Washington University

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT A major concern of the intestinal mucosa is to execute its role in absorption of nutrients while minimizing inflammatory or adverse immune interactions with the microbiome. Managing absorption entails not only sorting of nutrients by intestinal epithelial cells but also delivery of those nutrients to specialized blood and lymphatic vessels present in each villus of the small intestine. In turn, the blood and lymphatic vessels that collectively manage outflow from the gut may need to rely on specialized mechanisms that operate to limit dissemination of microbial signals to distal sites like the lung or the liver to avoid downstream organ injury. It is now appreciated, for instance, that alcoholic and nonalcoholic liver damage is driven substantially by microbial transit from the gut to the liver via the portal vein. Likewise, pulmonary damage can ensue in response to injurious cargo in lymph that gains access to blood via the thoracic duct and quickly next flows to the lungs. Yet mechanisms protecting against dissemination of microbial signals from the intestinal mucosa remain incompletely understood, possibly making planned manipulations of the mucosal barrier, as in vaccination or disease therapy, riskier than needed or resulting in surprises. For instance, our preliminary data suggest that the documented and seemingly counterintuitive liver damage that can result from anti-TNF neutralizing antibody therapy to treat inflammatory disease of the bowel may be due, at least in part, to disruption of leukocyte-mediated surveillance of the draining venous vasculature that removes microbes that escape the intestinal mucosal before they arrive to the liver. To fill in these basic knowledge gaps, we propose herein to delineate how different regions of the intestine program leukocyte-dependent and leukocyte-independent strategies to protect downstream cells and tissues against dissemination of microbial signals. In aim 1, we focus on mechanisms operative in phagocytic removal from gut-draining venous blood of whole microbes that escape the intestinal mucosal barrier and otherwise deliver the microbes to deeper tissues or distal locations. In aim 2, we will compare how the small bowel and colon may differentially transport and neutralize soluble microbial signals, like LPS, that can inadvertently escape the epithelial barrier to promote inflammation. This effort will include comprehensive proteomic and lipidomic evaluation of lymph and blood draining different regions of the gut mucosa, working with expert collaborators and taking advantage of our laboratory’s expertise in lymphatic biology and recent studies in the transport of intestinal cargo into gut-draining venous blood.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Depression and anxiety disorders are common in patients in the primary care setting and have clear evidence- based guidelines for screening, diagnosis, and treatment. However, rates of screening, detection, and treatment among Medicare beneficiaries remain low. Without proper treatment, these patients may experience persistent depression and anxiety symptoms, difficulty co-managing other chronic conditions, worsening functional status, and avoidable and expensive acute medical events. In 2017, Medicare launched the Quality Payment Program (QPP) to incentivize delivery of high quality, low cost, evidence-based care in the outpatient setting. The program covers a variety of alternative payment models (APMs) such as patient-centered medical homes (PCMHs) and accountable care organizations (ACOs). Across all payment models, clinicians are paid for their performance based on the quality and cost of care they deliver to patients. However, the effects of the QPP on treatment of depression and anxiety disorders by primary care providers (PCPs) are unknown. There is a critical need for research on the effect of the QPP on access to care and delivery of evidence-based treatment for depression and anxiety disorders in the primary care setting, as well as the subsequent outcomes for patients. Our scientific premise is that the QPP, which is a program targeted at the general patient population, is likely to produce mixed incentives and unintended consequences for primary care delivery to patients with depression and anxiety disorders. On one hand, the QPP incentivizes PCPs in higher-risk bearing APMs such as ACOs and PCMHs to adopt innovative and collaborative care models that may increase rates of evidence-based treatment. However, on the other hand, the QPP does not risk adjust for the most prevalent types of depression and anxiety disorders when judging clinician performance, which creates a financial disincentive to PCPs for caring for patients with these conditions, potentially threatening their access to care. The objectives of this R01 application are to conduct a longitudinal study using real-world data to evaluate the effect of the QPP on: 1) access to PCPs across payment models for patients with depression and anxiety disorders; and 2) delivery of evidence-based treatment for these conditions and subsequent patient outcomes. This study will pursue two specific aims. For aim #1, we will conduct a retrospective cohort study using longitudinal data from the Medicare Current Beneficiary Survey, Centers for Medicare and Medicaid Services Virtual Research Data Center, and Physician Compare for 2017-2020 to investigate two hypotheses: 1) beneficiaries with depression and anxiety disorders will have less access to PCPs in higher risk-bearing APMs; 2) PCPs who disproportionately treat beneficiaries with these conditions will receive lower QPP performance scores and payments. For aim #2, we will use the same data to investigate the hypotheses that beneficiaries with these conditions with access to PCPs in APMs, such as ACOs and PCMHs, will: 1) receive higher rates of evidence-based treatment; 2) have better health and cost outcomes.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract: In the past decade, checkpoint blockade immunotherapies have greatly improved the overall survival of advanced melanoma patients. However, these therapies have failed to treat many other cancer types, including cancers of the pancreas, liver, and stomach. Understanding the successful tumor protective immune responses in long term cancer survivors could promote the understanding of anti-tumor immune responses and the development of novel therapeutic strategies. Melanoma patients who developed dermal immune-related adverse events (irAEs), including rash and vitiligo, have better overall survival than those unaffected patients. However, the immune mechanisms linking dermal irAEs with exceptional anti-tumor immunity remain unknown. Thus, specific aim 1 will comprehensively characterize the phenotype, persistence, antigen specificity, and localization of anti-tumoral T cell responses in both vitiligo and rash affected melanoma survivors using single-cell RNA-seq, single-cell TCR-seq, bulk TCR-seq and 10X spatial transcriptomics. I hypothesize that compared to unaffected melanoma patients, dermal irAE patients maintain more durable proinflammatory T cell responses with a more tumor-focused TCR repertoire. This project will be conducted at the Norris-Cotton Cancer Center (NCCC), well supported by a collaborative team including medical oncologist, surgeon, dermatologist and immunologist. The Sponsor’s lab houses expertise in tumor immunology, memory T cell and translational research and the sponsor has rich experiences in mentoring graduate students. The trainings will be focused on knowledge and novel technical skills such as the 10X spatial transcriptomics to successfully finish the research project. In addition, developing professionals for the transition to the K00 phase is also an important training objective. Transitioning to the K00 phase, the research focus will be cancer immunotherapy resistance mechanisms and the development of novel immunotherapeutic strategies that leverage microbiome. Intratumoral microbiomes were recently found to promote successful tumor immunity even in ‘immune-cold’ cancer types, yet the exact molecular and cellular mechanisms remain unknown. I hypothesize that certain microbiomes could reprogram immune cells and the tumor cells themselves, leading to a more proinflammatory anti-tumor microenvironment. The research will be conducted in an outstanding cancer immunology lab combining leaderships in both translational human research and mechanistic fundamental studies in pre-clinical models. The research trainings will be focused on using mouse models, genomics, epigenomics, metabolomics and cellular immunology approaches to identify the critical mechanisms to overcome immunotherapy resistant cancer growth. The goal by the end of the F99/K00 trainings is to understand the features of tumor protective immune responses and the optimal design of novel cancer immunotherapies. These trainings will provide critical knowledge and skills for the ultimate career goal to establish a research group in academia focusing on developing successful immunotherapeutic regimens.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Heart failure (HF) is a major cause of mortality worldwide, and identifying novel therapies to treat HF represents an urgent clinical need. The long-term vision of my laboratory is that apolipoproteins can be used to treat HF. We have discovered that reduced circulating levels of apolipoprotein M (ApoM) are associated with increased mortality in human HF. Each standard deviation reduction in ApoM is associated with a doubling of mortality risk in HF, an association that is independent of B-type natriuretic peptide, coronary artery disease, and other known risk factors. ApoM is made almost exclusively by the liver, secreted by hepatocytes, and binds the bioactive lipid sphingosine-1-phosphate (S1P) on HDL particles in the circulation, ultimately activating G-protein coupled S1P receptors on various cell types; however, the precise mechanism by which ApoM may increase HF survival is unknown. To understand mechanisms of cardioprotection by ApoM, we utilized a doxorubicin cardiotoxicity (DoxTox) model. Dox is utilized to treat multiple human cancers, but its use is limited by DoxTox and long-term HF. We have discovered that Dox reduces ApoM in humans and mice. In DoxTox models, increasing ApoM improves survival and prevents Dox-induced cardiac dysfunction. In a clinically relevant acute myeloid leukemia model, preliminary studies indicate ApoM does not interfere with Dox anti-cancer efficacy. Our preliminary data suggest that ApoM attenuates Dox-induced autophagic impairment in the myocardium. We find ApoM increases autophagic flux and preserves nuclear transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis implicated in multiple cardiomyopathies. Our data suggest ApoM-driven autophagy and preservation of nuclear TFEB are protective mechanisms generalizable to other cardiomyopathies. This R01 proposal tests the hypothesis that ApoM, via canonical S1P signaling, enhances myocardial autophagy and preserves nuclear TFEB to attenuate DoxTox. Aim 1 tests whether hepatic S1P production is required for ApoM-mediated myocardial autophagy; Aim 2 tests whether the S1P receptor at the level of cardiomyocyte is required for autophagy, and Aim 3 tests whether cardiomyocyte TFEB is required for the cardioprotective effects of ApoM. Aim 3 also utilizes the innovative technique of CUT&RUN sequencing to determine whether ApoM directs TFEB to specific transcriptional targets, which will help elucidate or confirm other pathways downstream of TFEB directed by ApoM. Success of these aims will identify mechanisms by which ApoM can attenuate DoxTox and improve outcomes in HF.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT Our long-term goal is to employ innovative community based participatory research to establish a community advisory board, to collaborate with our community partners to recruit, enroll, and retain a cohort of Black participants and, then, to examine causal mechanisms that increase the risk of Alzheimer disease (AD) within the community cohort. The long preclinical stage of AD, as reflected in biomarkers among adults, is a key risk factor of symptomatic AD. However, despite Blacks having a higher risk of developing AD, recent studies suggest that they have less abnormal levels of biomarkers than Whites in cognitively normal samples. This study aims to examine other risk factors of cognitive decline and AD such as depression, stress, and social determinants of health (SDOH) in a representative sample of Black participants. This research is significant because there are nearly 46 million Black Americans, comprising 13% of the population in the United States. The Black older adult population is expected to increase, from 4.4 million older adults in 2016 to 12.1 million by 2060. Despite these demographic projections, Blacks are significantly underrepresented in AD research. An almost exclusive focus on Whites has created a knowledge gap in understanding how SDOH mechanisms affect diverse populations. Closing this knowledge gap soon is critical since epidemiological studies suggest that Blacks are at twice the risk of AD compared to Whites. Our Specific Aims will (1) Establish a cohort of middle-to-older age Black adults (N=300) using community- based participatory research to understand the unique social, environmental, and economic barriers related to AD risk, (2) Determine the impact of depression, stress, and a novel, theory-based SDOH composite index (CI) on cognitive functioning in participants who are cognitively normal with and without preclinical AD, and (3) Test the association between white matter hyperintensities (WMH) and hippocampal volume (HV) with the SDOH-CI in a subset of participants (N=150) with magnetic resonance imaging (MRI) data. To test our Aims, we have assembled a multidisciplinary team with expertise in AD, SDOH, community- based participatory research and system dynamics, community mobilization, stress and depression, plasma biomarkers, genetics, neuroimaging, neuropsychology, and biostatistical methods. Participants will undergo a one-time blood draw for AD biomarker profiling, cognitive assessment using a neuropsychological battery, and participate in one MRI scan session. Participants will also participate in workshops, complete a comprehensive battery of SDOH measures mapped onto the National Institute of Aging’s Health Research Disparities Framework, and clinical, neurological, and neuropsychological tests annually for up to five years. Once obtained, this knowledge of how within-group heterogeneity in cognitive functioning and AD risk is impacted by SDOH may better support effective AD intervention and treatment for Black Americans.

