Washington University

NIH Research Projects · FY 2024 · 2020-07

Abstract Chronic lower respiratory tract disease is a major cause of morbidity and mortality in the U.S. and worldwide. Currently there are no effective disease-modifying therapies and it remains unclear how to define and optimally treat disease endotypes within the spectrum of asthma and COPD (chronic obstructive pulmonary disease; chronic bronchitis and emphysema). This mechanistic research aims to define new pathways amenable to therapeutic intervention based on the role of diseased airway epithelial cells as an upstream driver of chronic airway disease. As a foundation for this proposal, multiple human clinical and translational studies as well as allergen- smoke- and virus-induced animal models have solidified the relevance of the pathogenic epithelial- derived cytokine IL-33 in COPD and asthma. However, a major knowledge gap that remains is understanding the mechanism by which nuclear-sequestered IL-33 can be activated and secreted from diseased airway cells to drive inflammation. Here we present preliminary data that demonstrates human COPD airway epithelial cells express increased levels of a truncated, spliced IL-33 isoform, which is capable of escaping nuclear sequestration to be abundantly secreted. Our analysis further revealed novel features of this secreted IL-33 isoform including post-translational modification, interaction with exosome-associated chaperones, and utilization of exosome trafficking pathways for secretion. Accordingly, this study aims to elucidate the impact of these newly-discovered features of IL-33 biology on the pathogenesis of chronic airway disease. Aim 1 will define how IL-33 interaction with exosomal chaperones enhances cytokine secretion and receptor activation to drive airway disease, using human cellular and mouse airway disease models coupled with validation in human airway disease specimens. Aim 2 will investigate the role of post-translational modification in augmenting IL-33 secretion and receptor activation to propagate disease, through an analogous approach using human cellular and mouse models with validation in human specimens. Together, these aims will address key steps in the pathologic sequence that initiates and sustains chronic airway disease, illuminating novel ways to target exosome-mediated cytokine secretion at the mucosal interface.

NIH Research Projects · FY 2026 · 2020-07

Project summary Diabetes is characterized by progressive loss or dysfunction of pancreatic insulin- producing β-cells. Previous efforts demonstrated loss of β-cell identity, rather than cell death, as a primary contributor in loss of functional β-cell mass in diabetes progression. Strikingly, this process is reversible, challenging the notion of permanent β-cell damage in diabetes. However, underlying mechanisms remain elusive, and we have poor understanding of the window for intervention to preserve or restore functional β-cell mass. The long-term goals of this project are to understand temporal progression and underlying mechanisms of loss of pancreatic β-cell identity in diabetes. In this proposal we seek to understand β-cell adaptation to metabolic changes and conditions, determine the temporal progression and underlying mechanisms of loss of β-cell mass and identity in diabetes in vivo, and ways to prevent or restore functional β-cell mass. Proposed studies will utilize novel inducible mouse models of monogenic neonatal diabetes and of polygenic type 2 diabetes to understand β-cell adaptation to changes in metabolism, determine the temporal progression and underlying mechanisms of loss of functional β- cell mass and identity in different forms of diabetes, the role of autophagy in this process, and mechanisms to mitigate glucotoxicity-induced β-cell failure. Successful completion of the proposed aims will provide new molecular and mechanistic insights into the adaptive potential of β-cells and will shed light on mechanisms to preserve β-cell mass and identity.

NIH Research Projects · FY 2025 · 2020-06

ABSTRACT: Uropathogenic Escherichia coli (UPEC) is the causative agent of urinary tract infection (UTI) in 75-95% of all cases. UTI is a major health problem in the United States and globally. In the United States, UTls decrease quality of life for 15 million people (mostly women), cause significant economic loss (>S9.7B when including sequelae), and drain our antibiotic arsenal yearly (15% of all antibiotics prescribed in the US go to treating this disease). Sequelae ofUTI include: i) chronic recurrent UTI (rUTI); ii) complications during pregnancy; iii) kidney infection; and iii) sepsis (25% of sepsis cases are traced back to urinary origin. Yearly, over 1 million women in the US are referred to urologists because of rUTI treatment difficulties, which are being exacerbated by the rapid spread of antibiotic resistance in UPEC. Few studies have considered the role of multiple habitats UPEC colonize including the gastrointestinal tract (GIT) and urogenital sites (periurethral area and vagina) prior to entering the bladder and causing UTI. The GIT and vagina have long been considered reservoirs for UPEC, but surprisingly, little is known regarding how UPEC establish and maintain residence throughout and between these body habitats. This proposal uses innovative technologies to investigate UTI holistically, elucidating molecular details of the dynamics of E. coli in its multiple host habitats. Previous work revealed that the microbiota of the GIT is dysbiotic in rUTI patients. E. coli residing within the dysbiotic GIT of rUTI patients have a distinct transcriptional profile, compared to E. coli in the GIT of healthy controls. Importantly, the transcriptional state of a UPEC inoculum matters for bladder infection, suggesting that the GIT transcriptional state may serve to prime successful adaptation of E. coli in the bladder or other host reservoirs. Thus, this proposal investigates the "whole body problem" of rUTI involving multiple host reservoirs of E. coli, which serve as "staging areas" for uropathogens, and the gut-bladder axis, which manifests as dysbiosis in the GIT microbiota influencing the local and systemic immune state. This proposal seeks to: i) characterize the role of the GIT microbial community, inflammation and antibiotic treatment in invoking a distinct uropathogen transcriptomic state observed in rUTI women (Aim 1); ii) assess whether the transcriptional state of E.coli and dysbiotic microbiota that is associated with rUTI result in a differential ability to colonize the bladder (Aim 2); and iii) assess the role of intermediate bacterial urogenital reservoirs, specifically how the vaginal or periurethral microbiotas facilitate or preclude colonization and subsequent transition of E. coli to the bladder (Aim 3). Accomplishing these aims will greatly increase our understanding of rUTI, which afflicts millions of women, giving much needed insight into the role of transcriptional adaptation of E. coli in different reservoirs including the GIT and urogenital sites. This work will reveal how adaptations affect or prime E. coli residence in subsequent reservoirs and/or in the bladder and the role of GIT inflammation and antibiotic treatments on the gut-bladder axis, which will lead to new and better antibiotic-sparing therapeutics.

