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NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Project Summary/Abstract Epigenetic modifiers govern cell fate transition during animal development and their mutations drive multiple human congenital disorders; however, the molecular mechanisms underlying the roles of epigenetic modifiers in these normal and pathological processes remain poorly understood. It is widely believed that epigenetic modifiers function through the epigenetic marks they catalyze. Nevertheless, the discoveries of catalytic- independent role of epigenetic modifiers challenge this view, raising the question about the biological function of epigenetic marks. Mono-methylation of histone H3 at lysine 4 (H3K4me1) is a reliable mark of enhancers that shape cell identity, and its reconfiguration accompanies the differentiation of pluripotent stem cells, suggesting that the regulation of H3K4me1 plays an instructive role in cell fate transition. To examine this hypothesis, we investigated the catalytic function of LSD1 and LSD2, two paralogous histone demethylases targeting H3K4me1, in regulating gene expression during cell fate transition. Using state-of-the-art approaches such as precise genome engineering, epigenetic and transcriptomic profiling, and stem cell differentiation, we demonstrate functional synergism between the demethylase activity of LSD1 and LSD2 in regulating cellular differentiation. Based on these compelling preliminary data, here we propose to dissect the molecular mechanisms underlying how the demethylase activity of LSD1/2 regulates cell fate transition. The results generated from our proposed studies will not only reveal novel molecular mechanisms underlying the roles of H3K4me1 in gene regulation and cell fate transition, but also provide insights into understanding the pathogenesis of diseases driven by LSD1/2 loss-of-function. This research aligns with the NIH mission to advance our understanding of fundamental biological processes and contribute to knowledge relevant to developmental disorders and regenerative medicine.

NIGMS - National Institute of General Medical Sciences

Project summary/abstract. Proteins orchestrate cellular processes as dynamic ensembles of interconverting conformations, characterized by the underlying free energy landscape (FELs). Understanding these FELs is paramount for deciphering biological mechanisms, elucidating disease pathogenesis, and engineering novel therapeutics. However, resolving complete FELs, predicting how they respond to perturbations like mutations or ligand binding, and designing them de novo present formidable challenges, limiting our ability to rationally control protein function. This application seeks to bridge this critical gap by developing an integrated computational and experimental platform for the comprehensive decoding, modulation, and de novo design of protein FELs. I am a postdoctoral researcher in Dr. Anum Glasgow’s laboratory at Columbia University, with a strong background in computational biophysics, protein engineering, and advanced hydrogen-deuterium exchange mass spectrometry (HX/MS) analysis. My development of PIGEON-FEATHER, a state-of-the-art Bayesian framework for deriving site-resolved energetics from HX/MS data, exemplifies my commitment to advancing methods for studying protein ensembles. Building on this foundation, my K99 research will establish a transformative framework for resolving, manipulating, and designing protein FELs, providing fundamental insights and practical tools for protein science, drug discovery, and synthetic biology. Aim 1 will develop PF- MetaD, a novel enhanced sampling approach that incorporates HX/MS-derived protection factors (PFs) into meta dynamics simulations. This will be enabled by two deep learning tools I propose to develop—PFNet and PFBoost—for accurate, residue-level PF determination. Together, these will allow the reconstruction of complete protein FELs. Aim 2 will apply these landscape insights to a critical biomedical challenge by designing state- selective protein binders to modulate the FEL of BRAF kinase, aiming to rationally control its activity in cancer- associated mutants by reshaping its conformational ensemble. Aim 3 will push the boundaries of protein engineering by pursuing the de novo design of a universal, ligand-responsive allosteric protein switch based on the PAS domain scaffold, programming its FEL for custom molecular recognition and regulation. Under the primary mentorship of Dr. Anum Glasgow and Dr. Barry Honig, and with the support of collaborators and the rich research environment at Columbia University and affiliated New York City institutions, I will train in single- molecule FRET, high-throughput screening methodologies, advanced machine learning for integrating multimodal biophysical data, scientific leadership, and grant writing. These skills will enable my long-term goal: an independent multidisciplinary lab at a leading R1 institution, focusing on FEL-guided design of functional and therapeutic proteins. This K99/R00 award is critical for my transition to an independent investigator, transforming our ability to rationally program biomolecular behavior.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Influenza viruses cause seasonal and epidemic outbreaks that pose a recurring burden on global public health systems. Despite annual vaccination efforts, severe influenza virus infections occur each year and disproportionally impact children, the elderly, and those with pre-existing conditions. Influenza-induced lung damage and persistent inflammation are highly variable across infected individuals, with limited understanding of the pathogenic signals that contribute to these processes. This proposal hopes to shed light on the pathogenic signals that promote the development of damage-associated niches in the lung leading to more severe disease outcomes. Prior members of the Thomas laboratory discovered a subset of damage- responsive fibroblasts (DRfibs) that reside in damage-associated lung niches and uniquely contribute to influenza-induced lung damage. DRfibs produce high levels of ADAMTS4, an enzyme that degrades versican, an extracellular matrix component produced in the lung during development and infection. Interestingly, when mice lack ADAMTS4, they are protected from influenza-induced mortality compared to wildtype littermate controls. It was found that a dense versican barrier prevented CD8 T cell: DRfib crosstalk, leading to fewer IFNg-producing CD8 T cells, less lung damage, and improved hypoxemia in ADAMTS4 KO mice. This proposal seeks to exploit ADAMTS4 KO mice as a model of damage-associated niche disruption to elucidate how preventing T cell: DRfib communication affects T cell phenotype, clonality, and specificity using spatial transcriptomic, scRNAseq, and TCR sequencing approaches. Low blood oxygen saturation (hypoxemia) is included as a predictor of poor outcomes in 9 out of 12 influenza and pneumonia severity scores, highlighting a strong correlation between impaired oxygenation and influenza severity. Tissue-level hypoxia in the lungs is also a characteristic of influenza illness. Previous research has shown that a cell’s microenvironment can significantly impact its phenotype; however, the impact of hypoxia on immune and stromal cell subsets in the lungs during and following a respiratory virus infection has not yet been explored. This F32 proposal will employ a unique mouse model to reveal how hypoxia in the lung microenvironment during severe respiratory viral infection influences the phenotypes of T cells and fibroblasts. Successful completion of the proposed will generate a unique atlas of hypoxic cell phenotypes that could have broad implications for the field as many severe respiratory viruses induce hypoxemia and lung damage. Dr. Paul G. Thomas, a well-established influenza immunologist and member of the Center of Excellence for Influenza Research and Response, and St. Jude Children’s Research Hospital will be integral to achieving the goals outlined in this proposal by providing technical training, practice in scientific communication, mentorship experience, on-site access to state of the art resources, and networking opportunities with influenza experts. Completion of the research and training goals outlined in this F32 proposal will unveil novel mechanisms of influenza pathogenesis while supporting the development of the applicant’s independent research career.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The mammalian heart undergoes profound transcriptional and phenotypical remodeling during postnatal development, a process known as cardiac maturation. However, the molecular mechanisms driving this transition is not fully understood, posing a major challenge in cardiac regenerative medicine, where induced cardiomyocytes from pluripotent stem cell differentiation or non-myocyte reprogramming exhibit an overall immature phenotype that severely limits their application in cell therapy and in vitro disease modeling. In this K99/R00 application, I propose to integrate cutting-edge single cell multiomics with state-of-the-art computational methods to unravel the cell-type-specific gene regulatory networks governing cardiac maturation, and develop a novel dual-reporter system to model and enhance cardiac maturation in vitro and in vivo. During the K99 phase, I will characterize the epigenomic changes of the mouse heart during postnatal development at a single cell resolution using various single cell multiomic technologies (Aim 1), and construct cell-type-resolved gene regulatory networks underlying cardiac maturation using bioinformatic approaches coupled with deep learning (Aim 2). I will also establish cell culture and mouse models with CRISPR-mediated knock-in of dual-fluorescent reporters to track and assess cardiomyocyte maturation (Aim 3a). During the R00 phase, I will experimentally characterize key regulatory elements and novel transcriptional regulators using functional genomic approaches (Aim 3b). I will also leverage these findings to enhance the maturation of in vitro-derived cardiomyocytes for improved therapeutic potential (Aim 3c). The expected outcomes of my proposed research will deepen our understanding of postnatal cardiac development and uncover new therapeutic strategies to improve cardiac function after injury. My career goal is to lead an independent research group that develops and employs innovative technologies to study the regulatory mechanisms underlying cardiac development, regeneration, and disease. In my K99 phase, I will acquire crucial knowledge and skills in advanced single cell genomics and computational biology to complement my previous expertise in developmental biology and cardiac research. My career development will be supported by an exceptional mentoring and advisory committee from UCSD/Salk/HHMI, along with world-class resources, training opportunities, and institutional support at UC San Diego. These elements will provide a strong foundation for my successful transition to an independent tenure- track faculty position.

