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Triangle Community Foundation

Triangle Community Foundation ($600M in assets) supports nonprofits in NC through grants focused on education, community development, arts, environment. Awards range from $2,500 to $150,000.

National Institute of Food and Agriculture

The TCEG program (assistance listing 10.221) is designed to help build Tribal education capacity in the food and agricultural sciences for Native Alaskan/American Indian students. This NOFO will begin a new four-year continuation funding cycle. The six legislatively mandated priorities are: Curricula design and materials development. Faculty development and teacher preparation. Student experiential learning Equipment and instrumentation for teaching science, technology, engineering, arts and math. Student recruitment and retention. Instruction delivery systems and strategic partnerships.

Friends of the Troll's Knoll

The Friends of the Troll's Knoll is a community group dedicated to promoting and strengthening the safety and security of the community by maintaining and activating the green space right-of-way on the east side of the Fremont Troll, located at N 36th St and Winslow Pl N Seattle WA 98103 and turning it into a public park. The group will hire a designer to create a design plan for the space and hire an artist to create and install artwork. The anticipated end date for the project is August 31, 2020.

Friends of the Troll's Knoll

The Friends of the Troll's Knoll (FOTK) is a neighborhood group dedicated to engaging the community in beautifying and activating the urban forest around the Fremont Troll. This grant will fund: (1) site survey of the land, (2) arborist analysis, (3) detailed planting plan, (4) installation of a willow art feature, (5) addition of a Japanese style shade structure, known as a hokora, and to the existing moss turtle artwork (installed by us in 2020). The anticipated project close out is May 31, 2022

Friends of Troll's Knoll

The funding will cover benches, decommissioning of the damaged Mushroom Lantern sculptures (funded by DON in 2021), replacement art with stainless steel sculptures, and erosion control. Currently, we do not have seating for mobility impaired visitors and would like to purchase and install sturdy horizontal basalt column benches. Our site is open 24/7 and free and open to the public. The anticipated project completion date is December 31, 2026.

Tulsa Community Foundation

Tulsa Community Foundation ($4.5B in assets) supports nonprofits in OK through grants focused on education, arts, social services, community development. Awards range from $2,500 to $250,000.

NCI - National Cancer Institute

PROJECT ABSTRACT This proposal seeks to overcome critical limitations in personalized therapeutic strategies for triple-negative breast cancer (TNBC), an aggressive subtype marked by poor prognosis. We propose developing personalized cancer treatment, designed to stimulate antigen-presenting cells and induce robust, antigen-specific T cell responses against TNBC cells. We develop cost-effective Tumor-derived Cell-membrane Coated NanoVaccine (TCC-NV) armed with adjuvants, that bypass limitation of resource-intensive neoantigen prediction, prioritization, and validation process. Our proposed TCC-NV integrated with radiotherapy (RT) addresses the challenge of low mutation burden in TNBC that often limits personalized neoantigen-based treatments. This multifaceted approach leverages immune-competent mouse models that closely mimic human TNBC, focusing on the innate and adaptive immune system's role in anti-tumor responses. Specific Aims are: 1) Evaluate antitumor activity of TCC-NV in TNBC tumor models. 2) Investigate radiation impact on increasing neoepitope presentation and determine whether immune checkpoint inhibitors (ICI) therapy can synergize with TCC-NV to eradicate TNBC tumors. The TCC-NV platform's production simplicity facilitates clinical translation and increases the potential for future clinical success in personalized treatment of primary and metastatic TNBC. This mechanistic study is related to the mission of the NCI as it aims to contribute to closing the knowledge gap in understanding the role and function of neoantigen-based cancer vaccine therapy and to provide survival benefits for TNBC patients who urgently require more effective treatment strategies beyond traditional chemotherapy. Dr. Yazdimamaghani’ s career goals are to integrate research from nanomedicine, radiation biology, oncology, and immunology to launch a robust and successful research program by receiving a tenure track faculty position. By leading multidisciplinary research to push the boundaries of personalized cancer immunotherapy and mentoring the next generation of scientists in immunoengineering, he aims to build a successful career as an assistant professor. His vision is to establish a highly collaborative research team focused on unraveling the complex dynamics of immune cell-biomaterial-tissue interactions within the TME, driving the development of innovative immunotherapeutic strategies for effective disease treatment. The NCI Transition Career Development Award is a vital cornerstone for successfully launching his independent research laboratory. It provides essential support for developing expertise in grant writing, effective research management, communication skills, and fostering constructive collaborations. Closely collaborating with Dr. Charles Perou, a world-renowned expert in breast cancer genomics and molecular subtypes, and Dr. Benjamin Vincent, a specialist in cancer immunogenomics, ensures comprehensive expertise, support, and access to state-of-the-art resources for the proposed experiments. Both collaborators are affiliated with the UNC School of Medicine and the UNC Lineberger Comprehensive Cancer Center, fostering an ideal environment for advancing this research.