NIH Research Projects · FY 2024 · 2020-06

The functional architecture of the nervous system is usually thought of in terms of groups of neurons with similar functions being clustered together. However, groups of synaptic connections with similar functions also cluster together resulting in a fine-scale (1-10 μm) functional architecture of nervous tissue. The significance of this fine-scale microcircuitry has been difficult to asses because it can involve dozens of neurons computing signals that are not readily detectable at the level of neuronal cell bodies. Here we propose an approach to studying developmental and functional principles of local microcircuits in the mouse visual thalamus. In the dorsal lateral geniculate nucleus (dLGN), axons from the retina (RGCs) form glia encapsulated clusters of synapses (glomeruli) with thalamocortical relay cells and inhibitory neurons. The functional significance of these local microcircuits is unknown. Based on the diverse patterns of glomerular connectivity we observed in our previous studies of mouse dLGN, we believe these microcircuits are preforming visual-channel-specific computations ranging from simple relay of signals to complex feature detection. We also hypothesize that, central to the organization of these microcircuits, is the use of developmental retinal activity to group functionally related RGCs and inhibitory neurites together in the same glomeruli. Our first step in testing the above hypotheses is to create a detailed mapping of the development of mouse dLGN glomerular microcircuits using serial section electron microscopy that will reveal which cell types initiate glomerulus formation and which aspects of the microcircuit’s connectivity appear during activity dependent synaptic remodeling. We will then test whether visual deprivation or transgenic silencing of subsets of RGC inputs alters the grouping together of neurites or the specificity with which they form synapses. The results of these developmental studies will reveal the extent to which dLGN microcircuit structure is the result of visual experience. We will then probe the function of dLGN glomeruli by first recording the response properties of thalamocortical cells and then reconstructing the glomerular microcircuitry of those same neurons with electron microscopy. By matching receptive field properties to their microcircuit configurations, we will learn whether different glomerular types are specific to different channels of visual processing and will gain insight into the computations likely to be executed by different local microcircuits. We will next use optogenetic stimulation of RGCs to determine how different glomerular configurations integrate signals from converging inputs. The proposed experiments will reveal the origin, organization and function of the fine-scale microcircuitry of an important model system for vision and circuit development.

NIH Research Projects · FY 2025 · 2020-06

This is a competitive renewal of an R01 that initially was funded on validation of the first positron emission tomography (PET) radiotracer, [18F] ASEM, which binds specifically to α7 subtype of nicotinic acetylcholine receptors (α7-nAChR) successfully in human studies. Our development and validation of [18F] ASEM in our prior funding period was critical for future and in the current application we proposed in vivo investigation of α7-nAChR in schizophrenia (SCZ), and cognitive function. Deficits in cognitive function are a core feature of SCZ. People with SCZ show marked deficits in attention, working and long-term memory, and executive functioning. A strong association between low α7-nAChR density and severity of cognitive deficits has been demonstrated in animal models, and with SCZ diagnosis in human post-mortem studies. The overarching goal of the current proposal is to determine α7-nAChR availability in SCZ in vivo in connection with cognitive deficits. In the prior funding period, we established age effects, excellent test/retest reproducibility, and specificity of binding of [18F] ASEM to α7- nAChR. In this renewal, we propose to enroll 60 patients with SCZ and 60 controls matched for smoking status, age, sex, race, and parental socioeconomic status. Subjects will be genotyped for a SCZ-linked polymorphism of α7-nAChR gene (rs3087454) which will be included as a covariate in our analysis. The majority of our planned SCZ subject population is anticipated to be on atypical antipsychotics (AP). To anticipate possible confounds of medication we will test the effects of 3 of the most common atypical APs of our SCZ population in baboons as a SubAim to confirm our no-effect of atypical AP findings in mice and 1 SCZ patient on- and off-Risperidone. Aim 1 We will test the hypothesis demonstrated in post-mortem studies and in our preliminary data, that [18F] ASEM binding will be decreased in SCZ. Effect of AP on [18F] ASEM binding will be tested in baboons as a SubAim1. Aim 2 Because of the high comorbidity of SCZ and tobacco use, and association between severity of cognitive deficits in SCZ and availability of the high-affinity α4β2-nAChR subtype, we will examine in the same SCZ and control subjects for α4β2-nAChR characteristics using our α4β2-nAChR selective PET tracer, [18F] AZAN. Our hypothesis is that [18F] AZAN binding will be lower in the brain regions of smoking participants with SCZ, when compared to matched smoking controls. In Aim 3 we will examine negative symptoms and cognitive deficits using a validated cognitive battery and in clinical scales for SCZ as primary outcomes. We will determine whether cognitive deficits are related to the α7- or the α4β2-nAChR availability, or, possibly, synergistically with these two subtypes. This will be the first study of cognitive deficits in SCZ using two human PET tracers with high selectivity for α7- and α4β2-nAChR. The long-term results is to determine a relationship between these two nAChR subtypes and cognitive function in SCZ, which ultimately will provide a better understanding of nAChR pathophysiology and guidance for future nicotinic drug development for SCZ.

NIH Research Projects · FY 2026 · 2020-05

Overall: Project Summary – 2P30AG066444-06 The Washington University Charles F. and Joanne Knight Alzheimer Disease Research Center (Knight ADRC) initiates, fosters, and supports the performance of innovative, cutting-edge research on Alzheimer disease (AD) and related dementias (ADRD) with regard to the etiology, pathogenesis, diagnosis, treatment, and prevention of disease. We provide well-characterized research participants (persons with symptomatic AD and age-matched controls), their clinical, cognitive, and imaging data, and their biospecimens (DNA, CSF, plasma, dermal fibroblasts, iPSCs, brain tissue) to research projects. We also provide intellectual and financial support to scientists at Washington University, other Alzheimer Disease Research Centers, and the research community nationally and internationally and engage in formal and informal collaborations, including multidisciplinary/multi-Center studies and the initiatives sponsored by the National Institute on Aging and the National Alzheimer Coordinating Center. Historically, our Center has focused on the earliest stages of dementia to identify the initial clinical and pathologic changes that distinguish AD from normal aging. Our approach is balanced between clinical and basic science domains with emphasis on interdisciplinary efforts. We will continue our training of students, fellows, and junior faculty in clinical and basic science research skills. We will continue to engage in outreach activities to transfer information on ADRD to lay and professional audiences. We are committed to assuring that our research cohort includes individuals at higher risk for dementia from the greater metropolitan St. Louis area. This renewal application includes eight Cores, plus the Research Education Component (REC): A: Administrative, B: Clinical, C: Data Management & Statistics, D: Neuropathology, E: Outreach, Recruitment, & Engagement, F: Biomarker, G: Genetics & High Throughput -Omics, and H: Health Disparities Engagement.