NIH Research Projects · FY 2025 · 2021-09

Participant engagement and genomic sequencing research from the Washington University Participant Engagement and Cancer Genomic Sequencing Center (WU-PE-CGS) will fill critical gaps in knowledge, methodology, and characterization of cancer populations requiring more research, leading to optimal approaches for participant engagement, outreach, and communication in genomic characterization studies. Our overall goal is to build a rigorous, scientific evidence base for approaches on direct engagement of patients and post-treatment cancer survivors as research participants. Our focus is on rare cancers in populations with significant adverse outcomes (cholangiocarcinoma, multiple myeloma, and colorectal cancer). Our Center will be housed in an environment that fosters multidisciplinary collaboration, catalyzes new ideas, and ensures support that finds solutions for complex recruitment and engagement challenges. The specific aims are to: (1) Advance the field of participant engagement to study adverse cancer outcomes by conducting innovative and impactful direct stakeholder engagement with continuous evaluation and research; (2) Expand an exceptional, broad team of investigators, patients, and patient interest stakeholders; (3) Address adverse cancer outcomes by understanding challenges and improving the ability for low-resourced populations requiring more research to encounter, use, and benefit from genomic sequencing and analysis; (4) Organize and integrate Center units to facilitate team science within our Center and across the Network. WU-PE-CGS builds on a long, outstanding leadership record in cancer and genomic research across the cancer continuum and will enable a significant return on scientific investment in several ways. First, our Center has distinctive features, including a combined focus on adverse cancer outcomes, application of strategies to increase participant engagement, biospecimen acquisition success, and exceptional genomic sequencing expertise. Second, we have assembled a broad, world-class team with strong links to multiple rare cancers. Third, we engage investigators from different disciplines and invest in developing early career scholars. Fourth, we strategically and creatively disseminate products in ways that benefit researchers, practitioners, and community members. Fifth, we will partner with patient-centered interest groups to engage patients, optimize recruitment, and seamlessly return results. Finally, we developed a focused strategy for collective integration of our units. These synergies will allow us to become a national resource for optimal participant engagement, outreach, and communication approaches in genomic characterization studies, accelerating progress for researchers, patients, and their communities.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Children with Callous-Unemotional (CU) traits, which are defined by impairments in empathy, prosociality, and guilt, represent a uniquely at-risk subset of children with conduct disorder. These children have worse prognoses and response to treatments than children with conduct disorder alone. As a result, many of them suffer from poor educational achievement, substance use disorders, and antisocial behaviors. Genetic and environmental factors both affect the development of CU traits, which can occur as early as age 3 years. Twin studies place the heritability of CU traits around 58-81%, with the most implicated genes coding for neurotransmitter receptor variants. Due to the potential genetic effect on brain development, prior studies in adolescents have investigated and found alterations in emotion processing and emotion regulation areas of the brain. However, it is unknown whether these brain changes precede the early development of CU traits or occur later as a result of the pathology. To address this gap, we will leverage a unique, prospective, longitudinal cohort of 385 mother-infant dyads (recruited through R01 MH113883) that includes state-of-the-art neonatal neuroimaging, parental measures of CU traits (added by the applicant), and measures of offspring CU traits at age 3 years (added by the applicant). We hypothesize that altered functional brain connectivity in the newborn period, which occurs prior to postnatal environmental exposures, will mediate the heritable link between parent’s and children’s CU traits. To test this hypothesis, we plan to link CU traits in both mothers and fathers to CU traits in their children at age 3 years. Second, we will explore the relationship between neonatal functional connectivity in emotion processing and emotion regulation regions and CU traits at age 3 years. Finally, we will examine the role of neonatal functional connectivity in mediating heritable relationships between parent and children CU traits. Critically, this proposed project builds upon the candidate’s strong background and experience in neuroimaging analyses and behavioral assessments, enabling development of new skills across the domains of quantitative methods and atypical child development. Finally, not only will this project improve the understanding of aberrant brain development underlying early CU traits, but it will also provide the necessary background for the candidate to become a highly successful academic physician-scientist investigating the biological underpinnings of CU traits and treating patients with externalizing disorders.

NIH Research Projects · FY 2025 · 2021-09

Summary/Abstract Tremendous progress on cancer has been made at the molecular level over the past decade, largely due to the broad application of high throughput, large-scale bulk whole genome, exome and RNA sequencing. In particular, the discovery of numerous medium to high-penetrance drivers, characterization of pathogenic germline variants, and the revelation of many-to-many relationships of genes and pathways, have brought a fuller view of the combinatorial complexity of cancer. Indeed, newer technologies, like single-cell and spatial genomics methods, are now augmenting bulk sequence data to power deeper studies of cancer dynamics, such as heterogeneity, evolution, and interaction with the microenvironment. The current view is that such advanced data, augmented by improved bioinformatics analysis tools and larger, well-curated cohorts will enable medicine to push beyond statistical descriptions toward a genuine deterministic understanding of cancer. Toward this goal, our proposal seeks to extend and apply established bioinformatics systems to integrate the above technologies and leverage our broad range of capabilities and to support the NCI Genomic Characterization Network (NCI-GCN) and Center for Cancer Genomics (CCG) via three specific aims: (1) annotating and interpreting coding and non-coding somatic and germline alterations, (2) characterizing tumor cell populations, evolution, and the tumor microenvironment, and (3) unlocking biological and clinical insights at both the individual and cross-cancer (Pan-Cancer) levels to discern basic themes across the major human cancers. Our approach involves fluencies in four areas of core competence outlined in the program RFA: DNA mutations, long-read sequence analysis, scRNA-Seq analysis, and spatial genomics data analysis (with connection to digital imaging analysis).

NIH Research Projects · FY 2025 · 2021-09

Hypertrophic Cardiomyopathy (HCM) is the most common inherited heart disease and the most common cause of sudden death in young people. While genetic studies have identified specific sarcomere genes associated with HCM, they fail to predict which patients will develop HCM. This proposal is motivated by mounting clinical and animal model evidence for mechanical epigenetic factors possibly explaining this variance. These data suggest that mechanical overload on the heart, caused by hypertension, can act together with sarcomere mutations to cause maladaptive hypertrophic remodeling in HCM. We are also motivated by the need to identify the factors underlying the failure of drug treatments to reverse HCM: although medicines that reduce blood pressure can reverse idiopathic (non-genetic) hypertrophy, they fail to reverse the course of symptomatic HCM. Based upon these prior data, we hypothesize that HCM mutations alter the magnitude of cardiac overload required to induce hypertrophic remodeling and shorten the timeframe over which remodeling is reversible. We aim to dissect the molecular mechanisms through which mechanical loading integrates with sarcomere mutations to cause structural and functional pathology in HCM linked to mutations in Myosin Binding Protein C (MYBPC3). This is possible for the first time because we have developed a medium-throughput, human induced pluripotent stem cell (iPSC) derived micro-heart muscle model system that allows us to apply a controlled magnitude of mechanical overload to iPSC-derived cardiomyocytes. This system will enable us to characterize the effects of overload on micro-heart muscle derived from both iPSC without disease mutations, and from iPSC engineered to harbor HCM patient specific MYPBC3 mutations (Aim 1). We will extend our magnetic hydrogel technologies to dynamically control the magnitude of mechanical overload on micro- heart muscles in situ, enabling us to determine mechanisms through which HCM mutations render cardiomyocytes resistant to blood pressure reducing therapeutics (Aim 2). Finally, we will determine molecular mechanisms linking mechanical overload and MYBPC3 mutations with hypertrophic remodeling (Aim 3).