NIAID - National Institute of Allergy and Infectious Diseases

During suppressive antiretroviral therapy (ART), HIV-1 persists in long-lived resting memory CD4+ T cells of children and young adults with perinatal HIV-1 as both intact and defective proviral genomes. The intact, replication-competent proviruses contribute to the latent reservoir and are a lifelong barrier to cure. In perinatal HIV-1, the reservoir is established early and shaped by unique immunologic factors. A growing body of evidence suggests that while defective proviruses cannot contribute to rebound in the absence of ART, these proviruses are transcriptionally and translationally active, potentially leading to adverse immune effects. However, the frequency, composition, and potential immunologic effects of defective proviruses across pediatric age groups remain poorly understood. In this proposal, we aim to characterize the defective proviral reservoir in children and young adults living with perinatal HIV-1 by determining the abundance and sequences of proviruses that are maintained for years despite ART and assessing their ability to produce viral mRNA and proteins. This project leverages well-characterized, bio-banked peripheral blood mononuclear cell (PBMC) and plasma specimens from pediatric HIV-1 cohorts to systematically characterize the landscape of defective proviruses in perinatal infection from infancy through adolescence. Our hypothesis is that in longstanding treated perinatal HIV-1, defective proviruses are transcriptionally and translationally active and drive persistent residual HIV-1 viremia during ART, promoting immune activation and exhaustion despite replication incompetence. Defective proviruses may also serve to produce decoy viral proteins that elicit autologous neutralizing antibodies, thereby reducing the efficacy of autologous neutralization of the latent reservoir. We propose three specific aims. In Aim 1, we will quantify and characterize intact and defective proviruses across pediatric age groups using near full-length single genome sequencing. In Aim 2, we will assess the transcriptional activity of defective proviruses following ex vivo stimulation in co-culture for HIV-1 mRNA analyses and their correlation with immunologic and clinical measures, including markers of immune activation and exhaustion. We will then compare it to sequences from low level plasma viremia to determine whether defectives are the source. In Aim 3, we will perform the quantitative viral outgrowth assay (QVOA) with the ultrasensitive p24 Simoa assay to identify if high- and low-level p24 producing wells are harboring intact or defective proviruses. We will then determine whether env-pseudotyped virus derived from intact or defective proviral sequences can be neutralized with autologous plasma IgG. By integrating molecular virology and immunology profiling in a pediatric context, this study will generate novel insights into the role of defective proviruses in HIV-1 persistence in children. Our findings will inform the design of age-specific cure strategies and contribute to the broader goal of ART-free remission in children with perinatal HIV-1 towards a life free of co-morbidities.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Although effective therapeutics that target the dysregulated kinase activity of BCR::ABL1 have been developed for patients with chronic myeloid leukemia (CML), acquired resistance remains an important clinical issue. Additionally, problematic side effects plague a considerable proportion of patients who are expected to require lifelong therapy. The first five approved tyrosine kinase inhibitors (TKIs) for CML target the ATP binding pocket of BCR::ABL1 (“orthosteric” TKIs). Asciminib is the first active “allosteric” TKI for CML and was recently approved as a frontline therapy based on high response rates and excellent tolerability. Asciminib is rapidly being adopted as a preferred treatment in all lines of therapy. We have demonstrated that several mutations that confer resistance to orthosteric TKIs unexpectedly confer in vitro and/or clinical resistance to asciminib. We have further demonstrated that a clinical variant of BCR::ABL1 lacking ABL1 exon 2 is uniquely and highly resistant to asciminib. Notably, these isoforms retain asciminib binding affinity, thereby invoking a novel molecular mechanism of resistance. Our central hypothesis is that asciminib will be vulnerable to multiple resistance- conferring mutations that disrupt its allosteric effect on kinase conformation, in addition to a limited number of mutations that impair its ability to bind BCR::ABL1. Our rationale is that pioneering work on orthosteric TKI resistance mechanisms in CML have informed kinase conformational dynamics, optimal CML patient management, development of next-generation TKIs and successful prediction of TKI resistance mechanisms in several other malignancies. We propose to (i) employ orthogonal approaches to identify and validate single point mutants in BCR::ABL1 that can confer resistance to asciminib, and compound (≥2 on one DNA strand) mutants that arise following subsequent orthosteric TKI therapy, (ii) assess their sensitivities to a novel active investigational allosteric inhibitor, combinations of TKIs, and a novel bitopic TKI, (iii) determine mechanisms of resistance through computational and structural studies, (iv) define residues necessary for adoption of the closed ABL1 kinase conformation, and (v) assess the ability of asciminib-resistant mutants to pathologically activate ABL1 kinase activity. The proposed research is significant due to its potential to rapidly impact clinical investigation and optimize patient management, inform understanding of kinase regulation and other malignancies. The proposed research is innovative because it applies state-of-the-art methodologies to comprehensively define and characterize a novel mechanism of resistance to a first-in-class highly clinically active allosteric TKI and thereby establish a new paradigm. Additionally, it will assess the promise of emerging agents, TKI combinations, and an innovative bitopic TKI with best-in-class features for treating asciminb-resistant single and compound mutants.