NIH Research Projects · FY 2024 · 2020-05

SUMMARY To prepare for the next emerging infection, our goal is to establish a state-of-the-art Emerging Infectious Diseases Research Center with surveillance for the key disease syndromes – respiratory disease, encephalitis and fever of unknown origin– that have been observed most frequently associated with emerging viruses in the past few decades. In parallel, surveillance of animal and insect vectors will be performed to identify the origins of and define transmissions patterns associated with, these novel emerging viruses. The Center includes four international surveillance sites–China, Hong Kong, Nepal and Ethiopia– which were carefully selected on the basis of having either an established history of viral emergence or high potential to capture such events. China, Hong Kong and Nepal are all situated in Southeast Asia, which has historically been a nidus for many emerging viruses such as H5N1 influenza, SARS Coronavirus, Severe Fever and Thrombocytopenia virus, and the very recently reported Alongshan virus. Ethiopia, along with Northeastern Africa, is at high risk for emergence of MERS Coronavirus (MERS-CoV) due to endemicity of MERS in camels, a key reservoir in the region. To identify novel or emerging viruses, we will use complementary virus family- specific consensus PCR and unbiased next generation sequencing approaches and then sequence their complete genomes. Subsequently, we will generate key reagents essential for establishment of diagnostic assays and the study of fundamental aspects of viral pathogenesis, epidemiology, and immune control. These include development of cell culture systems, targeted RT-PCR/PCR assays, serological assays, monoclonal antibodies for antigen detection and potential therapeutic applications, mouse models of infection, and if appropriate, ferret models of virus transmission. An additional component of the center is international capacity building. Initial efforts focus on two exemplar viruses: MERS-CoV, a recently emerged virus, and a highly variant Dengue virus, Dengue virus 5, which is has prevalence and emergence potential. MERS-CoV is a deadly zoonotic respiratory pathogen with a case fatality rate of ~35% to date. We will implement surveillance for these viruses and develop additional reagents and assays to characterize their epidemiology and pathogenic potential. These efforts will establish and validate the critical infrastructure necessary to respond to a new emerging infectious disease. In the event of a new outbreak, efforts will be reprioritized to focus on response to the new emerging threat. The priorities, in order, are: (1) Human and animal surveillance for novel/emerging viruses; (2) Assay and reagent development; (3) Define epidemiology of novel/emerging viruses; (4) Pathogenesis, immune control, transmission and treatment of novel/emerging viruses.

NIH Research Projects · FY 2026 · 2020-05

Project Summary Members of the voltage-gated ion channels (VGICs) are critical for electrical and chemical signaling throughout the three kingdoms of life. Dysfunction of ion channels underlie a wide range of pathophysiology and they are one of the primary targets for new drug development. Although they share a common membrane architecture, the channels in this superfamily exhibit surprising diversity of function. Most open in response to a membrane depolarization but some open on hyperpolarization. Many of them are also polymodal- their activity is regulated by second messengers such as cyclic nucleotide or a physical stimulus such as temperature. The main objective of this proposal is to probe the molecular driving forces in order to understand the fundamental mechanisms of voltage-gating and its modulation by temperature and ligand. Current mechanistic approach tends to be structure focused to the extent that protein dynamics is either ignored or treated as secondary. Although the structures of many highly temperature-sensitive ion channels are now available, our understanding of the mechanism of tem- perature-sensitivity remains limited, in large part, due to our inability to directly probe the molecular forces. To address this issue, we are using a multi-pronged approach that combines new and existing tools to systematically characterize the molecular interactions that determine polarity of voltage-gating, exquisite temperature-sensitiv- ity and unusual allostery in VGICs. We are using the HCN channel as a model system to study gating polarity and ligand activation. Using zero model waveguides and newly developed high-throughput analysis algorithms we were able to probe the cooperativity of ligand binding in a model system. We are now poised to extend these studies to full-length channels and receptors. With regards to mechanisms of gating polarity, we have made a surprising discovery that a bipartite switch regulates gating polarity in HCN channels. Microsecond scale simu- lations in Anton supercomputer suggest a gating model which we will be tested further. We will carry out structural studies and combine it with voltage clamp fluorometry in order to annotate these structures. Next, we will also use ancestral protein reconstruction approach, to identify the deep allosteric networks that regulate gating po- larity in these channels. Our studies on temperature-dependent gating is based on two model systems: a) Tem- perature-sensitive Shaker mutant and, b) archaeal MthK channel. In order to determine the essential elements that are responsible for “sensing” temperature, we have to measure the thermodynamic properties such as heat capacity. We propose to develop a new approach involving single molecule force spectroscopy to extract these energetic parameters. Overall, our “molecular forces” focused approach has the potential to provide unparalleled insights into the mechanisms of voltage gating and its regulation by temperature in VGICs.

NIH Research Projects · FY 2026 · 2020-05

Project summary Peter Mitchell’s chemiosmotic theory postulated that the inner mitochondrial membrane (IMM) must have very low conductance for physiological ions to prevent dissipation of the voltage across the IMM (ΔΨ), which is used by ATP synthase to generate ATP. Interestingly, studies conducted in the 1960s and 1970s suggested that, contrary to the chemiosmotic theory, the IMM does conduct K+ and Cl-. Since then, there has been a significant push to identify and characterize the proteins responsible for these conductances, as their existence in the IMM despite possible ΔΨ dissipation suggests crucial physiological roles. However, even after many decades, the molecular identity, significance, and basic functional properties of the K+ and Cl- conductances of the IMM remain elusive. A major barrier to understanding these conductances was the inability to apply direct electrophysiological methods for their functional identification and characterization. This limitation led to the exclusive use of indirect methods, which produced conflicting results and caused significant confusion. In this project, we successfully applied whole-IMM patch-clamp electrophysiology to achieve the first direct measurement of the mitochondrial K+ and Cl- conductances. We named these conductances the mitochondrial uniporter for monovalent cations (UMC) and the mitochondrial voltage-gated Cl- channel (mClv). Direct electrophysiological analysis helped us identify their basic functional properties and eventually develop high- throughput assays to identify their inhibitors. Using these newly developed pharmacological tools, we demonstrated that UMC boosts mitochondrial capacity for oxidative phosphorylation, and its inhibition leads to an approximately five-fold increase in ATP production. In contrast, mClv supports mitochondrial anionic homeostasis and mitochondrial integrity by extruding excessive anionic substrates and byproducts of the Krebs cycle to the cytosol. In a comparison of mitochondria to an internal combustion engine, mClv is its exhaust system. In this project, we propose to achieve comprehensive electrophysiological characterization of UMC and mClv to define their functional properties. This will be followed by a detailed investigation into their mechanisms of action in intact mitochondria employing a combination of classical methods of mitochondrial bioenergetics (respiration, swelling, and ΔΨ measurements), electron microscopy (including cryo-tomography), and mitochondrial metabolomics. Finally, we will explore various pathways for the molecular identification of UMC and mClv using genetic, omics, and biochemical approaches. Accomplishing these goals is expected to yield transformative discoveries in the field of mitochondrial bioenergetics, advancing our understanding of this organelle beyond the original postulates of the chemiosmotic theory.