The U District Partnership

The U District Mural Program is an initiative to build vibrancy in Seattle's University District by adding new works of public art. Murals will be installed in the following locations: 1313 NE 42nd Street, 1319 NE 43rd Street, and 4261 Roosevelt Way NE. This project will be completed by August 31, 2021.

U.S. Mission to Colombia

PAS Bogota invites proposals for programs that strengthen societal and cultural ties, shared values, and bilateral cooperation between the United States and Colombia in support of U.S. Embassy Strategic Goals. All programs must include a U.S. cultural or educational element or connection with American expert/s, organization/s, or institution/s in a specific field that will promote increased understanding of U.S. policy and perspectives, and build partnerships between our citizens. Examples of PAS Small Grants Program programs include, but are not limited to: Academic and professional lectures, seminars and speaker programs; Artistic and cultural workshops, joint performances, and exhibitions; Awareness-raising programs on the challenges persons with disabilities face accessing the democratic process, including the lack of reasonable accommodations and information to exercise their political rights. Cultural heritage conservation and preservation programs; Policy advocacy programs that promote and advance the human rights of historically underserved and marginalized groups such as Afro-Colombians, Indigenous, lesbian, gay, bisexual, transgender, queer, and intersex (LGBTQI+), persons with disabilities, and persons otherwise adversely affected by persistent inequality. Workshops to strengthen networks of U.S. government (USG) programs alumni, CSO/NGO networks, entrepreneurial networks, and/or educational groups. Priority Program Areas: The Embassy s Cultural and Educational Grants Program supports U.S. Mission Bogota s strategic objective of enhancing opportunities for citizen participation in support of peace through cultural and educational program and advances the diversity, equity, inclusion, and accessibility (DEIA) and climate and environment priorities. Diversity, Equity, Inclusion, and Accessibility (DEIA) Disability Rights: o Projects that advance the rights of persons with disabilities, build capacity of organizations that advocate for enforcement and effective implementation of disability-inclusive legislation and policies, and promote democracy and political participation of persons with disabilities, among others. o Projects focused on improving access to educational opportunities for persons with disabilities. LGBTQI+: o Policy advocacy programs that advance the rights of lesbian, gay, bisexual, transgender, queer, and intersex (LGBTQI+) persons, including efforts to safeguard LGBTQI+ youth from harmful practices (e.g., so called conversion therapy ). o Programs that improve the quality of investigative journalism and transparency, increase awareness of the impact of stereotypical and biased reporting on LGBTIQI+ persons and women, and help counter disinformation. Climate and Environment Environmentally-focused activities addressing the climate crisis, combating wildlife trafficking, fostering resilience, conserving nature, water security, and reducing harmful pollutants, including, but not limited to, awareness raising campaigns, leadership, or capacity-building training workshops for youth and underserved communities, a recycled art installation or competition, among others. In addition to the outlined priority program areas, the Public Affairs Section may give consideration to project proposals focusing on the following topics: Science, Technology, Engineering, Arts, and Mathematics (STEAM) Economic empowerment of women, girls, Afro-Colombians, Indigenous communities, the Venezuelan diaspora, LGBTQI+ persons, and other underserved populations. Any other initiatives supporting Colombia s transition to a sustainable and inclusive peace. Project proposals managed by teams of U.S. government (USG) program alumni or designed to strengthen the USG alumni network in Colombia are highly desirable and will be given priority.