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Antiretroviral therapy (ART) effectively suppresses HIV replication, yet treatment interruption results in rapid viral rebound due to persistent viral reservoirs. A major barrier to eliminating these reservoirs is the progressive loss of HIV-specific CD8+ T cell effector function. Although immune checkpoint blockade can partially restore T cell function in cancer, these approaches show limited efficacy in people living with HIV (PLWH), underscoring the need to identify alternative mechanisms contributing to HIV-specific CD8+ T cell dysfunction. Extracellular adenosine (ADO) is a potent immunosuppressive metabolite generated from inflammatory ATP by the ectonucleotidases CD39 and CD73 and signals through the A2a adenosine receptor to inhibit CD8+ T cell function. Under physiological conditions, ADO levels are tightly regulated by adenosine deaminase-1 (ADA-1). In PLWH, increased expression of ADO-generating enzymes and A2aR, together with reduced ADA-1 expression, promotes ADO accumulation and is associated with immune dysfunction. However, the contribution of the ADO/ADA-1 axis to antigen-specific CD8+ T cell impairment in PLWH remains poorly defined. This project will define the role of ADO signaling in HIV-specific CD8+ T cell dysfunction (Aim 1) and evaluate whether targeted ADA-1 supplementation can improve antiviral function (Aim 2). We hypothesize that ADO/ADA- 1 axis impacts antigen-specific CD8+T cell function in PLWH and that restoring ADA-1 activity will improve HIV- specific function. Aim 1 will quantify ADO-driven suppression of antigen-specific CD8+ T cell function in PLWH. Aim 1.1 will determine whether HIV-specific CD8+ T cells are more susceptible to ADO-mediated suppression than CMV-specific CD8+ T cells within the same donor. Preliminary data demonstrate epigenetic and transcriptional repression of ADA-1 and enhanced ADO-pathway signaling in HIV-specific CD8+ T cells, supporting increased vulnerability. Aim 1.2 will determine whether chronic HIV infection broadly increases ADO sensitivity by comparing antigen-specific CD8+ T cell responses from PLWH and HIV-negative donors. Established MDDC–T cell coculture assays will be used to model antigen-specific function ex vivo. Aim 2 will test whether targeted ADA-1 supplementation improves HIV-specific CD8+ T cell function. Building on preliminary feasibility data, ADA-1 mRNA will be delivered selectively to CD8+ T cells using CD8- targeted lipid nanoparticles, alone or in combination with PD-1 blockade, to assess functional improvement. Overall, this exploratory study will define a mechanistically distinct pathway contributing to HIV-associated CD8+ T cell dysfunction and evaluate ADA-1 supplementation as a targeted strategy to restore antiviral immunity, informing future immune-based HIV cure approaches.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT People with HIV-1 (PWH) on antiretroviral therapy (ART) are prone to experiencing chronic inflammation despite effective viral suppression. This sustained inflammation has been linked to an elevated risk of developing a variety of age-associated comorbidities, including chronic kidney disease (CKD). Preliminary data supporting this study demonstrates multiple distinct inflammatory endotypes among aging PWH on ART, several defined by levels of chemokine C-C motif ligand 2 (CCL2), a critical mediator and biomarker of kidney injury and disease. However, the precise cellular and molecular inflammatory immune endotypes in PWH that could lead to disease remain undefined. Moreover, how endotypes defined by circulating inflammatory markers impact organ- compartmentalized inflammation and the functional and molecular states of immune cells are unknown. The overall objective of this project is to define the early cellular and molecular signatures of inflammation associated with the progressive development of CKD in PWH on ART in both blood and urine. This will be achieved by comprehensively defining plasma inflammatory endotypes in a retrospective cohort of aging (50+ years) PWH on ART, sampled as they progressed from early to later stage kidney disease, with comparison to PWH on ART with normal renal function. In addition, systems immunology will be used to characterize the soluble and cellular inflammatory profiles of peripheral blood and urine in a prospective cohort of 200 aging PWH on ART. Urine, a readily accessible non-invasive biofluid, contains proteins and viable cells originating from the kidney that can serve as indicators of renal inflammation and overall kidney function. A combination of advanced machine learning approaches, clinical tests, and human kidneys-on-chips models will be applied to define the relationship between systemic, urinary, and renal cell inflammation and dysfunction in PWH on ART that are associated with onset of CKD. This project will test the hypothesis that specific inflammatory immune endotypes can be identified in PWH on ART that promote the activation and dysregulation of immune and kidney cells, contributing to the development of CKD. The hypothesis will be tested, and the overall objective achieved, with completion of three Specific Aims. Aim 1 will define plasma inflammatory endotypes of aging PWH on ART and identify signatures that predict CKD. Aim 2 will identify how plasma inflammatory endotypes impact the cellular, metabolic, and functional programs in blood and urine of aging PWH on ART. Finally, Aim 3 will determine the mechanisms of activation of renal inflammatory programs using kidneys-on-chips. This research will identify specific endotypes of inflammation associated with development pf CKD and uncover pathways driving chronic inflammation in the blood and urine that promote kidney injury and disease. This knowledge will enable the development of CKD risk prediction tools and of therapeutic interventions like CCL2 signaling inhibitors to reduce inflammation-related kidney disease and other comorbidities in this expanding population.