NIH Research Projects · FY 2025 · 2020-05

Abstract We are studying two classes of DNA binding proteins, DNA helicases and single stranded (ss)DNA binding (SS8) proteins, both of which are essential for genome maintenance in all organisms. DNA helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA replication, recombination and repair. SS8 proteins bind tightly to these ssDNA intermediates, protecting the DNA, but also bind directly to at least 20 other SS8 interacting proteins (SIPs) to bring them to their sites of action. Defects in DNA helicases are responsible for a number of human diseases. We are undertaking quantitative studies of the mechanisms of DNA unwinding and ssDNA translocation of several superfamily 1, non-hexameric DNA helicases. E. coli Rec8CD functions in repair of DNA double strand breaks, E. coli UvrD and Rep helicases which function in several DNA repair pathways and M. tuberculosis (Mt) UvrD1 helicase. Rec8CD is a hetero-trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (Rec8, a 3' to 5' motor and RecD, a 5' to 3' motor). We have discovered that Rec8CD can unwind duplex DNA processively even in the absence of ssDNA translocation by the canonical Rec8 and RecD motors and that DNA melting and ssDNA translocation are separate processes, with DNA melting and DNA unwinding rates regulated by its nuclease domain. E. coli UvrD, Rep and Mt UvrD1 monomers are rapid ssDNA translocases, but do not possess processive helicase activity due to autoinhibition by a 28 sub-domain. Helicase activity requires activation by dimerization in the absence of accessory proteins. We have shown that the Mt UvrD1 dimerization involves a Cys-Cys disulfide bond between the 28 sub-domains of the two subunits and dimerization is under redox control. We have determined Cryo-EM structures of the UvrD1 dimer bound to a DNA substrate, the first such structures of an SF1 dimer. How this dimer translocates and unwinds DNA will be a focus of our studies. Rep and UvrD monomers can also be activated through interactions with the accessory proteins, PriC and MutL, respectively. Despite extensive study, the mechanism of helicase initiation and DNA unwinding is not well understood for SF1 helicases. We will determine what is needed to turn a ssDNA translocase into a helicase and how this is regulated. E. coli SS8 protein is a central player in all aspects of DNA metabolism. It can bind ssDNA in multiple binding modes that differ dramatically in their properties, in particular ssDNA binding cooperativity. We have shown that the intrinsically disordered C-terminal tails of SS8 regulate cooperative binding of SS8 to ssDNA as well as phase separation of SS8. We have shown that glutamate promotes both cooperative binding to DNA as well as phase separation of SS8 in the absence of DNA. We propose CryoEM studies to examine SS8-DNA cooperativity and the role of the C-terminal tails. An array of approaches, including thermodynamic, transient kinetic, Cryo-EM and single molecule approaches, will be used in these studies.

NIH Research Projects · FY 2026 · 2020-04

PROJECT SUMMARY/ABSTRACT Circadian rhythms are fundamental to proper organismal function, synchronizing gene expression and cellular function across organs to each other and to the external environment. These circadian rhythms are disrupted in Alzheimer’s Disease and other neurodegenerative diseases, leading to disturbed sleep-wake cycles and altered patterns of gene expression and cellular function and exacerbating pathology. In the brain, glial cells exhibit robust circadian rhythms which regulate critical processes such as neuroinflammation, phagocytosis of misfolded proteins, and lipid homeostasis. Moreover, our previous work has shown that Alzheimer’s Disease pathology can disrupt circadian gene expression patterns in glial cells. We hypothesize that manipulating the circadian clock in glial cells might be a powerful way to influence glial function to combat Alzheimer's Disease pathogenesis. Here, we focus on the core circadian clock proteins REV-ERBα and -β, which not only form a critical loop of the circadian clock, but are also nuclear receptors, transcriptional repressors, and metabolic regulators. Our data, collected during the previous funding period, show that REV-ERB proteins modulate several key aspects of glial function and have potent effects on brain homeostasis and Alzheimer’s Disease pathology in mouse models. Since REV-ERBs are nuclear receptors and are thus easily targeted with small- molecule therapeutics, it is important to understand their function in the brain in mechanistic detail, in order to facilitate therapeutic development. While REV-ERBs exert a mix of effects in different cell types of the brain, our data suggest that the inhibition of REV-ERB function in astrocytes, key glial support cells of the brain, holds the greatest promise for maximizing neuroprotective effects. In this renewal, we will focus on the roles of REV- ERBα and -β in astrocytes and how these influence Alzheimer’s-related protein aggregation and pathology in mouse models. In Aim 1, we will elucidate mechanisms by which REV-ERB proteins regulate astrocyte activation state and calcium signaling. In Aim 2, we will investigate the role of astrocyte REV-ERBs in controlling synaptic plasticity through regulation of extracellular matrix components. In Aim 3, we will determine how astrocyte REV-ERB manipulation affects pathology, including tau seeding and spreading, in mouse models of Alzheimer’s Disease. Together, these studies will illuminate novel mechanisms by which the circadian clock can be harnessed through manipulation of REV-ERBs to mitigate Alzheimer’s Disease pathology.

NIH Research Projects · FY 2026 · 2020-04

Abstract Adolescent Brain Cognitive Development (ABCD) is the largest long-term study of brain development and child health in the United States. The ABCD Research Consortium consists of 21 research sites across the country, a Coordinating Center, and a Data Analysis and Informatics Resource Center. In its first five years, under RFA-DA-15-015, ABCD enrolled a diverse sample of 11,878 9-10 year olds from across the consortium, and will track their biological and behavioral development through adolescence into young adulthood. All participants received a comprehensive baseline assessment, including state-of-the-art brain imaging, neuropsychological testing, bioassays, careful assessment of substance use, mental health, physical health, and culture and environment. A similar detailed assessment recurs every 2 years. Interim in-person annual interviews and mid-year telephone or mobile app assessments provide refined temporal resolution of developmental changes and life events that occur over time with minimal burden to participating youth and parents. Intensive efforts are made to keep the vast majority of participants involved with the study through adolescence and beyond, and retention rates thus far are very high. Neuroimaging has expanded our understanding of brain development from childhood into adulthood. Using this and other cutting-edge technologies, ABCD can determine how different kinds of youth experiences (such as sports, school involvement, extracurricular activities, videogames, social media, unhealthy sleep patterns, and vaping) interact with each other and with a child's changing biology to affect brain development and social, behavioral, academic, health, and other outcomes. Data, securely and privately shared with the scientific community, will enable investigators to: (1) describe individual developmental pathways in terms of neural, cognitive, emotional, and academic functioning, and influencing factors; (2) develop national standards of healthy brain development; (3) investigate the roles and interaction of genes and the environment on development; (4) examine how physical activity, sleep, screen time, sports injuries (including traumatic brain injuries), and other experiences influence brain development; (5) determine and replicate factors that influence mental health from childhood to young adulthood; (6) characterize relationships between mental health and substance use; and (7) specify how use of substances such as cannabis, alcohol, tobacco, and caffeine affects developmental outcomes, and how neural, cognitive, emotional, and environmental factors influence the risk for adolescent substance use.