U.S. Mission to Tunisia

U.S. DEPARTMENT OF STATE U.S. EMBASSY TO LIBYA, PUBLIC AFFAIRS SECTION Notice of Funding Opportunity (NOFO) Funding Opportunity Title: U.S. Embassy to Libya PAS Annual Program Statement Funding Opportunity Number: PAS Tripoli FY2024 CFDA Number: 19.040 Public Diplomacy Programs Maximum for Each Award: $25,000 USD PROGRAM DESCRIPTION The U.S. Embassy Tripoli Public Affairs Section (PAS) is pleased to announce that funding is available through its Public Diplomacy Small Grants Program. This is an Annual Program Statement, outlining our funding priorities, the strategic themes we focus on, and the procedures for submitting requests for funding. Please carefully follow all instructions below. The objectives of the Public Diplomacy Grant Program are to build capacity and community, promote social good, and enhance mutual understanding between the people of Libya and the United States. The U.S. Embassy to Libya is seeking projects that: Capitalize on arts initiatives to increase unity, social cohesion, and reconciliation that deepen Libyan national identity and are consistent with U.S. values. Promote leadership, positive community engagement, volunteerism, entrepreneurship, and soft skills development among youth, women, and underserved communities. Increase Libyan youth capabilities to help them explore and develop technological solutions for social problems through Science, Technology, Engineering, Arts, and Math (STEAM) programs. Projects that address environmental challenges to mitigate the effects of climate change are highly encouraged. Note: Alumni of U.S. Government funded exchange programs are encouraged to apply. Initiatives that support diversity and inclusion of minority groups and link with U.S. universities or organizations are also welcome. Additional information on this link: https://ly.usembassy.gov/notice-of-funding-opportunity-nofo/

U.S. Mission to Morocco

The U.S. Mission in Morocco s Public Affairs Office (PAO) is pleased to announce that funding is available through the Public Diplomacy Grant Program for projects ranging in value from $5,000 to $25,000. Projects for lesser or greater values will be considered on a case-by-case basis. The objectives of the Public Diplomacy Grant Program are to promote positive relations between Morocco and the United States; reinforce shared values; and connect Morocco s emerging leaders to the American people through projects that: Strengthen understanding of U.S. values and institutions; highlight U.S. culture, including American Studies, English language teaching/learning, and study in the United States; and support diversity, acceptance of minority groups, and other areas of mutual interest. Help Moroccan youth explore and discover their potential through innovative science, technology, engineering, arts, and math (STEAM,) programs, as well as entrepreneurship programs. Encourage Moroccan youth to participate in civic life through social entrepreneurship, volunteerism, and community engagement. APPLICATION PROCESSApplication DeadlinesApplications will be reviewed three times during Fiscal Year 2020. The deadlines for application are:Round 1: November 30, 2019Round 2: March 31, 2020Round 3: June 30, 2020Proposal FormatTo apply, please complete these forms in English:The Project Narrative (DOC 47 KB)The Budget Proposal (XLSX 22 KB)SF424 (Application for Federal Assistance Must be signed) (PDF 265 KB)SF424A (Budget Information) (PDF 1 MB)SF424B (Assurances Must be signed) (PDF 70 KB)Submit all forms in electronic format to: Rabatgrants@state.gov

U.S. Mission to Australia

The U.S. Mission to Australia s Public Diplomacy Section (PDS) announces an open competition to implement projects that advance U.S. economic, commercial, and security interests in Australia. This Annual Program Statement (APS) outlines strategic goals, expected outcomes, target audiences, eligibility criteria, and application guidelines for cooperative agreements ranging from $25,000 to $100,000, with a project duration of up to 24 months. Project proposals must address at least one of the following goals: Promote flag football in Australia; Combat antisemitism through Holocaust education; Strengthen Pacific Islands partnership by highlighting shared cultural values among the peoples of the United States, Australia, and the Pacific Islands focusing on arts, cultural heritage preservation, education, and training.In addition to aligning with one of the strategic goals, applicants should clearly explain how they advance American leadership and excellence and how the projects deliver measurable results. All programs must include a clear connection to or inclusion of American expert(s), organization(s), or institution(s) or cultural elements in a specific field that will promote increased understanding of United States policy and perspectives.Please read the entire APS package before submitting an application. Applications must be submitted by September 20, 2026, for projects beginning as early as October 1, 2026. For more information, contact PASGrantsAustralia@state.gov. Applications that do not meet the eligibility criteria and do not contain all of the required information will not be considered.