NIH Research Projects · FY 2024 · 2021-09

Samantha Chin (PD/PI) PROJECT SUMMARY/ABSTRACT Osteoporosis is the most common bone disorder in the world and represents a significant clinical and societal burden due to related bone fractures. Although classically viewed as an age-related disorder, osteoporosis now more commonly describes a general condition of low bone mineral density that can arise in children as well as adults. The mainstay of osteoporosis treatment is anti-resorptive therapy, which seeks to curb bone resorption. This approach, however, is plagued by undesirable side-effects and concerns regarding long- term efficacy, particularly in children, underscoring the pressing need for alternative therapeutic targets and strategies to effectively treat osteoporosis. Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis; however, the underlying pathophysiology is completely unknown. This is due in part to the fact that the role of PLS3 in bone health remains to be identified. This proposal will address these questions and build a basis to develop PLS3 as a novel anabolic anti-osteoporosis target. Genetic studies in mouse and zebrafish models suggest that PLS3 plays a role in osteoblast-mediated bone formation; however, it remains unclear how PLS3 mechanistically contributes to these processes. Aim 1 will employ pathogenic PLS3 mutants that are defective in either actin-bundling or calcium-regulation to elucidate how PLS3 promotes osteoblast differentiation and mineralization in cultured osteoblasts. In addition, RNA-seq analysis will also be used to identify novel pathways that contribute to mineralization in order to provide insight to the specific role of PLS3 and actin dynamics in osteoblast mineralization. To better understand how PLS3 contributes to regulation of bone development in vivo, Aim 2 is focused on developing a zebrafish model system to study the effect of pathogenic PLS3 mutations on bone formation. Taken together, this proposal will fill major gaps in our understanding of how PLS3 and its regulation of actin cytoskeleton dynamics contribute to bone health and the development of osteoporosis. The applicant has assembled a multi-disciplinary mentorship team with experts in the actin cytoskeleton, bone biology, cell biology, and zebrafish model systems that will support the applicant in completion of these aims. The proposal also takes advantage of the Washington University’s strengths in musculoskeletal and zebrafish research including the institution’s cutting-edge cores and facilities. Additionally, Washington University Medical Scientist Training Program has a rich history of supporting physician scientists at various stages of training that will also be invaluable to the applicant’s development. This training and mentorship will provide the applicant critical skills that will facilitate the transition to independent researcher and physician-scientist.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Precisely regulated gene expression is essential for photoreceptor development and maintenance. This process is governed by a genetic program centered on the cone-rod homeobox transcription factor CRX. Mutations in the human CRX gene have been associated with dominant retinopathies with a wide-range of phenotypes and ages of onset. A poor understanding of the mechanism of each individual mutation has made it difficult to develop treatment strategies. To address these challenges, our lab has defined four classes of disease-causing CRX mutations and made mouse models carrying a representative mutation(s) of each class. Up to now, we and others have characterized and reported findings on mouse models for three such classes, proving concordance between the mouse and human conditions due to each mutation. These studies have already provided a deep knowledge of disease pathogenesis. However, the pathogenic mechanism of mutations in the remaining class (Class II) remains to be determined. Class II mutations are linked to the early-onset dominant retinopathies Leber congenital amaurosis (adLCA) and cone rod dystrophy (adCoRD). We have generated mouse lines carrying two individual Class II mutations, Crx-K88N and Crx-E80A, and find that each develops a dominant LCA or CoRD- like phenotype associated with misregulation of photoreceptor gene expression. Because these mutations are located in the CRX homeodomain responsible for DNA binding, we hypothesize that the disease proteins misregulate gene expression by altering CRX’s DNA binding specificity, leading to CRX malfunction at target sites. In Aim 1 of this proposal, we will test our hypothesis in both cell culture and mouse models using cell biology, molecular and functional genomics approaches. Using unbiased high-throughput DNA binding and regulatory function assays, we will determine how these mutations alter CRX’s regulatory activity, leading to misregulation of gene expression and functional deficits in photoreceptors. In Aim 2, we will address the lack of treatment strategies for CRX diseases. We hypothesize that exogenous introduction of the proper amount of normal CRX during a therapeutic window can improve the photoreceptor phenotype in diseased retinae. We have designed a tunable gene augmentation approach that incorporates a tetracycline (doxycycline) switch to turn-on or turn-off therapeutic CRX produced by a transgene integrated within the genome or carried by an adeno associated virus (AAV). We will evaluate phenotypic improvement using established multidisciplinary approaches and expect to see varying degrees of phenotype rescue in different mouse models by CRX augmentation. The outcome of this research will advance our understanding of CRX disease and photoreceptor development, and inform future efforts to treat patients with CRX disease.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Cancer is a leading cause of death and disease. The recent success of immune checkpoint therapy (ICT) has revolutionized tumor therapy, indicating that manipulation of the immune system is an effective strategy to treat cancer. MAbs inhibiting CTLA-4 and PD-1 have been extensively shown to unleash T cell effector functions to control tumors in both mice and some cancer patients. However, ICT is incompletely effective for certain tumors, which escape using multiple mechanisms, one of which is the generation of a tumor microenvironment rich in immunosuppressive myeloid cells. TREM2 is an immune receptor expressed by tissue macrophages that binds phospholipids and lipoproteins and transmits intracellular signals through the ITAM pathway. Recently, TREM2+ macrophages have been reported in many human tumors. In our preliminary data, we demonstrate that TREM2-deficiency or mouse TREM2 blockade with the mAb 178 curbs subcutaneous tumor growth of the 3- methylcholanthrene (MCA) cell line and leads to complete tumor regression when associated with suboptimal PD-1 immunotherapy. Furthermore, high-resolution analysis of the tumor cell infiltrate in the MCA model reveals complex remodeling of the myeloid cell landscape in Trem2–/– and anti-TREM2 treated mice. The overall goal of this application is to advance our understanding of the therapeutic impact of TREM2 blockade in mouse models and human cancer. In Aim 1 we show that TREM2 targeting enhances ICT mediated by anti-PD1; we propose to determine whether TREM2 deficiency or blockade impact other tumor therapies, such as anti-CTLA4 and chemotherapy, which elicit different types of immune responses. The impact of TREM2 will be assessed using injected MCA cell lines and the spontaneous MMTV-PyMT model of breast cancer. In Aim 2 we will define the mechanisms through which anti-TREM2 impacts the tumor microenvironment. Given that a) immunosuppressive macrophages depend on lipid metabolism and accumulate lipid droplets; b) TREM2 promotes foam cell formation by binding lipoproteins; and c) anti-TREM2 mAb blocks lipid binding to TREM2, we will test the hypothesis that TREM2 blockade converts tumor macrophages from immunosuppressive to immunostimulatory by blocking lipid droplet accumulation and foam cell formation. We will also test an alternative mechanism based on the observation that TREM2 is cleaved from the cell surface by ADAM metalloproteases, generating soluble TREM2 (sTREM2), which promotes survival of macrophages in various disease models. We will test the hypothesis that lack of sTREM2 in a transgenic mouse with uncleavable TREM2 prevents survival of immunosuppressive tumor macrophages. In Aim 3, we show unpublished data indicating that anti-human TREM2 mAb 21E10 delays tumor growth of an injected MCA cell line in mice expressing human TREM2 in place of mouse TREM2. Therefore, we will determine whether TREM2 blockade with a specific mAb can be extended to a preclinical model expressing the human TREM2 receptor. Overall, this proposal will advance our knowledge of a novel therapeutic approach based on TREM2 that broadens our armamentarium for targeting immunosuppressive myeloid cells in tumors.