NIH Research Projects · FY 2024 · 2020-04

PROJECT SUMMARY The resistance of PDAC to multiple agents, has been linked in part to its unique tumor microenvironment (TME), which is characterized by a desmoplastic stroma composed of dense collagen-rich extracellular matrix (ECM), abundant and diverse populations of cancer associated fibroblasts (CAFs), and resultant tumor cell hypoxia. PDAC’s fibrotic stroma contributes to poor drug delivery, and deprived infiltration and function of anti- tumor immune cells. These three aspects have been linked to PDAC resistance to both chemo- and immunotherapy. However, it is not clear how the PDAC-associated desmoplasia and fibrosis might impact resistance to RT. Historical studies have focused on radation therapy (RT) as a direct mechanism to damage proliferating tumor cells leading to the accumulation of double-strand DNA breaks and cell death. It is also appreciated that RT can prime anti-tumor immunity by releasing tumor-derived antigens and danger signals, and that this likely plays a critical role in RT efficacy in multiple cancer types. However, it is unclear if these immune priming functions of RT are intact in a highly fibrotic and immunosupressive cancers like PDAC. Previous work from our lab demonstrated that inhibition of Focal Adhesion Kinase (FAK), which is hyper activated in PDAC, reduced tumor-associated fibrosis and thus improves responses to chemo- and checkpoint immunotherapies14,15. These studies have moved to clinical trials with promising early results. However, our recent data suggest FAK can synergize even more effectively with RT plus T cell checkpoint combinations. Based on these data, we hypothesize that fibrotic stroma contributes to PDAC resistance to RT and RT- induction of tumor immunity. To test this we will: Aim 1. Determine the mechanism(s) by which fibrosis impairs RT efficacy and how FAK inhibition overcomes this. Aim 2: Determine if FAK inhibition enhances RT-induced anti-tumor immunity and disease control in locally advanced PDAC patients. Aim 3: Determine the mechanism(s) by which inhibition of FAK signaling improves RT-induced checkpoint immunotherapy response. Impact: Studying how fibrosis negatively impacts RT efficacy in PDAC will further our understanding of how to integrate a stromal targeted agents into current RT regimens with the ultimate goal of improving efficacy of RT.

NIH Research Projects · FY 2026 · 2020-04

PROJECT SUMMARY The overarching goal of this proposal is to understand how two highly homologous epigenetic regulators, MLL3 and MLL4, coordinate critical cell fate decisions during adult blood development. Considerable prior work has gone into understanding the transcription factor networks that coordinate blood development, particularly at the level of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and lineage committed progenitors. These networks are important because they maintain blood and immune system homeostasis, and they touch on essentially every human blood disorder. Like transcription factors, epigenetic regulators are also critical for blood development and homeostasis, as evidenced by the fact that they are frequently mutated in clonal hematopoiesis and various leukemias. However, unlike transcription factors – which often have well- characterized DNA binding motifs, binding partners, and cis-regulatory elements – we currently have only limited insight into how epigenetic regulators are deployed to effect specific cell fate decisions. The case of MLL3 and MLL4 illustrates both the importance of epigenetic regulation in blood development and our limited understanding of their underlying mechanisms. MLL3 and MLL4 each nucleate a multiprotein, chromatin-bound complex called the Complex of Proteins Associated with SET1(COMPASS). MLL3 and MLL4 are highly homologous, and both bind enhancer elements to promote gene expression. However, the proteins have very distinct functions in HSCs and MPPs despite their shared homology. MLL4 maintains HSC self-renewal capacity while opposing myeloid differentiation, whereas MLL3 antagonizes HSC self-renewal while promoting differentiation. These observations create a unique opportunity to learn how structurally similar epigenetic regulators can be selectively recruited to discrete cis-regulatory elements to convey specific hematopoietic fates. In new preliminary studies, we made additional discoveries that shape the aims of this proposal. Specifically, we discovered that MLL3 and MLL4 have redundant roles in licensing HSC identity and myeloid potential despite their apparently antagonistic functions. Inactivating both proteins causes HSCs/MPPs to adopt a B-lymphoid primed state. We propose the following two aims to investigate the underlying causes of these cell fate changes. Aim 1 is to define mechanisms by which MLL3 and MLL4 license HSC identity, as well as mechanisms that account for their distinct functions in HSC self-renewal and differentiation. Aim 2 is to define MLL3/4 COMPASS- independent mechanisms of B-lymphoid development. We will use a combination of genetically engineered mice and various genomic and proteomic techniques to identify cis-regulatory elements, transcription factors and chromatin binding proteins that mediate MLL3- and MLL4-specific cell fates. In each aim, we will focus on understanding the regulatory logic that defines MLL3/4 COMPASS usage or COMPASS independence. The proposed work will create a paradigm for understanding transcription factor/co-factor interplay during blood development.

NIH Research Projects · FY 2026 · 2020-04

Anxiety disorders are the most common pediatric psychiatric illness, affecting up to 30% and severely impairing up to 20% of all youth prior to age 18. Unfortunately, up to 50% of children remain symptomatic even with the best available treatment, making anxiety disorders a major public health problem. Clarifying how the brain develops differently in children who end up developing an anxiety disorder could help us devise new, more effective treatments based on correcting or preventing these underlying brain differences. Prior studies had indicated that anxiety disorders are associated with changes in a part of the brain that directs attention to new, unexpected stimuli (the “ventral attention network”, VAN) and in a part of the brain that is involved in staying focused on current goals and not being distracted (the “fronto-parietal network”, FPN). Problems in the VAN and FPN may decrease the ability of individuals with anxiety disorders to maintain attention on current goals and cause them to instead be distracted by new stimuli that are interpreted as threatening. Work from the original grant (which this current renewal proposes to extend) indicated for the first time these VAN and FPN changes are present in neonates (age < 6 weeks) at high risk for developing an anxiety disorder based on family history or observed infant behaviors at age 1 year. Thus, the altered brain development associated with developing an anxiety disorder may start already by the time of birth. The goal of this proposed renewal is to continue to track the infants from the original study at two more visits: at preschool (3-5 years) and at school- age (6-8 years), thus being able to track how the brain continues to develop up until the time when many of these children are expected to have developed anxiety disorders. At each visit, we will use brain scans to measure VAN and FPN function, perform psychiatric interviews to determine whether an anxiety disorder is present, and observe how parents and children interact together in challenging situations (like doing a hard puzzle together). The goals of the study are to learn (1) how the VAN and FPN develop from birth to age 8 years, (2) how the VAN and FPN develop differently in children who end up having an anxiety disorder, and (3) to learn if there are specific types of interactions between children and their parents that are associated with changes in the VAN and FPN and in the child developing anxiety. Past work has shown that when parent-child interactions include a lot of “parental overcontrol” in which parents are highly controlling, the children are more likely to develop problems with anxiety. Overall, this study represents a time-limited opportunity to follow a group of children from birth up until the age by which many of them are likely to develop an anxiety disorder. Using the results of this study, we hope in future work to design new strategies that might prevent changes in the VAN and FPN and thus reduce risk for children to develop anxiety disorders. These strategies could include teaching parents and children how to interact in challenging situations to minimize parental overcontrol or training children to stay focused and avoid distractions by new, unexpected stimuli.

