; and 3) We showed preliminarily that Nicotinamide phosphoribosyltransferase (NAMPT) is likely a key upstream molecule driving the neurovascular protection afforded by HPostC. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN) enabling biosynthesis of NAD+, which is an essential co-factor of SIRT1 leading to its activation. In the present grant, we will test our central hypothesis is that NAMPT-driven NAD+ production plays a causal role in the neurovascular protection afforded by HPostC in SAH, and that this protection is either partially or completely SIRT1-mediated. The Specific Aims are (1) Test the hypothesis that NAMPT is necessary for the EBI and DCI protection afforded by HPostC in SAH; (2) Test the hypothesis that therapeutic strategies designed to augment NAMPT activity or increase NAD+ levels mimic the EBI and DCI protection afforded by HPostC in SAH; and if so, determine if this protection is partially or completely SIRT1-mediated; and (3) Determine the translational potential of therapeutic strategies targeting NAMPT and NAD+ by assessing their impact on long- term cognitive deficits after SAH. Methods used include: (a) Two complementary mouse models of SAH; (b) Assessment of NAMPT, NAD+, and SIRT1 levels; (c) Assessment of neuroinflammation, neuronal cell death, vasospasm, microcirculatory deficits, and short- and long-term neurobehavioral deficits; (d) Pharmacologic and genetic inhibition of NAMPT and SIRT1; and (e) Pharmacologic and genetic augmentation of NAMPT or NAD+. Overall, the work proposed in the present grant has the potential to identify an entirely new therapies for the treatment of patients with ruptured brain aneurysms – NAMPT activation or NAD+ augmentation. If successful, these studies will result in an improved understanding of the breadth, mechanism, and sustainability of HPostC- induced neurovascular protection in SAH and determine the translatability of NAMPT- and NAD+-directed therapeutics.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Multiple sclerosis (MS) is an immune-mediated, demyelinating, and degenerative disease of the central nervous system and is a leading cause of disability in young people. Most patients begin with a relapsing- remitting (RRMS) course. Clinical relapse and new white matter lesion formation is dramatically reduced by current therapies. However, despite evidently efficacious treatment of RRMS, many patients eventually develop progressive MS (PMS) wherein disability accumulates refractory to treatment. Progressive disability is driven by neurodegeneration which begins early in the disease, occurs independent of relapses, and is poorly understood. Two potential contributors to neurodegeneration are subclinical inflammation and metabolic stress. Clinical relapses and new lesion formation are driven by inflammatory demyelination, but additional inflammation is present outside of discrete lesions. This inflammation is associated with chronic demyelination and increased metabolic demand. Both inflammation and metabolic stress may lead to neurodegeneration, either alone or in combination. This study will use a novel positron emission tomography (PET) imaging agent, 11C-CS1P1, which binds to sphingosine-1-phosphate-receptor 1 (S1PR1) as a marker of inflammation. S1PR1 signaling is critical to MS as evidenced by four FDA approved S1P-modulators with high efficacy for reducing relapses in RRMS. Additionally, we will use metabolic imaging with 18F-FDG PET and advanced, oxygen-sensitive magnetic resonance imaging (MRI) to measure cerebral glucose and oxygen utilization, respectively. These measurements will be made in a cohort of heathy control participants, RRMS patients who will be initiating an S1P-modulating drug and RRMS patients initiating a similarly efficacious non-S1P-modulating drug (B-cell depleting therapies). Patients will be imaged prior to and 3-months following initiation of treatment. These data will allow for testing the hypotheses that subclinical inflammation and metabolic stress are present in RRMS and are incompletely mitigated by current treatments. This study will address three Specific Aims. The first aim tests the hypothesis that inflammation is increased outside of MS lesions in seemingly normal appearing tissue and is reduced by S1P-modulation. The second aim will examine the metabolic needs and stress of normal appearing white matter and gray matter in MS patients compared to controls. The first two aims will compare their respective biomarkers pre- vs. post-initiation of an S1P-modualting drug. The final aim investigates how S1PR1 expression and metabolic stress are modulated by an efficacious, but not S1P-modualting therapy. Completion of this study will provide new understanding of processes that drive neurodegeneration in MS and contribute to clinical morbidity and disability.