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY MPIs: de las Fuentes, Lisa; Davila-Roman, Victor G.; Málaga, German; Hartinger, Stella D43 Project Title: “Research Training: Chronic Non-communicable CVDs and Comorbidities in Peru” Project start / end: 9/1/2021-8/31/2026 Chronic cardiovascular disease (CVD) is the leading cause of M&M worldwide and in Peru. In addition to the usual CVD risk factors, social factors such a low socioeconomic status and environmental exposure further contribute to chronic CVD burden. Among non-communicable diseases (NCDs), high prevalence of hypertension with low rates of awareness, treatment and control represent an important health care gap that contributes to CVD burden in Peru. This application for D43 Training Grant represents a collaboration between US investigators (Washington University in St. Louis and Johns Hopkins University in Baltimore) and Peruvian investigators (Universidad Peruana Cayetano Heredia in Lima, Universidad Nacional del Altiplano de Puno, AB PRISMA in Lima and Puno). The D43 training program will leverage existing research projects and infrastructure in Peru to train Peruvian scientists and health professionals in chronic NCDs/CVD. Our group has been successful in securing grant support (i.e., NIH, Gates Foundation); however, the limited number of Peruvian investigators conducting and leading NCD/CVD studies represents a significant barrier to implementing an effective and sustainable NCD/CVD research program. The proposed training program will provide support (tuition, stipend, research funds) for 14 trainees over five years: 8 master’s, 4 PhD, and 2 postdoctoral Peruvian trainees and/or junior faculty. The objectives of this structured program are to develop the research careers of trainees in the areas of chronic CVD, stroke, implementation science, and environmental exposure; to provide mentoring and support by an internationally renowned faculty with multidisciplinary expertise; and to provide opportunities for career advancement and engagement in research projects. The program will provide intensive training opportunities and build capacity in a range of scientific disciplines and skills relevant to achieving research independence. The long-term goal of this program is to build a sustainable and collaborative research-training infrastructure to develop Peruvian research scientists capable of designing and executing interventions addressing unmet healthcare needs, including the translation of evidence-based interventions and the implementation and dissemination of effective policies to improve public health in Peru. This application is responsive to partnering institutions of PAR-18-901 including the NHLBI (HTN/CVD), Fogarty International Center (global health and LMIC), National Institute of Aging (NCDs including CVD/HTN), National Institute of Neurological Disorders and Stroke (stroke), and National Institutes of Environmental Health Sciences (environmental exposure as risk factors for NCDs/CVD). The ultimate goals of this D43 training program are twofold: 1) to equip outstanding junior investigators with the necessary expertise to solve challenging problems presented by the burden of chronic NCDs/CVD in Peru; and 2) to complete a full transition of program administration and leadership from the US to Peruvian MPIs, faculty, and institutions by D43 Year 5.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Each year, over 800,000 people in the United States suffer from a stroke. Although the vast majority survive the acute event, over half of survivors suffer moderate to severe impairment in motor, sensory, or cognitive function. As a consequence, stroke remains the leading cause of long-term disability, costing over $34 billion annually in direct medical costs and indirect costs (lost productivity) in the United States. In the face of this enormous disease burden, there are few therapies to improve stroke recovery. The brain has some intrinsic capacity for repair, but our understanding of the underlying mechanisms remains very limited. Recent studies suggest that a successful recovery from stroke injury requires neurovascular remodeling to reorganize the damaged brain network. Indeed, circuit repair and the resultant remapping is essential for stroke recovery. Moreover, cerebrovascular remodeling and changes in cerebral oxygen metabolism are observed in animals and patients after stroke and are associated with improved outcomes. Tight coordination of neural repair and cerebrovascular remodeling is likely required to meet energy requirements of brain repair. However, the spatiotemporal coordination of neurovascular repair and the attendant changes in oxygen metabolism after stroke remain incompletely understood. We seek to answer these important questions by developing a new dual-modal intravital imaging technique that integrates 2-photon fluorescence microscopy (TPM) and multi-parametric photoacoustic microscopy (PAM) for high-resolution, time- lapse and comprehensive imaging of neurovascular repair and metabolic changes after stroke. To this end, we have developed a prototype TPM-PAM system and a new cranial window with dual transparency (i.e., light and ultrasound), long lifetime, and compatibility for awake-brain imaging. Building on the strong scientific basis, this proposed project will focus on the development of a high-sensitivity TPM-PAM system for longitudinal imaging of the spatiotemporal interplay of post-stroke neural repair and cerebrovascular remodeling, as well as dynamic imaging of the coupling between neuronal activity, blood flow, and blood oxygen supply, at single-neuron single- capillary level in the awake mouse brain. The proposed research has three specific aims: (1) develop an optically transparent and acoustically sensitive microresonator for integration of TPM and PAM with high sensitivity, (2) develop and validate the microresonator-based TPM-PAM for neurovascular imaging in GCaMP mice, and (3) determine the spatiotemporal relationship between functional vascular repair and neuronal circuit repair after stroke. Advancing our understanding of stroke repair through the development and application of TPM-PAM may reveal promising new therapeutic targets to enhance functional recovery.