NHLBI - National Heart Lung and Blood Institute

Project Abstract Pneumonia is a leading cause of infectious deaths worldwide; over 2 million people die of pneumonia each year. The leading bacterial cause of pneumonia is Streptococcus pneumoniae (Spn), an opportunistic pathogen that colonizes the human respiratory tract. While there are >100 known serotypes of Spn worldwide, current immunization strategies protect only against a limited few. Recent advances in our understanding of mucosal immunology have identified lung-resident CD4+ memory T (TRM) cells as critical determinants of broad protection against multiple serotypes of Spn. However, despite their clinical value from the public health perspective, little is known about mechanisms that drive the establishment of these CD4+ TRM cells in the lungs. Furthermore, it is unclear whether Spn may alter CD4+ TRM cell formation in the lungs using its own virulence factors. Understanding these mechanisms is instrumental for development of next generation cross-protective immunization strategies against this pathogen. Relevant to this, our preliminary data suggest that the Spn toxin pneumolysin (Ply) and bacterial sensing by NLRP3 are both key to recruitment and establishment of CD4+ TRM cell in the lungs. However, it remains unclear whether pore-forming activity or complement-activating biology of Ply is required for CD4+ TRM cell formation nor is it known how NLRP3 sensing of Spn may coax CD4+ TRM cell formation. In this proposal we will test the hypothesis that Spn drives CD4+ TRM cell formation via Ply’s pore forming activity and induction of macrophage-epithelial crosstalk via NLRP3. This hypothesis will be tested through two specific aims: Aim 1 will determine whether pore formation activity of Ply drives CD4+ TRM cell formation by boosting T cell recruitment, and Aim 2 will determine whether NLRP3 sensing of Spn is required for macrophage-epithelial crosstalk to drive CD4+ TRM cell formation. These studies will be accomplished by using isogenic Spn mutant strains, genetically engineered mice, intratracheal murine infection models, adoptive transfers, spectral flow cytometry, and single cell- and bulk-RNA sequencing. Findings from these innovative studies will guide development of more effective, broadly protective Spn immunization strategies that will prevent life-threatening Spn-pneumonia and subsequent diseases. This proposal will support the applicant with her scientific, technical, personal, professional, and career development which includes courses and workshops, guidance from a strong mentoring team and dissertation advisory committee, opportunities to develop science communication skills, and opportunities to mentor junior students in the lab and classroom. The University of Michigan offers top academic training, connections with esteemed faculty in pulmonology, microbiology, and immunology, and state-of-the-art resources to achieve the proposed aims. Completion of this proposal will also support the applicant’s rigorous training in experimental design, microbiology and immunology techniques, and data interpretation that will usher her towards becoming a successful, independent scientist.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Asthma is an inflammatory disorder of the respiratory tract triggered in response to inhalation of stimuli like allergens. It is the most common chronic disease in children worldwide, making it a major public health concern. A hallmark feature of asthma is the excessive eosinophil recruitment to the airways (referred to as allergic airway eosinophilia) which inflicts tissue damage, airway wall remodeling, and reduced gas exchange in the affected lungs. The hygiene hypothesis posits that exposure to commensal microbes may educate our immune system and direct it away from development of allergies and asthma. Relevant to this, our preliminary data also suggests that inhalation experience with the frequent human nasopharynx-colonizing bacteria Streptococcus pneumoniae (Spn) confers protection against allergic airway eosinophilia. How such inhaled microbial experience may mechanistically preclude allergic airway eosinophilia, however, remains unclear. In this proposal we will test the hypothesis that Spn experience protects against allergic airway eosinophilia by potentiating a Treg- and a cDC1- dependent immunoregulatory axis; to be tested as part of Aims 1 and 2 respectively. Aim 1 will determine whether inhaled Spn experience is protective due to the production of IL-10 by regulatory T cells (Tregs) formed during bacterial encounter, and Aim 2 will determine whether Spn confers protection through the activity of an enriched cDC1 pool during allergen encounter. These studies will be accomplished using novel genetically engineered mouse lines, human relevant murine models of pneumococcal experience and asthma, spectral flow cytometry, and single cell RNA sequencing. Findings from these innovative studies will delineate pathways that may be exploited as targets for next-generation preventative and treatment strategies against asthma. What is more, this proposal will also support the applicant in scientific, professional, technical, career, and personal development through guidance by an expert mentoring team and dissertation committee, use of state-of-the-art experimental techniques, enrollment in a tailored course curriculum, engagement in workshops and seminars, and engagement in an array of science communication and mentorship opportunities. The University of Michigan boasts elite academic training; connections and collaborations with experts in the fields of immunology, microbiology, pulmonology, and allergy; and cutting-edge facilities to achieve the proposed aims. Completion of this proposal will additionally support the applicant’s thorough and rigorous training in creative experimental design, microbiological and immunological techniques, and data interpretation and presentation that will ensure her future success as an independent researcher and principal investigator.