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Achieving noninvasive, cell-type-specific, and spatially precise neuromodulation remains to be a major challenge in the development of neuromodulation technologies. The objective of this project is to develop Sonogenetics 2.0, the next-generation sonogenetic technique for cell-type-specific, spatially precise neuromodulation in the whole brain of freely behaving animals without intracranial surgery. Sonogenetics 2.0 employs low-intensity focused ultrasound (FUS) combined with microbubbles to deliver intranasally administered adeno-associated viruses (AAVs) to the FUS-targeted brain region with minimal systemic exposure. It then utilizes FUS sonication to remotely activate the expressed ultrasound-sensitive ion channels encoded by the AAVs and thereby controls the activity of AAV-transduced neurons. Sonogenetics 2.0 addresses three critical barriers to developing sonogenetics: the lacks molecular probes with optimized ultrasound sensitivity (Aim 1), requires surgical injection of viral vectors to express the probes (Aim 2), and has a low spatial resolution in delivering ultrasound in the mouse brain (Aim 3). Sonogenetics 2.0 will be independently validated by neuroscience laboratories and benchmarked with optogenetics (Aim 4). The proposed Sonogenetics 2.0 is significant because technological breakthroughs are urgently needed to fulfill the great potential of sonogenetics. Sonogenetics 2.0 provides a complementary tool to existing neuromodulation techniques with the potential to be translated to large animals and humans. A multidisciplinary team with combined expertise in ultrasound device design, ion channel engineering, neuromodulation, and neuroscience is well suited to this project. This project is innovative because Sonogenetics 2.0 is the first-in-class ultrasound tool for completely noninvasive and cell-type-specific neuromodulation by combining noninvasive genetic construct delivery with noninvasive activation of transduced neurons. The proposed research is expected to have a sustained, powerful impact in the research field of sonogenetics and provide the neuroscience community with a transformative tool that can be widely used to advance our current capabilities in investigating cell-type-specific processes in intact mammalian brains.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT Annual influenza vaccination is recommended for patients with ESRD undergoing dialysis by the Advisory Committee on Immunization Practices (ACIP). Non-experimental studies suggest that inactivated, seasonal, standard-dose vaccines are not effective or minimally effective at reducing influenza-related hospitalizations or mortality in the dialysis population, and the high-dose vaccine provides little to no additional protection beyond the standard-dose vaccine for these outcomes. Two vaccine formulations using advanced technologies have been licensed in recent years. The adjuvanted influenza vaccine, licensed in 2015, enhances the magnitude of the immune response to vaccine antigens and broadens the response to include similar mismatched viruses through heterotypic protection. The recombinant influenza vaccine, licensed in 2013 (trivalent) and 2017 (quadrivalent), uses the baculovirus-insect cell system to produce recombinant influenza antigens. Evidence suggests that both the adjuvanted and recombinant influenza vaccines have higher vaccine effectiveness compared to standard-dose vaccines in the general population. Despite increasing uptake of the adjuvanted and recombinant influenza vaccines among the dialysis population in recent years, no large-scale studies have been conducted to evaluate the utilization, effectiveness, or safety of these newly licensed vaccines in the vulnerable population of patients on dialysis. A large study is needed to compare the newly licensed adjuvanted and recombinant vaccines versus other vaccines and to inform influenza prevention approaches in the dialysis population. We propose a large, non-experimental study using existing data from the United States Renal Data System (USRDS) to examine the use, effectiveness, and safety of various influenza vaccines among adult patients with ESRD undergoing dialysis. The large size of our study will allow us to characterize small differences in vaccine effectiveness and safety between adjuvanted or recombinant vaccines vs. standard or high-dose vaccines during the same influenza seasons. Vaccine effectiveness analyses will incorporate a broad range of influenza-related outcomes, including markers of severity of illness. Safety analyses will focus on a priori identified adverse events after influenza immunization. Study design and analyses will use robust methods to measure and account for confounding bias. Our findings will inform dialysis patients and their providers about the risks and benefits of different vaccines and influence vaccine policy, with the ultimate goal of reducing the burden of respiratory disease in this vulnerable population.