NIH Research Projects · FY 2025 · 2020-03

PROJECT SUMMARY AND ABSTRACT The RAS genes KRAS, NRAS, or HRAS, are commonly mutated in human cancers. Clinically inhibiting RAS has proven challenging and RAS-mutant cancers remain some of the most intractable diseases, even to immunotherapies. It is thus critical to elucidate oncogenic RAS signaling, not only to better understand the tumorigenic process, but also to identify new potential therapeutic targets. To this end, I exploited the novel technique of BirA-mediated proximity labeling to identify proteins within the immediate vicinity (interactome) of each RAS isoform. I then screened an sgRNA library targeting interactome components for genes promoting RAS transformed cell growth, identifying the druggable phosphatidylinositol phosphate lipid kinase PIP5K1A as specifically driving KRAS oncogenesis. PIP5K1A represents an entirely new therapeutic target in KRAS-mutant cancers, and suggests that other proteins in the RAS interactome may similarly mediate RAS oncogenesis. I will capitalized on these discoveries in three aims. As PIP5K1A is a druggable kinase it provides a way to specifically inhibit KRAS oncogenesis, which could be exploited to enhance the antineoplastic activity of drugs targeting RAS effector pathways. Thus, in aim 1 I will elucidate the role and therapeutic potential of targeting PIP5K1A in KRAS-mutant cancers. The identification of PIP5K1A promoting KRAS oncogenesis suggests that other interactome proteins may similarly mediate RAS function. Thus, in aim 2 I will mine the RAS interactome for novel modifiers of RAS oncogenesis, focusing on the interactome protein EFR3A as a potential general mediator of oncogenic RAS-driven tumorigenesis. Finally, the RAS interactome is most certainly dynamic, varying under different conditions. Determining the content of the RAS interactome under distinct settings may thus identify new vulnerabilities specific to diverse cellular conditions. Thus, in aim 3 I will probe the RAS interactome in response to cellular perturbations. In sum, I will expand upon my discovery that PIP5K1A promotes KRAS oncogenesis to explore this kinase as a new therapeutic target and identify other novel therapeutic vulnerabilities that exists within the RAS interactome. The K99 segment of this grant will complete my training in RAS signal transduction, extend my training into phosphoproteomics, xenograft and genetically engineered mouse models of tumorigenesis. The R00 segment will capitalize on the use of proximity labeling to study the dynamic nature of oncogenic RAS signaling. My long-term goal is to transition into an independent investigator and apply systems biology approaches to uncover the signaling circuitry of oncogene drivers with the objective of identifying novel therapeutic vulnerabilities in RAS-mutant cancers.

NIH Research Projects · FY 2026 · 2020-03

PROJECT SUMMARY/ABSTRACT Current immunotherapy strategies, including immune checkpoint blockade therapy targeting CTLA-4 and/or PD1/PD-L1, have yielded promising results in certain types of cancer patients. However, the overall success rates of these strategies still vary from 15% to 35%, which suggests that there are other mechanisms and/or checkpoint signaling involved that are unresponsive to therapy mediated by malignant tumors. Thus, alternative novel strategies targeting more specific checkpoint molecules or interrupting tolerogenic pathways are urgently needed. It is now well recognized that the suppression and dysfunction of tumor-reactive T cells induced by regulatory T cells (Treg) in the tumor suppressive microenvironment present a major barrier for successful anti-tumor immunotherapy. We recently discovered a novel suppressive mechanism whereby human Treg cells induce senescence in effector T cells that then exhibit potent suppressive activity and amplify immune suppression. Therefore, a better understanding of the cellular and molecular processes that control Treg-induced senescence in effector T cells is essential for the development of effective strategies to treat human cancer. We identified significantly increased activation of the energy sensor AMPK and dys- regulation of lipid metabolism in Treg-induced senescent T cells. Furthermore, ATM-associated DNA damage response and MAPK signaling were selectively involved in T cell senescence mediated by human Treg cells. In addition, we have discovered that human Toll-like receptor 8 (TLR8) signaling reverses the suppressive function and prevents the induction of T cell senescence mediated by both naturally occurring Treg and tumor- derived Treg cells. The central hypotheses of this proposal are that: 1) Human Treg cells can selectively modulate molecular programs that rewrite T cell lipid metabolism in treated naïve/effector T cells, resulting in their differentiation into senescent T cells; 2) Senescent and dysfunctional tumor-specific T cells can be rejuvenated via checkpoint blockages of ATM and MAPK signaling in responder T cells, combined with TLR8 signaling activation in Treg cells, resulting in enhanced anti-tumor immune responses. Specific Aim 1 seeks to identify the molecular mechanism(s) responsible for the induction of senescence and dysfunction in responder T cells after interaction with Treg cells. We will dissect how Treg cells molecularly rewrite effector T cell fate and lipid metabolism. Aim 2 will test the novel concept and strategy that TLR8-mediated reprogramming of glucose metabolism in Treg cells combined with checkpoint blockage of selective MAPK and/or ATM- associated DNA damage signaling in responder T cells can synergistically enhance anti-tumor immunity through reversing the senescence and dysfunction of tumor-specific T cells. A positive outcome of these studies should lead to novel strategies to reprogram Treg metabolism and control the fate and function of tumor-specific T cells for the treatment of human cancers.

NIH Research Projects · FY 2025 · 2020-02

PROJECT SUMMARY Rare, usually private, biallelic pathogenic variants in the ATP binding cassette transporter A3 gene (ABCA3) are the most common monogenic cause of neonatal respiratory failure in term infants and childhood interstitial lung disease (chILD). ABCA3 transports phospholipids required for surfactant assembly and function across the lamellar body membrane in alveolar type 2 cells (AT2s). Broadly, ABCA3 pathogenic missense variants encode mutant proteins that (1) disrupt intracellular trafficking or (2) impair ATP-mediated, phospholipid transport into the lamellar body. However, the mechanism of ABCA3 variant-encoded disruption is not reliably predicted by location in the gene or protein and does not reliably correlate with associated heterogeneous lung disease phenotypes. Lack of neonatal symptoms among many children with chILD due to biallelic ABCA3 variants suggests that variant-specific mechanisms chronically disrupt AT2 cell metabolism and trigger differing lung disease phenotypes. Pathogenic variants in SFTPC, which encodes surfactant protein-C, activate intracellular stress and degradation pathways. However, there are limited data regarding activation of cell stress and proteostasis pathways by ABCA3 variants. Pharmacologic therapies for ABCA3 deficiency remain limited, non-specific, and unpredictably effective, and the 5-year survival for lung transplantation remains stagnant at ~50%. Variant-specific modulator therapies like those developed for individuals with cystic fibrosis due to biallelic variants in CFTR, which encodes another ATP binding cassette transport protein (also known as ABCC7), are needed. Recently, we observed similar cell-based features when ABCA3 pathogenic variants were expressed in either human induced pluripotent stem cell derived AT2 cells (iAT2s) or human pulmonary epithelial cell lines (A549). Further, iAT2 cells that express ABCA3 pathogenic variants demonstrate upregulation of pro-inflammatory pathways and reduced progenitor potential, consistent with an epithelial-intrinsic aberrant phenotype. For efficient characterization of cell stress and proteostasis pathways and for efficient high throughput screening of FDA-approved compounds that rescue ABCA3-encoded disruption, we will use A549 cell lines that stably express individual ABCA3 pathogenic variants. For confirmation of pharmacologic rescue of ABCA3 mutant AT2 cell phenotype, surfactant phospholipid composition, and lamellar body phenotype by screen-identified, FDA-approved compounds, we will use isogenic iAT2 cells edited to express the same ABCA3 variants. These specific aims will test the hypothesis that variant-encoded disruption of ABCA3 trafficking or phospholipid transport activates pathogenic cell stress and proteostasis pathways and can be corrected with FDA-approved compounds.