NEI - National Eye Institute

Project Summary: Neurodevelopmental disorders, including syndromic disorders of retinal and brain development, are a major cause of morbidity in children. A subset of these disorders results from abnormal vascular development, and microcephaly and chorioretinopathy (MCCRP) is a recently identified disease that may belong in this category. It is characterized by small head circumference, brain anomalies, developmental delay, and vision loss due to chorioretinopathy and abnormal retinal vasculature. Autosomal recessive MCCRP results from defects in TUBGCP4 or TUBGCP6, which encode components of the gamma-tubulin ring complex (γ-TuRC), a ubiquitous structure necessary for microtubule nucleation and spindle formation in cells. However, γ-TuRC has not previously been implicated in vascular development and it is unknown why defects in γ-TuRC lead to blindness and microcephaly. We demonstrated that Tubgcp4 and Tubgcp6 expression is highly upregulated in murine vascular endothelial cells (EC) from the retina and brain (relative to EC from other tissues), and that murine EC deficiency of TUBGCP4 results in embryonic lethality, indicating a critical role for TUBGCP4 in EC. Our long-term goal is to identify the role of the γ-TuRC in retinal and brain development. The objective of this application is to define the pathophysiology of TUBGCP4 and TUBGCP6-associated MCCRP. We will test the hypothesis that TUBGCP4 and TUBGCP6 serve critical, EC-specific roles in the retina and brain, and that deficiency of these proteins results in MCCRP due to a primary vascular developmental defect. Since retinal and cerebral vascular development is not complete until several weeks after birth, we will use novel conditional knockout mouse models of Tubgcp4 and Tubgcp6 and a tamoxifen-inducible EC-specific Cre recombinase to eliminate expression of these genes in EC postnatally. Ophthalmic studies will demonstrate the necessity of EC-specific TUBGCP4 and TUBGCP6 in retina and retinal vascular development, including optical coherence tomography (OCT), OCT-angiography, electroretinogram, optokinetic response, and retinal histology (Aim 1). Neurologic studies in mice and/or embryos lacking TUBGCP4 or TUBGCP6 in EC will demonstrate the necessity of these proteins in cerebral and neurovascular development, including MRI brain imaging, neurobehavioral studies, and brain immunohistochemistry (Aim 2). Elucidating the pathophysiology of MCCRP will improve our understanding of the genetic mechanisms controlling retinal and brain vascular development, and may reveal new therapeutic targets for more common blinding retinal vascular diseases. The career development objective of this proposal is to develop the mentorship and expertise needed to become a productive independent clinician-scientist and international leader working at the intersection of inherited retinal diseases (IRD) and disorders of vascular development. OHSU is a center of renowned expertise in IRDs, in vivo retinal vascular imaging, and the neurosciences; it provides state of the art resources and world-class faculty to support Dr. Everett’s scientific and career development goals for this proposal.

Delaware Community Foundation

Delaware Community Foundation ($350M in assets) supports nonprofits in DE through grants focused on education, community development, health, arts. Awards range from $2,000 to $100,000.