U.S. Mission to Australia

<p>The U.S. Mission to Australia’s Public Diplomacy Section (PDS) announces an open competition to implement projects that advance U.S. economic, commercial, and security interests in Australia.&nbsp;This Annual Program Statement (APS) outlines strategic goals, expected outcomes, target audiences, eligibility criteria, and application guidelines for cooperative agreements ranging from $25,000 to $100,000, with a project duration of up to 24 months.&nbsp;Project proposals must address at least one of the following goals:</p><p>·&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Promote flag football in Australia;</p><p>·&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Combat antisemitism through Holocaust education;</p><p>·&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Strengthen Pacific Islands partnership by highlighting shared cultural values among the peoples of the United States, Australia, and the Pacific Islands focusing on arts, cultural heritage preservation, education, and training.</p><p>In addition to aligning with one of the strategic goals, applicants should clearly explain how they advance American leadership and excellence and how the projects deliver measurable results.&nbsp;All programs must include a clear connection to or inclusion of American expert(s), organization(s), or institution(s) or cultural elements in a specific field that will promote increased understanding of United States policy and perspectives.</p><p>Please read the entire APS package before submitting an application.&nbsp;Applications must be submitted by September 20, 2026, for projects beginning as early as October 1, 2026.&nbsp;For more information, contact PASGrantsAustralia@state.gov.&nbsp;<strong>&nbsp;Applications that do not meet the eligibility criteria and do not contain all of the required information will not be considered.</strong></p>

U.S. Virgin Islands Council on the Arts

Arts and cultural grants for organizations in U.S. Virgin Islands, supported by NEA federal funding.

Over Educated Minority Artists - OEMA

The Labateyah Harvest Feast scheduled for Saturday, November 18th at 9010-13th Avenue NW, from 3:00-8:00 pm will be a time of art, music, dance, poetry, and food. This gathering of people young and old, is an ancient tradition of the Northwest Indigenous tribes. History is told and artwork is traded in an effort to create relationships. Music and dance are performed to inspire and uplift.

OD - NIH Office of the Director

Project Summary/Abstract The newly established Andrew & Erna Viterbi Family Vision Research Center at UC San Diego currently does not have an ultracentrifuge. As research laboratories move into the building, there is a critical need for core instrumentation to support their work. We have selected the Beckman Coulter Optima XPN-100 IVD ultracentrifuge and three rotors (Type 70Ti, SW32Ti, SW41Ti). This instrumentation will support the work of nine Major users for applications such as density gradient preparation, high-titer virus production, lipoprotein isolation, vessel isolation and more. While similar ultracentrifuges exist on UC San Diego campus, they are typically located within locked lab spaces designated for specific departments or building occupants. In addition, transporting samples between buildings can disrupt density gradients, compromise sample integrity and delay downstream processing. It is therefore critical for researchers to have access to an ultracentrifuge within the building. The ultracentrifuge will be housed in the shared equipment room of Andrew & Erna Viterbi Family Vision Research Center. The new research center, opening in June 2025, will have state-of-the-art wet and dry laboratories for vision research and clinical trials for precision ophthalmology, as well as administrative and educational spaces. The ultracentrifuge is an integral piece of equipment for many research groups relocating to the building, and access to it will directly support efforts to understand and treat ophthalmologic diseases.

OD - NIH Office of the Director

ABSTRACT The University of Rochester has a long, internationally recognized history of research about the effects of airborne substances in the lungs and other organ systems. Indeed, Rochester is the birthplace of inhalation toxicology, as technology was developed here during the Manhattan Project to conduct studies on the effects of radionuclides. The infrastructure has grown over the decades into a dedicated Inhalation Exposure Facility (IEF) that supports basic and translational research about how airborne substances, by themselves or in combination with other stressors, contribute to cumulative health risk across the lifespan. Ambient air pollution is a mixture of particles, gases, and semi-volatile constituents that exhibits temporal and spatial variability in composition and concentration. It continues to be a significant threat to human health. Ambient pollutant fine and ultrafine (UFP, <100 nm in airborne diameter) particles are largely combustion- derived, e.g., from industrial and traffic sources, and have carbonaceous, inorganic salt, and metal/metal oxides compositional signatures. UFPs are of particular interest as drivers of adverse health effects due to their high number concentrations in air, large surface area-to-mass ratios, ability to deposit efficiently throughout the respiratory tract, and potential for transport to extrapulmonary tissues upon inhalation exposure. The study of UFP exposure-related health effects is an area of strength for the Rochester IEF. Its infrastructure has supported ground-breaking discoveries about the effects of UFP in the lung and extrapulmonary organ systems (cardiovascular, central nervous); transport of inhaled UFP to the brain via the olfactory system; perturbations in learning, memory, and impulsivity behaviors; and the mechanisms that lead to these adverse effects. The IEF tools for generating and characterizing UFP exposures are heavily utilized by multiple investigators who are funded by NIH and other agencies. To support these funded projects and to ensure that the research is conducted in a rigorous manner, replacement of and upgrades to the equipment that is used to generate, monitor, and characterize UFP-rich aerosols for inhalation exposures are urgently needed. Thus, this application seeks support to purchase a new integrated UFP aerosol generation and characterization system with modernized safety and monitoring features. This integrated system allows direct translation between species: knowledge about human exposures to UFP can be leveraged to conduct evidence-generating mechanistic studies in animal models or cell culture systems and, vice versa, health outcome data from basic science models can be translated to human-relevant exposures. Without this system that represents the state-of-the-art, the translatability of findings from UFP inhalation exposures is significantly weakened and, by extension, the impact of findings on evidence-based decision making.