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY The goals of the WashU-Northwestern Genomic Variation and Function Data and Administrative Coordinating Center (IGVF-DACC) component of the IGVF Consortium are to collect, store, curate, and display all data, metadata, and analysis tools generated by the IGVF Consortium. The DACC will assist in developing and disseminating metadata and standards to be adopted by the community at large, approaches for integrative analysis of a wide range of data types, and visualization and analysis tools to facilitate access and understanding of complex datasets to non-expert users. Ultimately, the IGVF Consortium will produce tools, analyses, models, and data that form the catalog of variants and their functional impact. We will develop the DACC into a substantial service organization allowing scientific research to take full advantage of the IGVF reference catalog or map. To support the IGVF Consortium, we will establish databases with an application framework to facilitate complex data loading. We will include detailed experimental descriptions and metadata. We will define and develop pipelines that connect all Consortium members to the data and create avenues of access that distribute the data to the greater biological research community. We will establish metadata requirements, controlled vocabularies, standardized data formats, and quality control metrics for all IGVF data. We will bring together laboratories that generate complex data types via experimental assays with laboratories that integrate these data using computational tools to define the effects of genomic variation on genome function and how these effects shape phenotypes. By creating structures and data flow pipelines for the verification and validation of all data and providing processes for the documentation of metadata, the DACC will enhance the IGVF data production. The DACC will also coordinate integrative data analysis by creating and adapting analysis pipelines and developing advanced Genome Browser functions for the visual integration of IGVF data. Also, we will make the IGVF Web Portal that will be the primary entry point to the wealth of experimental data and computational analyses. The Portal will integrate these data resources and make them available via enhanced search and browsing capabilities. Finally, the DACC will provide documentation, training, and outreach via many media, including written documentation, video tutorials, online books, webinars, and meeting workshops and presentations.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to describe a five-year training and mentorship plan to prepare Drew Schwartz, MD, PhD to become an independent physician-scientist investigator studying the effects of antibiotics on the preterm neonatal microbiome and host response. Dr. Schwartz obtained combined MD and PhD degrees in the Medical Scientist Training Program at Washington University School of Medicine in St. Louis where he studied how uropathogenic E. coli invade bladder cells to cause severe urinary tract infections. After clinical training in Pediatrics and Infectious Diseases at Washington University School of Medicine and St. Louis Children’s Hospital, he joined the lab of Dr. Gautam Dantas, PhD, a renowned expert on the microbiome and antibiotic resistance. Using fecal samples from an established repository of over 70,000 stools from hospitalized neonates, Dr. Schwartz developed a novel gnotobiotic mouse model of infant microbiome development and disruption. He colonizes germ-free dams with neonatal stool and treats microbiota-humanized pups with clinically relevant doses of antibiotics. He has identified that specific microbiome-antibiotic combinations result in mouse death concomitant with microbiome disruption and immune dysregulation. The central hypothesis is that antibiotic treatment predisposes infants with certain microbiome and resistome compositions to either bacteremic or culture-negative late-onset sepsis. The specific aims of the proposal are to: 1) Define the effects of early-life antibiotics on human infant microbiome maturation, gut mucosal immune response, and vulnerability to bacteremia, and 2) Determine microbial and immune mechanisms of bacteremic and culture-negative late-onset sepsis in a neonatal microbiome-humanized mouse model. The proposed studies will utilize microbiome sequencing, flow cytometry, host immune profiling, and computational modeling to identify modifiable risk factors to refine antibiotic prescribing in the neonatal intensive care unit with the overall goal of reducing incidence and mortality from late-onset sepsis. The mentor, Dr. Dantas, will primarily oversee the project providing training on microbiome analysis, resistome sequencing, and bioinformatic modeling. Dr. Schwartz has assembled an advisory committee with expertise on neonatal infections, antibiotic treatment, gut microbiome development and disruption, and mucosal and peripheral immune response to commensal and pathogenic bacteria. The training plan incorporates achievement of technical expertise, grant writing skills, responsible conduct of research, and mentorship through one-on-one learning, University-sponsored workshops, and presentation and workshops at international conferences. Washington University School of Medicine is the ideal training environment given its extensive track record, outstanding resources, commitment to physician scientists, and necessary expertise to complete the proposed research. This K08 award will provide the necessary skills and resources that Dr. Schwartz requires to become a successful physician scientist whose lab studies the infant microbiome host interface.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Worldwide, up to 3% of people will experience psychosis, a heterogeneous neurodevelopmental and neurodegenerative brain disorder typically characterized by delusions, hallucinations, and functional decline. Currently, clinicians can identify adolescents and young adults who are at clinical high risk (CHR) for developing psychosis. However, as the mechanisms leading to development of psychosis are not fully known, we have limited ability to predict who will develop psychosis. Safe intervention in this population requires high confidence in predictive biomarkers that can stratify individuals into likely clinical trajectories, and match them with effective treatments. Africa has a very limited early psychosis research effort, resulting in a substantial gap in our knowledge about the ethnic heterogeneity of the high-risk state. Recently, a multi-site international effort, the Psychosis-Risk Outcomes Network (ProNET), was funded by the NIH to analyze variation in a diverse set of biomarkers to predict individual CHR clinical trajectories. However, while countries in North America, Europe and Asia are included in this landmark effort, it includes no African country. This is relevant, as risk genes for psychosis as well as the clinical and cultural presentation of psychosis often differ across ethnic groups. This proposal aims to build research capacity in Kenya, using state-of-the-art multimodal methods in Kenya identical to that applied in the ProNET study, in order to map clinical outcomes in CHR populations (Aim 1). This involves building ERP/EEG infrastructure in Nairobi, by acquiring research grade acquisition equipment and software; MRI upgrades, including advanced diffusion and fMRI BOLD imaging capability; and elaborate research training. In Aim 2, we will collect multi-modal biomarkers over 24 months (eight timepoints) from 100 CHR participants (aged 15-22) including brain MRI, ERP/EEG, psychopathology, cognition, genetics, and cortisol. Healthy volunteers (N=50) will complete baseline assessment to quantify typical variation. Aim 3 will test the hypothesis that psychosis outcomes in Kenyan CHR populations will differ from the international ProNET CHR cohort, including a lower rate of psychosis conversion and improved functioning. MRI and ERP analyses are expected to find orbitofrontal cortical thinning and reduced P300 (auditory P3b) amplitude with psychosis progression. Together, this work would address key existing knowledge gaps in global CHR research and provide insights into ethnic heterogeneity of outcomes among CHR patients. By building capacity in CHR clinical and biomarker- based research in Kenya, we will facilitate sub-Saharan Africa joining future international research efforts.