National Park Service

This Funding Announcement is not a request for applications. This announcement is to provide public notice of the National Park Service s intention to fund the following project activities without full and open competition. OVERVIEW The Demonstration Arts and Cultural Preservation Project seeks to make possible the continuation of a long-held park goal to provide a venue and support for the Native artists to perpetuate and preserve their history, culture and craft through park or Visitors Center studiobased work on art forms including wood carving, metal-smithing, bead work, fiber arts such as weaving, story-telling and other elements of culture. The partner, Sitka Tribe of Alaska (STA) will provide staffing to conceive a detailed plan for the project, to publically solicit applications from Native artists who wish to participate in the project, to select participating artists, to schedule artists studio time, and to monitor and manage artists participation and fulfillment of mission. SITK will provide training for the artists on the park, its history and mission, so that as artists interact with visitors they will be able to provide information not only on their arts, but on the park itself. Park staff will work closely with STA staff on scheduling, training and support, so that the artists in studio are on site during peak visitor periods. The project will run from Maythrough September. STATEMENT OF JOINT OBJECTIVES/PROJECT MANAGEMENT PLAN The goals are to foster the preservation of Native art and culture, and to enhance visitor enjoyment of the park. STA will provide the management of the project, and SITK will provide the venue, training and liaison with the visitors. The two partners will work together to align the artists, the venue, the scheduling of demonstrations and the visitor interaction. Through the selection and presence of active Native artists in the park studios, STA and SITK will jointly make possible the continued perpetuation of a number of Native art forms that are in significant need of support. These art forms have been passed down through generations of artists for several millennia, but the lack of contemporary venues and support for artists threatens the continued existence of these forms of art. With input from SITK, STA will establish a calendar of artists participation in studio space, aligned with visitor flows, and establish a management team to manage the project, including a senior manager with project and budget oversight, and a scheduler who will track and manage artist time and attendance. SITK will designate a member of the Interpretive staff as project liaison. That person will work on scheduling, preparing the studio space for artists, arrange for training artists in SITK and NPS information that will be helpful to visitors, and provide such other support as to make the project run smoothly. Artists will be provided such materials and equipment as are needed to be able to practice their crafts. The artists will be scheduled in-studio during days when the heaviest load of summer visitors is projected, so that their work can be viewed by the largest number of park visitors. STA and SITK will work together on publicizing the demonstration arts project, with a special emphasis on the key goal of preservation of culture and art forms. The partners will have scheduled check-in points through the life of the project to review its progress and to address any problems that may arise. The partners also will do a formal review when the project is completed so that lessons learned about success and challenges can be captured. RECIPIENT INVOLVEMENT STA will manage this project, and that management and associated costs will be funded by this project. STA will assign a senior manager to oversee its execution, including the management of the budget. The manager in turn will hire a part-time scheduler to ensure artists are scheduled at times appropriate to the studio space and visitor influx. The scheduler will track time and attendance, and provide that information to the manager so that the artists can be paid for their time in the studio. Program Point of Contact: Becky Latanich 907-747-0132.

Dentsu Foundation

Funds creative education, arts, and social good communication programs globally.

NCATS - National Center for Advancing Translational Sciences

Project Summary Transcription factors (TFs) are critical regulators of gene expression, serving as master switches that control cel- lular processes such as development, homeostasis, and response to external stimuli. Dysregulation of TF activity is implicated in a range of diseases, including cancer, fibrosis, and immunological disorders, yet TFs remain an underexploited class of therapeutic targets. Their intrinsic structural complexity, including large multimeric com- plexes and intrinsically disordered regions, has rendered them largely ”undruggable” by traditional approaches. This project seeks to overcome the challenges of targeting TFs by advancing Strategian, an artificial intel- ligence (AI)-powered drug discovery platform built on our proprietary TF-Scan technology. TF-Scan is a high- throughput functional proteomics platform that measures the effects of compounds on TF activity across thou- sands of targets in live cells. Leveraging data from TF-Scan, Strategian uses state-of-the-art machine learn- ing models, including protein and chemistry foundation models, to predict the effects of small molecules on TF- chromatin interactions. In this project, we propose to: (1) Evaluate and optimize cutting-edge protein and compound representations to enhance the predictive performance of Strategian. (2) Discover novel inhibitors for three high-priority TFs using active learning, a method that iteratively improves the AI model by validating its predictions through experimental testing. (3) Develop scalable data infrastructure to support real-time AI-driven drug discovery, including automated data integration pipelines and a web application for internal and external use. The research design integrates functional proteomics, advanced AI methodologies, and scalable cloud in- frastructure to systematically address the challenges of targeting TFs. By iterating through cycles of prediction, experimental validation, and model improvement, Strategian will identify novel TF inhibitors with therapeutic po- tential. The resulting platform will also be deployed to our internal programs, partners, and as a commercial software-as-a-service (SaaS) offering, enabling broad access to TF drug discovery tools. This project aligns with the mission of the National Center for Advancing Translational Sciences (NCATS) by developing innovative tools and approaches to overcome bottlenecks in drug discovery and development. By targeting transcription factors, this work has the potential to open new avenues for treating diseases that are currently intractable, ultimately improving human health and advancing translational science.

Tasveer

The Desi Improv Festival will include an improv workshop and a main improv event. The project is designed to introduce participants to the art of improvisation through engaging scenes, games and exercises in a fun, supportive environment. Research shows that improv is a powerful tool for team building and community engagement. By focusing on collaboration, creativity and spontaneous thinking, this workshop aims to break down communication barriers and foster acceptance and understanding. Participants will develop skills in adaptability, active listening and effective communication. Ultimately, this workshop will empower participants to feel more confident, connected, and better equipped to navigate diverse environments.

Tasveer

Tasveer will organize a free nine-day literary and storytelling festival focusing on South Asian culture in January 2019. It will feature presentations and workshops on writing fiction, comedy, screenplays, graphic novels, and other ways of telling a story. The events will take place at Seattle Art Museum Located at 1300 1st Ave and Northwest Film Forum located at 1515 12th Ave.