NIGMS - National Institute of General Medical Sciences

Project Abstract Despite over sixty years of research, many unanswered questions on Adeno-associated virus (AAV) biology exist. Following AAV delivery, a small fraction of AAV genomes persists as episomes, yet the mechanisms underlying this “Selective Persistence” remain poorly understood. Ours and others’ work has shown that AAV inverted terminal repeats (ITRs) play important roles in genome conversion, persistence, and safety. My recent work identified novel ITR design that attenuates toxicity and unique genetic “signatures” associated with long-term episomal persistence in mice, we have further shown proof-of-concept that grafting these persistence “signatures” onto synthetic substrates prolongs gene expression in mice. However, it remains to be determined whether the persistence “signatures” we observed in mice are also present in humans. To address this, we will answer three questions in this proposed work. First, what is the fate of AAV genomes across human tissues, and what molecular features the AAV episomes possess? Second, can genome fate be modulated by ITR engineering to enhance stable episome formation and reduce vector dose requirements? Third, to what extent can AAV-derived persistence mechanisms be grafted onto nonviral vectors to achieve durable expression? My lab integrates virology, genome engineering, genome–protein profiling and organ-on-chip systems with state- of-the-art techniques: long-read Sequencing, Spatial Biology, and Mass Spectrometry. Uniquely, through the human decedent H2H platform, we can directly examine AAV genome fate across human tissues—an unprecedented opportunity for the field. By combining these technologies, we may uncover how AAV episomes form, persist, and interact with host factors, expand our understanding of AAV biology, provide a foundation for safer and more efficient AAV therapies, and pioneer cross-platform strategies to enhance the durability of nonviral (including synthetic substrates) gene delivery.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT The filoviruses Ebola virus (EBOV) and Marburg virus (MARV), cause severe disease in humans with high case fatality rates. Advanced metagenomic analyses led to the discovery of previously unknown filoviruses from a range of animal species. Newly discovered filoviruses include Lloviu virus (LLOV) and Dehong virus (DEHV). While recent work on LLOV suggests that it might not pose a threat to human health, it is not known if DEHV can cause disease in humans. The discovery of novel filoviruses provides an excellent research avenue for determining the molecular correlates that define pathogenicity or protection by comparing closely related pathogenic and potentially nonpathogenic viruses. In this application, we propose to perform comparative studies with pathogenic (EBOV, MARV), likely nonpathogenic (Reston virus, LLOV), and filoviruses of unknown pathogenicity (DEHV). We will analyze potential determinants of filovirus pathogenicity across viruses, including replication kinetics (Aim 1), the magnitude of the virus-induced inflammatory response in macrophages (Aim 2), and virulence in two humanized mouse models (Aim 3). By integrating findings from all three aims, we will be able to define key molecular signatures of filovirus infection that have the potential to inform the assessment of the pathogenic potential of known and newly emerging filoviruses. In Aim 1, we will compare the replication kinetics of the various viruses in distinct cell types, which will inform about the virus-intrinsic and cell-dependent factors determining replication efficiency. We will perform RNA FISH analysis to gain insight into the earliest events of viral transcription and genome replication at single-cell level. Since viral replication kinetics determine the timing and rate of viral RNA production and protein expression, they may play a major role in shaping antiviral host responses and virulence. In Aim 2, we will comprehensively profile the phosphoproteomic/proteomic and transcriptomic changes in filovirus-infected human macrophages to map the differences in the host response signatures induced by pathogenic and nonpathogenic viruses. These analyses will be accompanied by mechanistic studies aimed to dissect the molecular mechanisms of filovirus-induced immune activation using knockdown cells and targeted inhibitor approaches. In Aim 3, we will comparatively assess the ability of the various filoviruses to cause disease in two human immune system (HIS) mouse models. This includes state-of-the-art histopathological analysis of the tissues including spatial transcriptomics. Our multidisciplinary team combines expertise in filovirus biology, virus-induced activation of innate immune cells, omics analysis, humanized mouse models, and histopathology of filovirus-infected tissues. We are therefore well positioned to perform the proposed work.