NIH Research Projects · FY 2026 · 2020-02

Summary During this funding period, we uncovered that the ATR signaling pathway is essential for safeguarding single- stranded (ss)-DNA gaps from being degraded by nucleases, as well as facilitating their effective repair when cancer cells undergo transient treatment with PARP inhibitors (PARPi). Moreover, we found that ssDNA gaps fail to be repaired in conditions where PARPi treatment is continuous, leading to their collision with DNA replication forks and the subsequent breaking of these forks. These broken forks are irreparable in cancer cells lacking the function of the breast cancer susceptibility genes BRCA1 and BRCA2. However, they are repaired by BRCA-proficient cells, indicating the presence of a fork recovery pathway that is absent in BRCA-deficient cells and whose absence contributes to the susceptibility of BRCA mutant cancers to PARPi. Based on these findings, we propose that the ATR signaling pathway has a previously underrecognized function in controlling the processing and repair of ssDNA gaps in cells treated with PARPi. The first aim will define the actual mechanisms by which ATR signaling limits the resection of ssDNA gaps by nucleases, as well as the mechanisms by which ATR signaling promotes the repair of gaps in cancer cells exposed to PARPi. We will achieve this goal by coupling our single-molecule DNA fiber assays with GAP-iPOND, a modified iPOND assay that we specifically developed to unbiasedly identify proteins associating with gaps in an ATR-regulated manner. Moreover, the electron microscopy approach available in Vindigni lab to directly visualize and measure the size of the ssDNA gaps on DNA replication forks provides a unique tool to study the formation, processing, and repair of ssDNA gaps. Using the same techniques, the second aim will test the innovative hypothesis that ATR, together with key recombination factors RAD51, BRCA1, and BRCA2, is also required to promote the repair of broken forks originating from ssDNA gap-replication fork collisions under conditions of continuous PARPi treatment. As tumors are exposed to PARPi repeatedly during cancer treatment, the abilities of tumor cells to repair both ssDNA gaps and broken forks are likely relavent to the therapeutic response to PARPi. Our studies will establish a new paradigm for the roles of the ATR signaling pathway and central recombination factors in the processing and repair of ssDNA gaps and broken forks in PARPi-treated cancer cells. They will also reveal how these pathways are deregulated in BRCA-deficient tumors and how their deregulation contributes to PARPi sensitivity. Interestingly, we also found that PARPi-resistant cells have fewer ssDNA gaps and DNA breaks compared to the PARPi-sensitive counterparts, and that inhibiting ATR activity restores ssDNA gap degradation and PARPi sensitivity. Thus, Aim 3 will test whether the pathways of ssDNA gap protection and fork repair are rewired in BRCA1-mutant cells when they become PARPi resistant. This knowledge is essential for a better understanding of the molecular mechanisms that dictate PARPi response in BRCA-deficient tumors and for developing new molecularly guided strategies to overcome PARPi resistance.

NIH Research Projects · FY 2026 · 2020-02

PROJECT SUMMARY/ABSTRACT Immunotherapy is a promising approach for treating patients with advanced breast cancer. However, immunosuppressive microenvironments induced by regulatory T cells (Treg) present a major barrier to successful anti-tumor immunotherapy. Defining the suppressive mechanisms used by different types of tumor- infiltrating Treg cells is essential for the development of novel strategies to treat human breast cancer. We recently discovered high percentages of γδ Treg cells existing among the tumor-infiltrating lymphocytes (TILs) of breast tumor patients, which are strongly negatively correlated with clinical outcomes. We further identified a novel suppressive mechanism whereby γδ Treg cells induce senescence in T cells and dendritic cells (DCs) that then also develop potent suppressive activity. Therefore, it is critical to further identify the molecular mechanisms responsible for γδ Treg-induced senescence in immune cells, and then to develop strategies to reverse senescence induction mediated by γδ Treg cells. Increasing evidence indicates that the ability of a lymphocyte to perform functional immune responses is controlled by pathways of energy metabolism. However, little is known about the regulation of energy metabolism in tolerogenic DCs and Treg cells. We recently found that γδ Treg cells dramatically reprogram DC lipid metabolism. In addition, we observed that TLR8 signaling significantly suppresses glucose metabolism in human γδ Treg cells via inhibition of both glucose transporters and glycolysis-related enzymes. The central hypotheses of this proposal are that: 1) breast cancer-derived γδ Treg cells rewrite lipid metabolism in DCs, resulting in DC senescence with tolerogenic phenotypes and functions; 2) reprogramming of metabolism in Treg cells and DCs can serve as a novel strategy to synergistically enhance anti-tumor immunity for tumor immunotherapy. Specific Aim 1 seeks to identify what lipid species are changed in γδ Treg-induced senescent DCs and whether the altered lipid components are causatively related to the DC senescence and impaired functions. We will then investigate the importance of transcription factor STAT and PD1-PDL1 signaling in controlling lipid metabolism disorder, senescence induction and impaired functions occurred in γδ Treg-treated DCs. Specific Aim 2 will identify the key glucose metabolites that involve γδ Treg-mediated immune suppression and are regulated by TLR8 signaling for functional reversal in human γδ Treg cells. We will then test the novel concept that TLR8 activation in γδ Treg cells combined with checkpoint blockade of PD-L1 in DCs can serve as novel strategies to reprogram their metabolism and synergistically enhance anti-tumor immunity for breast cancer immunotherapy. A positive outcome from these studies should lead to novel strategies to reprogram innate and adaptive immune cell metabolism for future breast cancer immunotherapy.