NHGRI - National Human Genome Research Institute

ABSTRACT Noncoding genetic variation that alters gene regulation is of paramount importance for health, disease, and evolution. Diseases ranging in incidence from the most common to the most rare all have substantial risk associated with regulatory variation; and most of the genetic differences between closely related species are noncoding. Whole genome sequencing can directly identify that variation but to realize its potential to elucidate the genetic determinants of health and disease, will require accurate annotation of this noncoding variation for functionality. In coding sequence, the genetic code allows variants to be annotated to a rough hierarchy of likely functional effects and pathogenicity. In noncoding sequence such annotation is less clear. Perturbation assays, i.e., assays that modify genetic or epigenetic states and measure the effect of those perturbations on regulatory endpoints, offer a possible path to annotating noncoding variation. However, to fully leverage this data, novel and sophisticated statistical and machine learning approaches are required to extract useful information from those assays, to integrate that information across regulatory endpoints, and to extrapolate findings so that annotation of previously unobserved (unperturbed) variation in diverse cell types is possible. The goal of the Duke Prediction Center is to develop the analytic approaches and tools that will allow for the routine annotation of noncoding variation for functionality and ultimately pathogenicity. Aim 1 is to establish best practices in perturbation assay design and analysis. This will allow IGVF characterization centers design their experiments so that, when coupled with optimized analyses, the data produced will be maximally informative for subsequent predictive modeling. Aim 2 is to develop novel mechanistic machine learning approaches for predicting the functional effect of noncoding variation on function in diverse cell-types. Aim 3 is to identify noncoding genomic regions that are subject to functional constraint which will be leveraged in prioritizing variants for pathogenicity. The expected outcomes of this project will be (i) robust estimates of optimal experimental design parameters and recommendations for analysis tools and best practices for the various assays used within the IGVF consortium, (ii) predicted functional effects of observed variation to be shared through the IGVF variant/phenotype catalog as well as a state-of-the-art machine learning method (and associated tools) that can identify previously-unknown interactions among genomic variants, both observed and novel, and predict their functional impact in diverse cell types, and (iii) a list of regulatory elements subject to functional constraint shared through the IGVF variant/phenotype catalog and a principled prioritization framework (and associated tools) for interpreting variation within patient genomes for pathogenicity. Due to the considerable success of genetics, there are thousands of unknown regulatory causes of disease. Each of those causes is an opportunity to improve treatment, diagnostics, or prevention. This project will be a major advance towards unlocking that potential.

Destiny Arts Center

Support for the organization’s programs and activities: martial arts, dance, theater, youth leadership, and violence prevention at their main site, and outreach classes in schools and community centers.

NIMH - National Institute of Mental Health

PROJECT SUMMARY/ABSTRACT This K23 award will facilitate Dr. Grau’s transition into an independent investigator who develops and tests scalable interventions with attention to health disparities to meet the variation in needs among individuals with post-traumatic stress disorder (PTSD) and hazardous alcohol use (HAU). Targeted research and training activities will extend his knowledge and build his expertise in developing and testing scalable interventions that integrate therapist-delivered and digital components to effectively engage and treat patients with PTSD+HAU in under resourced community settings like Federally Qualified Health Centers (FQHCs). Training: Dr. Grau proposes a comprehensive training plan with the following training aims: (1) Develop expertise in mobile health interven-tions, (2) Gain skills in qualitative data collection and analysis and (3) Acquire key skills in and knowledge of optimization trial design and conduct. Context: PTSD is an extremely damaging psychiatric condition when left untreated, especially for individuals with low-income who are served in FQHCs. Brief, modified evidence-based PTSD treatments are effective and feasible in FQHCs; however, co-occurring problems, most notably HAU, negatively impact treatment engagement and effectiveness. As such, there is a critical need to develop scala-ble interventions that treat PTSD+HAU while maximizing engagement in under resourced community settings such as FQHCs. Responsive to the NIMH Strategic Plan, Goal 3 (Strive for Prevention and Cures), Objectives 3.2 (Develop strategies for tailoring existing interventions to optimize outcomes), and 3.3 (Test interventions for effectiveness in community practice settings), the overall research objective is to develop and test a multimodal intervention that integrates therapist-delivered and digital interventions to treat PTSD+HAU in FQHCs. Re-search Plan: Aim 1 will assess the feasibility and acceptability of three stepped care, therapist-delivered inter-vention components for PTSD+HAU in our partner FQHC. Aim 2 will refine the therapist-delivered components and develop digital components (e.g., personalized text messages) in preparation for a PTSD+HAU hybrid ex-perimental design (HED). Aim 3 will assess the feasibility, acceptability, and preliminary effectiveness of a PTSD+HAU HED in FQHCs combining therapistdelivered and digital interventions to maximize engagement in treatment. Aim 3 results will inform a fully powered HED (future R01 submission) to create a multimodal adap-tive intervention for PTSD+HAU with patients in community health settings. Key innovations include state-of-the-art training, PTSD+HAU scalable intervention development with real world patients in community settings, and use of the HED design. Optimization of adaptive interventions for community patients with PTSD+HAU is a critical next step and will have significant impact on PTSD treatment in FQHCs.

NIMH - National Institute of Mental Health

Abstract Despite antiretroviral therapy (ART), HIV-associated brain injury (HABI) persists in over half of people with HIV (PWH), manifesting as chronic cognitive impairment. While HIV-1 primarily infects microglia, driving central nervous system (CNS) neuroinflammation, current preclinical models do not recapitulate the chronic, suppressed infection characteristic of the ART era. Furthermore, they do not capture complex patient genetics and multicellular, glial and neuronal, interactions in a scalable and efficient manner. To address this need for more physiologically relevant models, we propose the development of an AI-guided triculture platform comprising major CNS cell types. This platform will use induced pluripotent stem cell (iPSC)-derived microglia, astrocytes, and neurons, using both morphological profiling and other omics-based profiling to model HABI under ART suppression. AI/machine learning (ML)-driven analysis of cellular morphology, combined with multi-omic data integration, will facilitate rapid classification and prediction of microglial functional states and their impact on neuronal health. Leveraging Modulo's established triculture system, previously successful in yielding therapeutic candidates for amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) currently in Investigational New Drug (IND)-enabling studies, we will construct a scalable HABI model under ART suppression. Our objectives are to (1) develop and validate an HIV-infected, ART-suppressed triculture platform, utilizing AI/ML-driven morphological profiling to classify HABI-specific microglial states; and (2) comprehensively characterize this model through neuroinflammatory profiling, behavioral correlates, and integration with publicly available HABI patient datasets. We hypothesize that our combined computational lab-based triculture system can effectively model HABI pathophysiology under ART conditions, enabling both rapid disease state classification and identification of therapeutic targets. Through the integration of experimental and computational approaches, this platform will provide insights into HABI mechanisms and accelerate therapeutic development. We will disseminate this model to the scientific community through publication and collaboration. Connecting in vitro modeling with patient outcomes offers a powerful tool for investigating neuroimmune dysfunction in HIV and related neurological disorders. Successful implementation will yield a platform for modeling neuroHIV under ART suppression, advancing our understanding of disease mechanisms and facilitating the discovery of novel therapeutic strategies for PWH with cognitive impairment.