NIH Research Projects · FY 2025 · 2021-08

Exercise and mindfulness are believed to be effective stress reduction interventions, but research to date has not been able to assess their benefits while individuals are coping with a major stressor in real time. The recent pandemic is an unwanted natural experiment in the deleterious effects of stress – especially social isolation (social disconnectedness and loneliness), a stressor particularly strongly associated with the pandemic - on older Americans’ cognitive and emotional health and risk for Alzheimer’s Disease (AD). This project will elucidate whether exercise and mindfulness can mitigate the effects of pandemic stress on cognitive function and emotional health in later life, including neurobiological measures of risk for AD. We will leverage a unique resource: the NIH-funded trial, “Mindfulness-Based Stress Reduction and Exercise for Age-Related Cognitive Decline” (MEDEX). By leveraging MEDEX and following these participants, who continue to attend monthly booster sessions of their randomized condition remotely during the pandemic, we will have repeated sets of clinical, cognitive, molecular, and neuroimaging measures covering 7.5 years during the pre-, during-, and post-pandemic period. We can examine intervention effects, as well as individual factors such as resilience, on long-term outcomes. Among other innovative aspects of the project, we will analyze effects on two novel peripheral biomarkers: Senescence-Associated Secretory Phenotype (SASP), which measures mechanisms of biological aging, and plasma amyloid Aβ42 and Aβ40, which measure AD risk. In the proposed project, (1) during the pandemic, we will use novel methods such as Ecological Momentary Assessment (EMA) to characterize social isolation both objectively (e.g., number of social contacts) and subjectively (e.g., loneliness), and its biological mechanisms on aging (such as elevations in SASP and plasma amyloid); (2) post-pandemic, we will assess downstream effects on cognitive function, emotional well-being, and brain health, including AD risk, using neuropsychological assessments, EMA, and neuroimaging. Outcomes include (Aim 1) changes in cognitive performance and emotional well-being, and decline in emotional well-being measured by positive and negative affect and sleep quality; increases in biological aging and decreasing AΒ42/40 ratio in the post-pandemic phase, indicating higher risk of AD; atrophy in hippocampal and prefrontal volume (structural MRI) and reduced global functional connectivity (resting-state fMRI). Modifiers of these effects (Aim 2) include exercise and mindfulness; psychological resilience; medical morbidities; and APOE genotype. Mechanisms of cognitive, emotional, and brain health changes (Aim 3) include amyloid (Aβ40 and Aβ42), SASP, DNA methylation, and cortisol during the pandemic. This project will advance our knowledge of the impact of social isolation and other stressors on older adults, including mechanisms by which these stressors produce deleterious cognitive, emotional, and brain health changes over time, and whether exercise and mindfulness have durable protective effects.