NIH Research Projects · FY 2026 · 2023-07

Low back pain is one of the most common disabling conditions in the world. The worldwide prevalence of activity-limiting (acute and chronic) low back pain is approximately 12%, which equates to approximately 933 million people globally suffering with low back pain at any given time. Chronic low back pain (cLBP) refers to pain lasting at least twelve weeks or longer, and it is consistently among the top five most common reasons for primary care physician visits. Although some individuals with cLBP have clear pathoanatomic causes of pain, the vast majority of cLBP is “non-specific” and is not accompanied by readily identifiable pathology of the spine or related tissues. Without a clear target for treatment of cLBP, effective pain management can be difficult to achieve. Because objective measures of disease activity have not consistently been strong predictors of clinical symptoms, cLBP provides an excellent model for investigating the influence of social determinants such as racial background and social stress on the progression of pain and disability over time. During our previous funding cycle, we found evidence that non-Hispanic Black (NHB) individuals with cLBP reported significantly greater pain severity and disability than their non-Hispanic White (NHW) counterparts. In addition, we observed racial differences in pain-related psychosocial and biological measures, which were significantly associated with racial group disparities in cLBP severity. Furthermore, racial disparities in cLBP were exacerbated by low socioeconomic status (SES), such that NHBs with low SES demonstrated the absolute greatest burden of cLBP. While our findings demonstrate clear racial differences in cLBP severity and disability between NHBs and NHWs, the findings are largely indirect and cross-sectional. Differences in how cLBP progresses over time between NHBs and NHWs, as well as factors that contribute to cLBP progression, remain poorly understood. Among the multiple factors that inevitably contribute to racial group differences in cLBP, social stress represents a potentially important social determinant of greater cLBP severity and disability in NHBs. For this proposed follow-up study, we will elucidate the contribution of social stress and its biological consequences (i.e., allostatic load) to racial group disparities in cLBP by prospectively assessing the two-year progression of clinical pain and disability, as well as pain-relevant psychosocial functioning among NHBs and NHWs with cLBP. We will use Social Safety Theory (developed by our co-investigator, Dr. George Slavich) to guide the study aims related to the progression of racial group disparities in cLBP; however, we will also use a recently developed resilience framework to guide inclusion of psychosocial resilience factors that may mitigate the effects of social stress on cLBP in NHBs and/or NHWs.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Conventional prostate adenocarcinoma (PCa) is the second leading cause of cancer death in American men. Patients with organ-confined disease are candidates for potentially curative treatment by either radical prostatectomy or radiation therapy. However, 20-40% of patients undergoing radical prostatectomy and 30-50% of patients undergoing radiation therapy can experience biochemical recurrence within 10 years. These findings indicate that there is suboptimal identification of lethal PCa at the time of diagnosis. Therefore, identification of aggressive disease at the time of diagnosis could stratify patients, develop more effective therapy options, and extend survival. In the clinical setting, noninvasive imaging biomarkers are routinely measured with multiparametric magnetic resonance imaging (mpMRI). However, mpMRI has multiple limitations that result in reduced sensitivity and specificity for PCa, in part from obscuration from inflammatory or stromal cells in the prostate. This proposal advances the use of a clinical magnetic resonance imaging (MRI) sequence, diffusion basis spectral imaging (DBSI), that has the ability to detect structural and cellular changes in the PCa microenvironment (e.g., stroma, inflammation, tumor), that cannot otherwise be determined with conventional mpMRI, a significant advancement. In parallel, our team has discovered a panel of extracellular proteoglycomic biomarkers in lethal forms of PCa (i.e., fucosylated glycans and modified collagens—“FuCol” biomarkers) with Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry imaging of histologic specimens. These molecular markers provide insight into the structural derangements of lethal PCa and because structural changes affect water diffusion, it suggests that these structural changes may actually be detectable with DBSI. We hypothesize that MALDI-detected proteoglycomic markers, expressed as the FuCol score, are associated with structural and metabolic changes in lethal PCa that can be visualized with DBSI to better identify aggressive, potentially lethal PCa at the time of diagnosis. In the first Aim, we will continue to validate our FuCol score as a predictor of disease recurrence and metastasis in a large institutional biorepository. In this Aim, we will investigate the effects of race and diet on the FuCol score and its ability to predict poor outcomes. We will also establish the ability to measure a FuCol score as part of a “noninvasive liquid biopsy” to predict outcomes. In Aim 2, we will enroll a prospective cohort of prostatectomy patients to develop “Diffusion Molecular Imaging (DMI)”; an AI-driven tool that generates in vivo FuCol scores using in vivo DBSI as its input prior to prostatectomy, hence a non-invasive imaging readout of lethal disease. In Aim 3, we will develop an augmented risk prediction model that incorporates novel DBSI imaging, the clinical Decipher genomics platform, and conventional clinical metrics (grade, stage, PSA) to better predict lethal disease at prostatectomy. In summary, these experiments will result in rapid acceleration of a clinically-ready workflow to detect molecular biomarkers associated with poor outcomes. This will dovetail with parallel strategies that our group is developing to treat these cohorts of patients with lethal prostate cancer variants.