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary The overarching goal of this R03 proposal is reduce the moisture sensitivity of dental adhesives by incorporating cellulose nanocrystals (CNCs). This work is motivated by prior research in related engineering applications (e.g., pressure sensitive adhesives, coatings) demonstrating that CNCs decrease moisture sensitivity of polymeric matrices. Despite this prior art, systematic investigation of CNC impact on dental adhesive materials has yet to be undertaken. This constitutes a major gap, as moisture sensitivity is a significant contributor to the high rate of secondary caries and therefore identifying benign materials that can address this issue is of utmost importance. Furthermore, the proposed specific aims will determine fundamental interactions between CNCs and water, determining if ‘bound’ water within a polymerized adhesive delays or eliminates water-mediated degradation mechanisms. I am qualified to lead this project, as previous and ongoing work from my research team investigates reduced water sensitivity in food packaging and adhesive materials modified with cellulose nanomaterials. Furthermore, my expertise in photopolymerized polymer networks employed as dental materials complements this expertise and makes me uniquely qualified to oversee this investigation. Building upon this foundation and expertise, the overarching hypothesis for this proposal is that long-term stability of the adhesive layer will be enhanced when CNCs are uniformly incorporated and distributed within a photopolymerized dental adhesive. This project consists of two Specific Aims. In Specific Aim 1, I will determine how the distribution of CNCs varies based on the composition of model self-etch adhesives, and how this distribution impacts moisture sensitivity, network properties, and adhesive performance when exposed to moisture. This is motivated by the high degree of heterogeneity associated with currently employed adhesive systems. In Specific Aim 2, different surface functionalizations will be explored to optimize and modify the distribution of CNCs within adhesive networks and potentially improve the impact of these additives on adhesive performance. Given the heterogeneous nature of adhesive materials, I expect surface functionalization will enable more effective distribution. This area of research constitutes a new domain at this early-stage of my career, and thus the results from this award will serve as motivation to investigate a more diverse range of bio-sourced nanomaterials (e.g., functionalization, geometries) for restoration systems where biomaterial-water interactions need to be tailored.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Cocaine use disorder (CUD) is highly comorbid in people with HIV (PWH) and can accelerate infection, alter neuropathology, and exacerbate cognitive decline despite antiretroviral therapy (ART). Many of these effects are due to the infection and dysregulation of CNS-associated myeloid cells, especially microglia, which comprise a significant reservoir in this compartment. However, the precise mechanisms by which cocaine (Coc) dysregulates microglia to enhance HIV infection are unclear, partly due to the lack of translationally relevant human microglial models suitable for mechanistic evaluation of Coc-mediated changes in viral dynamics. Classically, Coc has been thought to act by blocking dopamine transporter (DAT) activity, exposing microglia to aberrantly high dopamine concentrations. Our data show that dopamine can increase HIV infection and inflammation in microglia and other myeloid cells. However, recent data show that Coc has other mechanisms of action beyond the modulation of dopaminergic tone, involving the ER protein sigma1 (σ1), which has diverse cellular functions including the modulation of cellular stress pathways such as the unfolded protein response (UPR). Viruses, including HIV, can exploit the UPR to amplify stress-induced protein production in the host cell, enhancing viral replication. Our preliminary studies indicate that Coc’s effects on σ1 may drive a Coc-mediated increase in HIV infection in microglia, potentially through increased stress response and independent of dopamine’s effects. My preliminary data show that both Coc and σ1 agonists increase HIV replication in human inducible pluripotent stem cell (iPSC)-derived microglia (iMg). These effects are blocked by σ1 antagonism but not by inhibition of DAT or dopamine receptors. We also show increased σ1 protein expression and recruitment to the ER/nuclear envelope space in HIV-infected iMg treated with Coc, and preliminary single-cell RNAseq data suggest changes in the UPR. Therefore, we hypothesize that Coc-mediated activation of σ1 increases HIV infection of microglia via activation of the UPR. In Aim 1, we will test the involvement of σ1 in driving Coc-mediated changes in HIV infection of iMg using pharmacological and genetic modulation, and we will also confirm the absence of dopaminergic involvement. We will assess changes in viral dynamics using AlphaLISA and immunofluorescence (IF) high-content imaging. In Aim 2, we will test the hypothesis that Coc induces greater σ1 activity in the presence of HIV infection utilizing confocal and high-content IF imaging of σ1 subcellular localization in cellular compartments like the nuclear envelope, ER, and mitochondria-associated ER membrane. Movement of σ1 to these compartments is a feature of σ1 activation. In Aim 3, we will use single-cell RNAseq to test the hypothesis that Coc-induced σ1 activity drives increased HIV infection in iMg via upregulation of UPR genes. The results from these experiments will not only define novel interactions between HIV and σ1 that could reveal new antiretroviral targets but will also broadly inform on the role of σ1 in microglia and potentially identify biomarkers for prevention strategies against CUD and its associated comorbid diseases.