NIA - National Institute on Aging

Project Summary Anti-aging antibody research, including strategies targeting interleukins and other antigens, shows promise in rejuvenating the immune system, improving metabolic functions, and extending healthy lifespans. AI-driven platforms are revolutionizing antibody development by accelerating affinity maturation and optimizing developability properties, enabling simultaneous optimization of multiple characteristics. These advancements could lead to more effective treatments for age-related diseases and a significantly improved quality of life for the growing aging population. However, zero-shot predictions for antibody affinities using pretrained models without additional target-specific data remain challenging. In this project, we propose a new strategy to address this challenge by generating diverse antibody-antigen interactions at an unprecedented scale (Aim 1) and training new AI models using these generated data in combination with data collected from literature and public databases (Aim 2). We will rigorously evaluate the performance of the new models and benchmark against the state-of-the-art methods. We will test the generality of the new models on a diverse set of antigens and experimentally validate the prediction accuracy (Aim 3). We will apply the models to identify new antibodies against new therapeutic targets associated with ageing or age-related diseases. Once complete, the proposed research will provide a powerful tool for accelerating antibody discovery and optimization as well as new antibody candidates for anti-aging treament.

Food and Drug Administration

For generic drug development, population pharmacokinetics (popPK) analysis is a critical part of the emerging technology of model-based bioequivalence (BE) analysis. PopPK models provide support for generalizing the conclusion of BE to groups that were not included in a BE study. The popPK model selection is essentially a multiple-objectives/variables optimization problem. Recent years have witnessed the overwhelming success of the reinforcement learning (RL) approaches in addressing optimization problem. Thus, the objective of this project is to develop a model selection method for the popPK analysis using the deep-learning based RL algorithm. Specific Aim 1: Develop a model selection method using a deep-learning based RL algorithm. A thorough survey should be conducted to gain a good understanding of the current state of the art for deep-learning based RL algorithms and their applications. The most appropriate algorithm/pipeline should be adopted to develop the model selection method. Specific Aim 2: Design simulations reflecting different scenarios of PK data, such as independent/correlated covariates, simple/complex (e.g., multiple peaks) time-concentration profiles and sparse-sampling design. The simulated datasets should be used to conduct systematic performance checks. Specific Aim 3: Identify proper metrics for performance evaluation. The selected metrics should be unbiased and mathematically/statistically meaningful. Specific Aim 4: Conduct performance evaluation. The developed model selection method and at least a stepwise regression and a genetic algorithm-based approach should be applied to the simulated datasets to perform popPK model building. The selected performance evaluation metrics should be used to compare the performance of the different methods. Specific Aim 5: Use real PK dataset(s) to demonstrate the applicability and advantage of using the developed method in popPK model building.

NIGMS - National Institute of General Medical Sciences

Optical microscopy is a cornerstone of biomedical research, enabling detailed visualization of biological structures and processes. However, traditional refractive microscopes are inherently limited by chromatic aberrations, group delay dispersion, and narrow spectral ranges, while existing reflective designs suffer from central obscuration, reducing contrast and efficiency. These limitations restrict the ability to perform high-resolution, broadband, and multimodal imaging, hindering advancements in biomedical research. To address these challenges, we propose the development of an ultra-broadband microscope based on an innovative obscuration-free, off-axis freeform reflective optical architecture. This system is designed to achieve diffraction-limited performance across an ultra-broadband spectral range — from the ultraviolet (UV) to the infrared (IR) — while eliminating chromatic aberrations and minimizing optical dispersion. By leveraging freeform optics and an unobscured reflective design, this technology will provide superior contrast, higher optical efficiency, and enhanced imaging capabilities beyond the limitations of conventional refractive or reflective microscopes. A key innovation of this project lies in the development of off-axis freeform reflective configurations and novel optomechanical integration strategies to create a compact, ultra-broadband-compatible microscope. This project will focus on three key objectives: (1) Design and optimization of a compact, high- performance off-axis reflective microscope with diffraction-limited imaging across a wide spectral range. (2) Prototype fabrication and assembly, advancing high-precision diamond-turning techniques to ensure superior optical quality and robust system integration. (3) Performance validation through rigorous experimental testing across UV, visible, and infrared spectra, benchmarking the prototype against state-of-the- art commercial refractive and reflective microscopes. The anticipated outcome is a transformative microscope platform that enables high-contrast, ultra- broadband imaging with an unprecedented working spectrum. This technology will establish a new paradigm for high-performance optical microscopy, unlocking new opportunities in biomedical research and expanding imaging capabilities across multiple disciplines. This proposal directly aligns with the objectives of NIGMS NOFO (PAR-25-203) by advancing a demonstrated proof-of-concept ultra-broadband microscope into a fully functional prototype with broad applicability in biomedical and biological sciences research. By overcoming fundamental optical constraints, this microscope has the potential to transform biomedical imaging and biological research, facilitating new discoveries and expanding the frontiers of optical microscopy.