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The nervous system is an electrical system, and neurons are the archetypal electrically active cell, but ion transport is fundamental to the regulation of every cell in the body, whether excitable or not, and almost 20% of approved drugs actually work on ion channels. Multiple neurological diseases, including movement disorders, migraine, and epilepsy, can clearly be caused directly by inherited mutations of specific ion channels and transporters, but defective ion transport is also associated with every big disease, from cancers to heart disease to Alzheimer's disease. We aim to generate a cohort of researchers with the appropriate in- depth understanding of membrane biology, in combination with state-of-the-art technical skills, to carry out basic and translational research in excitability. The intended result is researchers with the capability to transform excitability research – and hence therapies – by harnessing burgeoning advances in (i) membrane protein structure determination, (ii) measurement of excitability, by electrophysiology and imaging, from molecular to tissue levels, (iii) modeling, both biological and computational and (iv) modulation of excitability, both pharmacologically and physically. The program will support later stage pre-doctoral and post-doctoral trainees in dual-mentored research, with a primary mentor from one of the above fundamental focus areas, and a second mentor who could be from an applied focus area – including methods development or organismal and clinical studies. In addition to intensive research experiences, trainees will participate in a didactic course, geared towards in-depth research understanding, as well as activities devoted to training in grant writing, presentations, and career development. The program faculty mentors, chosen with regard to breadth and diversity of background and career stage, all with strong track records of funding and commitment to trainee development, the interdisciplinary nature of training opportunities, and institutional commitments combine to foster a unique environment suited to the goal of this TriMED program. The program will identify individuals with appropriate backgrounds, who are committed to a career in excitability research, and provide them with mentored pre- and post-doctoral research experiences that will establish a foundation for future careers capable of bringing new insights and tools to bear in neuroscience and disorders of excitability.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ ABSTRACT Deficits in motivated behavior and effort-cost decision-making (ECDM) are core to schizophrenia and related disorders (SZ). ECDM paradigms show that SZ are less likely to use contextual information (e.g., reward magnitude and probability) to drive decisions about whether to exert high effort for rewards. As such, they are less likely to expend effort in situations when it would benefit them the most to do so. These motivational deficits have been closely linked to functional outcomes and their treatment remains ineffective, highlighting the need to examine factors that could enhance ECDM in SZ. Social influence (e.g., the presence of information about peer decisions) has been shown to drive ECDM in healthy individuals (HC), particularly in conditions of low reward or probability (when the other contextual factors may be less likely to independently do so). While there is very little literature examining the role of social influence on effort in SZ, there is some evidence that SZ may increase effort expenditure in response to social encouragement and that (unlike in HC) this may particularly be the case in conditions of high reward magnitude. Thus, social factors could enhance ECDM in SZ by increasing effort in the conditions in which it would be most beneficial to do so-- conditions in which SZ demonstrate the most impairments. Consistent with behavioral findings that SZ are less likely to use reward-related information to inform effortful decisions, they also may show reduced reward-related modulation in neural regions associated with effective ECDM: ventral striatum, ventromedial prefrontal cortex, and anterior cingulate cortex. Interestingly, these regions overlap with those involved in response to peer information and social reward, raising the possibility that social information may modulate decisions about effort in SZ by activating neural systems that play a role in effective ECDM. Further, to understand whether and for whom these laboratory-based neural and behavioral markers of social influence on ECDM might lead to functional benefit in SZ, it is critical to examine how they relate to individual differences in real-world reports of social motivation. Thus, this proposal seeks to use a multimethod framework (e.g., behavioral task, fMRI, EMA) to examine whether social information modulates decisions about effort expenditure in SZ as it does in HC, the neural bases of these decisions, and real-world individual differences in social motivation that are associated with this relationship. Results from the proposed study could elucidate the nature of social and motivational impairments in SZ and inform intervention efforts to ameliorate these impairing deficits. The realization of this project will allow the applicant to receive training in: 1) neuroimaging techniques and analysis, 2) the link between social processes and motivated behavior in SZ, 3) EMA design, collection and analysis, 4) research rigor and reproducibility, and 5) professional development. This project will help the candidate accomplish her goal of becoming an independent principal investigator that uses multimodal approaches to investigate the mechanisms and real-world correlates of social and motivational impairments in SZ.

NIH Research Projects · FY 2026 · 2023-07

Project Summary The long-term goals of this joint project are to understand the mechanisms, role and significance of electrical control of insulin secretion in hyperinsulinism and diabetes. Previous efforts demonstrated the central role of the KATP channel in electrical activity of the pancreatic beta-cell and revealed how defective channel activity can have profound effects on insulin secretion, and cause hyperinsulinism and neonatal diabetes. This understanding now underlies the use of KATP channel activators and inhibitors, respectively, as first line therapies. However, a paradoxical, essentially unexplained, crossover to glucose-insufficiency, or outright diabetes, is seen in both animals and humans with hyperinsulinism. In Type 2 diabetes, a parallel crossover from initial hypersecretion to secretory failure, may reflect a similar progression. Proposed studies will utilize novel inducible KATP channel knockdown models and models of type 2 diabetes to comprehensively probe the mechanistic basis of this reversal, and thereby improve understanding and management of HI and other forms of long-term -cell secretory failure.