NIGMS - National Institute of General Medical Sciences

Abstract This proposal seeks to understand emerging non-canonical roles of phosphagen kinases in cellular and organismal stress responses. Phosphagen kinases are a class of enzymes that include creatine kinases. They are well-known to support energy balance, but other functions are less well-understood. Recent research suggests that augmenting the activity of these enzymes may be sufficient to increase cellular stress responses, which are important in preserving tissue health during aging and disease. This makes phosphagen kinases potentially exciting targets for aging and disease intervention, but we still don’t fully understand how they work. Furthermore, their functions may depend on which tissues different versions of the enzymes are present in and what they are doing in each tissue. Understanding tissue-specific roles of enzymes is challenging in humans and laboratory models such as rodents and cell culture. In contrast, the roundworm, C. elegans, is a well-established and efficient model system that is perfectly suited for addressing these questions, and it shares many genes and biological processes in common with humans. In this study, we propose to use this model to: 1) Identify which of three different versions of phosphagen kinases support tolerance to diverse stresses 2) Test the hypothesis that phosphagen kinases protect cells by acting similarly to antioxidants and preserving the function of energy machinery. Simultaneously, identify which tissues are most affected by phosphagen kinase function and sensitive to its loss through tissue-specific genetic manipulations. 3) Determine whether phosphagen kinase function changes the way other enzymes and genes act to support stress response using new genetic sequencing technologies. Altogether, this work will illuminate mechanisms of phosphagen kinase function under stress. paving the way for novel aging and disease interventions. Furthermore, through this funding, at least 9 undergraduate students and 2 graduate students will be trained in state-of-the-art biomedical science, fostering the next generation of scientists and America’s continued superiority in biomedical research.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract The forkhead box (FOX) family of transcription factors (TFs) regulate key biological processes across eukaryotes. One member, FOXG1, functions as a transcriptional repressor which regulates neurodevelopment within embryonic brains, and has been implicated in a range of neurodevelopmental disorders. The FOX family of TFs is defined by the presence of a conserved DNA-binding domain (DBD). The molecular mechanisms of this DBD on its own, and how mutations to this region impair its function and lead to disease, have been well studied. However, how the intrinsically disordered regions (IDRs) outside of the folded DBD influence FOX TF function remains understudied. There is a critical need to understand the role of these IDRs given that dozens of disease-associated mutations have been identified within the IDRs of FOXG1. In the proposed work, we aim to investigate the role of IDRs on FOXG1 chromatin binding, nuclear organization, and function in gene regulation. My preliminary data have revealed that FOXG1 forms nanoscale clusters in nuclei of live cells which drives its tight binding to chromatin. However, a disease-associated variant of FOXG1, which has truncations within its central IDR, no longer forms DNA-associated clusters. Additionally, I found that FOXG1 forms biomolecular condensates via phase separation which contributes to its association with DNA. Biomolecular condensates are dynamic structures often formed through IDR-mediated phase separation and have been recently implicated as an underlying mechanism for TF clustering in vivo to regulate gene expression at specific genomic loci. Intriguingly, we found that mutations within the same central IDR of FOXG1 which show a loss in nuclear clustering also show a loss of phase separation. We have identified a conserved enrichment of aromatic amino acids within the central IDR of FOXG1 using a state-of-the-art sequence analysis approach in collaboration with Dr. Rohit Pappu’s group at WashU. I have determined that these conserved aromatic residues