NIH Research Projects · FY 2025 · 2020-02

PROJECT ABSTRACT Toxoplasma gondii is a widespread parasite of animals that causes opportunistic infections in humans. Although healthy humans control the infection, they are not able to completely eliminate the bradyzoite stage that reside in neurons and muscle cells. Hence, they remain chronically infected. Complications occur due to reactivation of chronic infections in immunocompromised patients. Complications also can arise from new infections during pregnancy when the parasite can cross the placental barrier and infect the developing fetus. It is estimated that ~ 2 billion people worldwide are chronically infected with T. gondii and hence at risk of reactivation should their immune functions decline. Existing chemotherapy for T. gondii is only effective at suppressing acute infection due to the tachyzoite stage but it is unable to eradicate the chronic bradyzoite stage. The goal of this project is to identify preclinical drug candidates that show potent inhibition of parasite growth including elimination of bradyzoites. Preclinical candidates will be identified based on efficacy against chronic toxoplasmosis in vivo and appropriate metabolism and safety profiles for advancement to the clinic. We will focus on the highly promising bicyclic pyrrolidine scaffold that targets phenylalanine tRNA synthetase (PheRS) to develop inhibitors that are both potent and selective. We will design, synthesize, and test new analogs to develop structure activity relationships for on-target activity. We will optimize potency, selectivity, brain penetration, bioavailability, metabolism and pharmacokinetic (PK) properties of lead compounds. Specific go no/go criteria for potency, selectivity and PK properties will be used to advance compounds to in vivo testing. Genetic and genomic approaches will be used to identify potential resistance mechanism, and to address the mechanisms of action of leads. We will employ quantitative assays for monitoring inhibition of bradyzoite growth in vitro, and animal models for monitoring the efficacy of lead compounds against reactivated toxoplasmosis in the brain. Successful achievement of these goals will deliver preclinical candidate(s) for future IND-enabling studies with the eventual goal of curing chronic toxoplasmosis.

NIH Research Projects · FY 2025 · 2019-12

SUMMARY The success of Community Directed Treatment with Ivermectin (CDTI) in the Americas and Africa has led to a target of eliminating infection with Onchocerca volvulus, the filarial nematode that causes onchocerciasis, in 80% of endemic African countries by 2025. This ambitious goal depends on sustaining not only drug coverage but also sustaining drug sensitivity for treatment periods as long as 25 years in hyperendemic foci, and also on preventing post-CDTI recrudescence due to reinvasion of parasites from regions where elimination has not been achieved. These two requirements for successful and sustainable elimination require the development of new tools capable of routine monitoring of ivermectin susceptibility and of modelling parasite migration over several spatial and temporal scales so that the risk of post-CDTI recrudescence can be estimated objectively. We propose to extend our existing data on genetic associations for ivermectin response to develop a panel of genetic markers predictive of that response as the basis for a simple genotyping surveillance tool for ivermectin efficacy, and to extend our existing data on O. volvulus population structure to parameterize a mathematical model of onchocerciasis (EpiOncho) so that recrudescence risk can be estimated quantitatively. We will carry out genotype-by-sequencing and genetic association analysis on >300 adult female worms whose ivermectin response is known then test the ability of the resulting panel of genetic markers to accurately predict repopulation rates in the skin following ivermectin treatment in additional, previously uncharacterized foci elsewhere in Africa. Similarly, we will carry out genotyping-by-sequencing of a large, geographically diverse selection of microfilariae and infective larvae from throughout Africa to develop and test a panel of markers that define the boundaries of parasite transmission zones and can be used to assign parasites to a population of origin. These data will be used to parameterize a “patch model” version of EpiOncho. The expected outcome will promote development of much needed tools to (i) monitor ivermectin efficacy (ii) estimate risk of post- treatment recrudescence and (iii) facilitate successful elimination of onchocerciasis.

NIH Research Projects · FY 2026 · 2019-09

ABSTRACT Around 18,000 Americans suffer new spinal cord injuries (SCI) each year. Primary and secondary damages caused by SCI permanently impair sensory and motor functions, which require long-term therapeutic, rehabilitative, and psychological interventions. Thus, developing therapies to treat or reverse SCI is a pressing need in regenerative medicine. In contrast to mammals, teleost fish naturally regenerate functional neural tissue and reverse paralysis after complete spinal cord (SC) transection. Following SCI, pro-regenerative glial and neuronal responses distinguish the zebrafish SC from the mammalian SC and enable natural repair post-injury. Importantly, these pro-regenerative processes occur without the detrimental outcomes of reactive gliosis or neurotoxicity elicited by the mammalian SC. However, little is known about the cellular and molecular mechanisms that direct glial and neuronal regeneration in zebrafish. In this proposal, we will determine the potency of the progenitor cells that direct SC repair, and the regenerative roles of neurogenic and gliogenic progenitor cell types after SCI. These studies will provide a mechanistic understanding of glial and neuronal regeneration during zebrafish SC repair, and will guide approaches for manipulating SCI outcomes in mammals.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY (Overall: Human Pangenome Coordination Center) The human reference genome has been the cornerstone of human genetics and genomics research for over twenty years. However, there is a broad consensus that no single reference sequence can fully represent the spectrum of genomic diversity across global populations. Advances in sequencing technologies and a greater appreciation for the importance of genetic diversity make improving the human reference sequence both timely and practical. With advances in long-read sequencing technologies and computational methods, it is now feasible to construct a human pangenome reference that captures and represents the full compendium of genetic variation from a large collection of diverse genomes. We will extend the successful infrastructure developed by the Human Pangenome Reference Consortium Coordination Center. In the next phase, we will significantly improve the human pangenome reference by representing as much human genetic variation as needed to meet disparate scientific and clinical applications, while engaging with international partners and the global research community to provide a widely accessible and user friendly pangenomic resource and tool ecosystem. During the second phase of the Human Pangenome Reference Consortium, our Coordination Center will: i) Continue to maintain and improve the pangenome reference, including supporting the generation of additional diverse, reference quality sequence assemblies; ii) Facilitate adoption of the pangenome reference by the broad research and clinical genetics and genomics communities; iii) Foster the development and deployment of user-friendly informatics tools for the pangenome; iv) Facilitate embedded ELSI research; and, v) Develop international partnerships to ensure that the human pangenome reference engages with the populations it seeks to represent.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY/ABSTRACT Older adults, and particularly those with Parkinson disease (PD), may experience walking difficulties that negatively impact their daily function and quality of life. This project will examine the impact of music and singing on walking performance, with the goal of understanding what types of rhythmic cues are most helpful to people with Parkinson disease and older adults. Our pilot work suggests that imagined, mental singing while walking helps people walk faster with greater stability, whereas walking to music also helps people walk faster but with reduced stability. In Aim 1, we will compare walking while mentally singing to walking while listening to music, using personalized cues tailored to each person's walking performance. We will also test whether finger tapping, a rhythmic task similar to walking in many ways, responds similarly while mentally singing and listening to music. In Aim 2, we will investigate the brain mechanisms underlying the enhancements in movement performance seen with mental singing or music listening. We will use magnetic resonance imaging (MRI) to measure brain activity during finger tapping with and without various cues to understand which areas of the brain are more or less responsive to different types of cues. Using the information gained in the first two aims, we will then conduct an intervention study in Aim 3 to compare and contrast the effects of music-based vs. singing-based training for people with PD. We will determine which training method results in the greatest improvements in walking and tapping performance and measure changes in brain activity with training. We will also ask the participants how acceptable and usable the different training approaches are. This work is among the first to focus on singing as an intervention to improve walking in PD and is innovative in its use of this novel, untapped, highly accessible, adaptable, low-cost approach that has the potential to enhance walking, thereby improving everyday function and quality of life for people with PD.