NIH Research Projects · FY 2025 · 2021-08

SUMMARY The continuing saga of anti-Ab antibody aducanumab has produced the first positive phase 3 outcome for Alzheimer's disease (AD) since memantine was approved in 2003. This promising, albeit controversial, result has breathed new life into the therapeutic potential for Ab-lowering strategies and legitimized the ongoing exploration of other means to chronically and safely mitigate Ab. Past work had shown that small peptide inhibitors can be readily tailored to prevent Ab aggregation, but in vivo delivery of peptide-based drugs was limited by short half-life and poor brain penetration. We have identified two Ab sequence variants that meet criteria for potential therapeutic use, as they 1) prevent aggregation of WT Ab in vitro, 2) promote disassembly of Ab existing fibrils, 3) mitigate toxicity of Ab oligomers, and importantly, 4) do not self-aggregate. To deliver these peptides in vivo, we have developed a novel mini-gene to express our variant peptides at the plasma membrane where they are released into the extracellular space by g-secretase cleavage. By packaging this minigene into an AAV vector that is injected into APP/PS1 mice, our pilot data show that viral expression of variant Ab lowers Ab load and delays plaque formation. The current proposal will build on these results through the following specific aims. First, we will decipher the biophysical mechanism of interactions between variant and wild type Aβ peptides. We will use analytical methods of CD spectroscopy, SEC chromatography, EM, and antibody profiling to define the structural mechanism by which our variants prevent/reverse aggregation of wild-type Ab. Second, we will determine how dosage, timing and route of variant Aβ administration influence efficacy in vivo. We will use viral strategies to compare interventional treatment after amyloid onset with preventative treatment starting at birth, determine the lowest effective ratio of variant:wild- type Ab needed to modify plaque formation and cognitive function, and test whether delivery through the CSF can match the effect of neuronal transduction. Third, we will interrogate the neuroimmune reaction to variant Aβ as an accomplice to plaque reduction. We will use histological and transcriptional profiling to assess whether a neuroimmune response to either the variant peptide or its AAV carrier contribute to plaque prevention in vivo, and if the neuroimmune response changes with age. Finally, we will test the potential for variant Aβ to slow AD aggregate seeding. We anticipate that variant Ab will slow seeding by AD Ab extracts, but will more importantly test whether variant Ab can assuage cross-seeding of tau in amyloid-bearing mice. If successful, this strategy for self-inhibition may also be applicable to other protein misfolding diseases where peptide treatments have been eschewed for technical reasons that can now be overcome through expression engineering and viral technology.

NIH Research Projects · FY 2025 · 2021-08

Title: From 3D genomes to neural connectomes: Higher-order chromatin mechanisms encoding long- term memory Summary The Cremins Lab focuses on higher-order genome folding and how classic epigenetic modifications work through long-range, spatial mechanisms to govern genome function in the developing brain. Much is already known regarding how transcription factors work in the context of the linear genome to regulate gene expression. Yet, severe limitations exist in our ability to engineer chromatin in neural circuits to correct synaptic defects in vivo. At the lab’s inception, it remained unclear whether and how genome folding would functionally influence cell type-specific gene expression. Thus far, we have developed and applied new molecular and computational technologies to discover that nested chromatin domains and long-range loops undergo marked reconfiguration during neural lineage commitment, somatic cell reprogramming, neuronal activity stimulation, and in repeat expansion disorders. We have demonstrated that loops induced by cortical neuron stimulation, engineered through synthetic architectural proteins, and miswired in fragile X syndrome were tightly connected to transcription, thus providing early insight into the genome’s structure-function relationship. We will now focus on a fundamental mystery in neuroscience: how memory is encoded over decades despite rapid turnover of synaptic proteins/RNAs. We hypothesize that the 3D genome integrates molecular traces of synaptic plasticity written on chromatin to store long-term memory in neural circuits. We will employ single-cell genomics and imaging technologies to dissect the extent to which individual synaptic inputs create 3D epigenetic traces. We will perform genome-wide CRISPR screens to identify specific loops and epigenetic modifications functionally important for synaptic plasticity. We will also re-direct technologies used for genome architecture mapping to create molecular activity-dependent connectome maps, and computationally integrate neuronal connectome maps across length scales with 3D epigenetic data sets. Successful completion of this work will shed new light on the genetic and epigenetic mechanisms governing structural and functional synaptic plasticity in physiologically relevant in vitro and in vivo models of memory encoding and consolidation. Many neurological disorders exhibit synaptic defects, and alterations in neuronal activity-dependent gene expression underlie pathological neural phenotypes. Addressing this knowledge gap will provide an essential foundation for our long-term goals to understand how, when, and why pathologic genome misfolding leads to synaptic dysfunction, and to engineer the 3D genome to reverse pathologic synaptic defects in debilitating neurological diseases.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Although early stage colorectal cancer (CRC) is curable with surgery, there is a critical need to stratify high-risk early stage patients that would benefit from adjuvant treatment. In contrast to early stage CRC, late-stage metastatic CRC (mCRC) is usually lethal presenting a critical need to match treatment modalities to patients based on molecular phenotyping. To address these unmet clinical needs the proposed study aims to understand the molecular mechanisms enabling primary CRCs to metastasize with the longer-term goal of rationally guiding treatment decisions. While transcriptome sequencing has provided an unbiased method for discovering lncRNAs, existing large-scale sequencing projects are comprised of predominantly primary tumors without matched metastatic samples. This represents a critical barrier to studying lncRNAs involved in the progression of primary to metastatic disease. To address this gap, we conducted the first meta-analysis of normal, primary, and distant metastatic tissues across CRC patients to identify differentially expressed RNAs Associated with Metastasis (RAMS). We prioritized a previously uncharacterized nuclear localized lncRNA, RAMS11, since: (1) its expression correlated with metastatic progression, (2) its expression associated with poor disease-free survival across multiple independent patient cohorts, and (3) it promoted oncogenic phenotypes in vitro and in vivo. Further, subsequent mechanistic experiments demonstrated RAMS11- dependent recruitment of Chromobox protein 4 (CBX4) to transcriptionally activate Topoisomerase II alpha (TOP2α). This provides a strong rationale for our hypothesis that RAMS11 interacts with CBX4 to epigenetically regulate genes to promote oncogenic phenotypes and treatment resistance. This study will focus on dissecting how RAMS11 dependent CBX4 target gene regulation confers oncogenic phenotypes in vitro and in vivo. We will also assess whether RAMS11 can help identify high-risk CRC patients and its role in chemotherapy resistance. Overall, our proposal will significantly advance the lncRNA tumor biology field by providing mechanistic insight into RAMS11 epigenetic regulation to promote mCRC. Our research has translational impact by evaluating the potential role of RAMS11 to stratify CRC patients at high-risk of develop recurrent/metastatic disease that would benefit from specific adjuvant therapies.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Reading and spelling instruction typically focuses on single-syllable words, and children sometimes have difficulty when confronted with longer words. These difficulties can be especially severe in children with dyslexia. Previous research has concentrated on spelling– sound relationships and reading and spelling processes for one-syllable English words. Less is known about how children deal with longer words and how to teach them to do so. To fill the gap in knowledge about longer words, the current project studies two-syllable words—the most common type of longer word in English. We focus on three issues: which syllable of a word is stressed, whether a stressed first syllable has a short or long vowel, and whether a middle consonant is spelled with a single or a double letter. One set of studies examines the linguistic cues that could potentially help readers and spellers decide on stress, vowel length, and consonant doubling. To do this, we examine the spelling–sound relationships in comprehensive lists of disyllabic words that occur in reading materials at each of the kindergarten to university levels. We expect to find a number of probabilistic patterns that, taken together, help to predict stress, vowel length, and consonant doubling. We ask whether some of these patterns change across grade levels as children are increasingly exposed to words of Latin and Greek origin. To study how children are currently taught to read and spell longer words, we analyze teaching materials that are widely used in the U.S. for phonics and spelling instruction. We expect to find that children receive relatively little teaching about longer words and that the teaching that they do receive is sometimes incomplete or misleading. Finally, we conduct studies in which students in Grades 2, 5, 9, and university are asked to pronounce and spell selected words and nonwords. The goal of these studies is to examine whether learners pick up the patterns that are available in the language, including patterns that are not normally covered in phonics and spelling instruction. We expect to find that children do learn about some untaught patterns but that this process is slow and ultimately incomplete. Instruction could help to speed the process. The results of the project will provide a basis for improving the teaching of reading and spelling for children, including children who struggle with literacy learning. The findings should also be useful in designing instruction for adults who are learning English as a second language.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Human infertility is a global problem and failure of embryo implantation accounts for a significant percentage of pregnancy failure during both natural pregnancy and in vitro fertilization procedures. Implantation is an extremely complicated process requiring precisely controlled hormone signaling, growth factor signaling, and cell-cell interactions. Decidualization is a required step in the implantation process. It involves the rapid proliferation and differentiation of fibroblast-like endometrial stromal cells into epitheloid-like decidual cells. These cells become part of the decidual tissue that surrounds the implanting conceptus. Decidualization defects can directly lead to implantation failure. Moreover, early decidualization defects can cause other adverse pregnancy outcomes including abnormal placentation, restricted intrauterine fetal growth, and early parturition. Understanding decidualization is crucial for improving IVF success rates, developing novel contraceptives, and discovering new treatments for endometriosis. Despite its significance in reproduction, the genetic framework of decidualization had never been systematically studied until our recent development of a suitable high throughput screening tool, immortalized human endometrial stromal cells that carry the yellow fluorescent protein gene under the control of the progesterone-sensitive prolactin promoter (PRL-Y cells). We recently used PRL-Y cells to perform a whole genome siRNA functional screen to uncover novel regulatory genes for human decidualization. One major signaling pathway uncovered by the screen is the retinoic acid (RA) signaling pathway. Contrary to the current dogma that RA suppresses decidualization, we propose a paradigm-shifting hypothesis that RA signaling is absolutely required to initiate and promote decidualization, and is therefore required for female fertility. In this proposal, we will follow up on our exciting preliminary findings and study the function of RA signaling during decidualization through careful mapping of its downstream signaling pathways. In Aim I we will determine which RAR or combination of RARs is required during decidualization, and we will clarify the tissue-specific requirements for RA signaling during peri-implantation. In Aim II, we will identify RA downstream targets during decidualization in mouse and human. Finally in Aim III, we will investigate the interplay between RA and FGF signaling, and between RA and homeodomain proteins during decidualization. Successful completion of this project will dramatically increase our understanding of RA signaling during implantation/decidualization and will have an enduring impact on implantation biology and drug discovery.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Preterm birth and intrauterine growth restriction are significant public health problems in the United States, affecting 10% and 3-9% of births, respectively. These adverse pregnancy outcomes result in increased risk of mortality and lifelong morbidities, in addition to substantial monetary costs of >$26 billion annually. Intrauterine/placental inflammation contributes to more than half of preterm birth. Much of the research related to placental inflammation has focused on acute inflammatory processes, which are typically driven by bacterial infections in the amniotic cavity, and little is known about the triggers or mechanisms that initiate and sustain chronic placental inflammation (CPI). It is well known that some CPI is associated with viral infection, such as cytomegalovirus, but in most cases, the etiology of CPI remains unknown. We hypothesize that most CPI is a chronic inflammatory reaction to viral infection in the placenta. We will comprehensively analyze placental tissue from pregnant women to determine whether viruses and antiviral inflammatory profiles are associated with CPI. Furthermore, we will test maternal blood collected during pregnancy to determine whether we can identify a viral and/or host signal that will predict development of CPI. Lastly, we will test neonatal umbilical cord blood for the presence of viral infection or host signal consistent with that seen in the placenta or maternal blood. This study has the potential to lead to the development of a predictive or diagnostic test for CPI, and it will help us to understand how CPI is triggered and progresses so that we may ultimately be able to design a preventative or therapeutic treatment. Ultimately, our ability to predict and/or prevent CPI will improve pregnancy outcomes and child health.