are critical for FOXG1 phase separation, nuclear clustering, and chromatin association. From this preliminary data, I hypothesize that FOXG1 utilizes IDR-driven phase separation to form nuclear condensates which directly regulate its chromatin binding and function as a transcriptional repressor. We propose to test this hypothesis with a series of computational, biophysical, and cellular approaches designed to 1) identify critical sequence features within FOXG1 IDRs and predict their function on phase separation and chromatin binding, 2) quantify the effects that individual IDRs and critical amino acid residues have on FOXG1 phase separation, 3) determine how FOXG1 IDRs influence its DNA binding strength at a single-molecule level both in vitro and in cellulo, and 4) identify the role of FOXG1 IDRs in transcriptional repression. Collectively, the results from this study will provide critical insights from single-molecule to mesoscale levels into how IDRs regulate FOX TF activity and how mutations in these regions can lead to disease.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY mRNA & lipid nanoparticle (RNA-LNP) therapeutics are a versatile new drug modality used for vaccines, gene editing, cell reprogramming, & protein replacement. While vaccines aim to stimulate immunity, other applications must avoid immune activation. Achieving the right immune outcome is critical, as unintended responses can lead to harm or therapeutic failure. Much of the focus of RNA-LNP immunity has been on professional antigen presenting cells (pAPC), such dendritic cells (DC). However, RNA-LNP transfect diverse cell types, including hepatocytes & skeletal myocytes, yet little is known about the role, if any, these cells play in RNA-LNP immunity. To understand better the role of non-pAPCs in the immune response to RNA-encoded protein – with the goal of establishing rules for the design of RNA-LNP that can more precisely & safely control the direction of the immune responses – we generated RNAs that are selectively silenced in different cell populations. Our preliminary data indicate that RNA expression within hepatocytes greatly reduces the quantity of Ag-specific T cells, whereas RNA expression in muscle fibers boosts Ag-specific T cells; establishing that these cells are not bystanders, but active participants, in how RNA-LNP immunity unfolds. We also find that unrestricted RNA expression can lead to hepatic & muscle cell killing when there are pre-existing Ag-specific T cells, and this toxicity can be ameliorated using synthetic target sites for hepatocyte- or muscle-specific miRNA to silence the RNA in these cells. The objective of this project is to elucidate the mechanisms by which RNA-LNP transfection & expression in pAPCs and non-pAPCs, specifically myocytes & hepatocytes, modulate the T cell response to RNA-encoded protein, and to harness these insights to improve the safety and efficacy of cancer and CAR T cell vaccines. In Aim 1, we will determine how RNA expression within hepatocytes dampens T cell response, and test whether silencing RNA cancer vaccines in hepatocytes can enhance tumor immunity & reduce toxicity. In Aim 2, we will test the hypothesis that the T cell response to RNA-LNP is boosted by RNA expression in muscle fibers that serve as a prolonged Ag reservoir that maintains T cell stimulation. In Aim 3, we will test the hypothesis that RNA-LNP transfection of hepatocytes & myocytes turns on stress & infection response pathways that results in RNA-derived antigen being transferred from non-pAPCs to pAPCs for T cell education. These studies will utilize a variety of state-of-the-art models, methods, & technologies, including novel RNA formulations for tailored cell expression, genetic mouse models for immune analysis, spatial transcriptomics for identifying the pathways activated by RNA-LNP in tissues, & normathermic machine perfusion to study RNA-LNP in whole human livers. This project will: (1) help define our understanding of how myocytes & hepatocytes contribute to RNA-LNP immunity, (2) provide novel approaches for modulating the direction of RNA-LNP immunity, and (3) enable more tailored design of RNA cancer vaccines that maximize immune activation & minimize healthy cell killing, which can be used to improve immunotherapies.