NIH Research Projects · FY 2024 · 2021-08

ABSTRACT Macrophages serve as HIV reservoir, which is a barrier to cure. However, the molecular mechanism underlying how the virus establishes and maintains infection in this cell type is much less understood. Elucidating the mechanisms of HIV replication in macrophages is of vital importance to the HIV eradication efforts. We previously demonstrated that cyclin L2 is required for HIV-1 infection in macrophages, interacts with and targets the HIV restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) for degradation. However, the details of the cyclin L2-SAMHD1 interactions are unknown. In addition, how cyclin L2 is regulated in macrophages is unknown. In our recent studies we showed that cyclin L2 interacts with and is phosphorylated by the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A). While knockout of cyclin L2 decreased HIV infection 10-fold, depletion of DYRK1A increased HIV infection in primary macrophages up to 10-fold. The overarching objective of this proposal is to determine how the interplay between cyclin L2, DYRK1A and SAMHD1 regulate HIV replication in macrophages. Our specific aims are: Aim 1: Define the mechanism by which the kinase DYRK1A regulates cyclin L2 in macrophages. We hypothesize that DYRK1A inhibits cyclin L2 through phosphorylation or degradation. First we will determine whether DYRK1A-mediated phosphorylation of cyclin L2 prevents the promotion of HIV infection in macrophages. Second, we will define the molecular domains of cyclin L2 and DYRK1A required for their interactions. Third, we will determine the mechanism by which DYRK1A regulate cyclin L2 levels in macrophages. Aim 2: Determine how cyclin L2 and HIV-1 regulate SAMHD1 in macrophages during viral infection. HIV- 1 possesses no Vpx, but is able to establish infection in macrophages despite the abundance of SAMHD1. Therefore, HIV may orchestrate the degradation of SAMHD1 through cyclin L2 or other means. In this aim, we will (i) determine the molecular domains of cyclin L2 and SAMHD1 required for this interaction (ii) identify the viral or cellular factor(s) that induce elevation and reduction in cyclin L2 and SAMHD1 levels respectively during early HIV infection and (iii) determine if SAMHD1 degradation in macrophages is influenced by cyclin L2 phosphorylation by DYRK1A. These studies will reveal how the cyclin L2-DYRK1A-SAMHD1 complex regulate HIV infection in macrophages. Our efforts will be highly significant for understanding a novel pathway that determines whether HIV replication is restricted or enabled in macrophages. The potential impact is significant since the dissection of these protein interactions could reveal potential therapeutic targets against HIV infection.