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NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Cryptococcal meningoencephalitis (CM) is a devastating opportunistic infection of the central nervous system due to Cryptococcus neoformans (Cn), an encapsulated fungus that mainly occurs in people with HIV-associated immunosuppression. In 2020, globally, 4.4% of people with HIV (PWH) and <200 CD4 T cells/µl had cryptococcal antigenemia (CrAg) and 19% of all AIDS-related mortality was caused by 152,000 cases of CM. CrAg screening, antiretroviral therapy (ART), and intensified antifungal regimens have led to earlier diagnosis and improved CM outcomes. Yet, in 2019 the total economic burden of cryptococcosis in the United States was $534,594,527, and globally, mortality remains high, 13-40%. There is an urgent need for more effective therapy for CM+ PWH. While HIV-associated CD4 T cell loss is the main CM risk factor, other factors are at play since CD4 alone does not identify those who will develop CM or respond to treatment. Since antibody immunity is also impaired in PWH, we posit immunoglobulins (Igs) in human sera that bind Cn due to natural reactivity with fungal glucans, latency, and/or environmental exposure enhance control of Cn. We showed Cn capsular glucuronoxylomannan (GXM)-IgG is ubiquitous in adult and children’s sera, and PWH with reactivation (CrAg+) and disease (CM) have higher levels than those with latency (CrAg-, CM-). In marked contrast, levels of IgM/IgG/IgA that bind glucan constituents of the Cn cell wall; β-(1-3)-rare β-(1-6)-glucan (Lam(inarin), β-(1-3)-glucan (curdlan) and/or β-(1-6)- glucan (pustulan), were lower in PWH with reactivation (CrAg+) and disease (CM) than latency (CrAg-, CM-), suggesting a deficit of certain glucan-binding Igs in reactivation and disease (states of fungal growth) may impair Cn host defense. Pre-existing (natural, not elicited by infection) human Igs bind conserved glucan determinants on fungi, and IgG2 is their main subclass. We found plasma IgG2 was higher in CrAg+ than CrAg-, but lower (as was IgA) in CM+ than CM- PWH, suggesting reactivation elicits Igs that may contribute to control of Cn. The hypothesis of this application is: human Igs which target defined glucan epitopes modulate Cn virulence, enhance host control of Cn, and correlate with survival in CM+ PWH. Our specific aims are: 1. To identify Cn glucan epitopes targeted by human Igs and determine Ig antifungal activity in vitro. 2. To produce glucan-binding human monoclonal antibodies (huMabs) and identify their effects on Cn biology and functional efficacy in vitro and virulence in mice. 3. To seek associations between defined plasma glucan-specific Ig levels and survival in CM+ PWH in the Ambition CM treatment trial. This project leverages basic and translational approaches to identify human Ig-mediated effects on Cn biology and virulence characteristics and seek glucan-Ig correlates of CM survival in the landmark 844 participant Ambition trial, in which mortality was 25-29%. There is an urgent need for more effective and innovative approaches to CM therapeutics. Knowledge gained from this project will provide biomarkers of mortality risk, and the huMabs generated will be poised to advance CM therapy as novel agents to enhance antifungal drug efficacy and improve clinical CM outcomes.

NIDA - National Institute on Drug Abuse

Project Summary Compared to HIV seronegative persons, people with HIV (PWH) suffer from increased rates of comorbidities, including HIV-associated neurocognitive impairment (NCI). These HIV-associated comorbidities are driven, in part, by chronic inflammation and immune cell dysfunction. The drivers of this inflammation and exact mechanisms remain unknown. However, the inflammasome is increasingly recognized as an important mediator of the innate immune response and a driver of comorbid diseases via accelerated aging termed “inflammaging”. Indeed, HIV itself has been described as an accelerated aging process possibly mediated via its associated chronic inflammation. Supporting this theory are studies which have demonstrated that HIV proteins and nucleic acids can activate the NLRP3 inflammasome and IL-1β production in immune cells. However, co-existing comorbidities, such as drug use, are also immunomodulatory and introduce confounding immunological effects that make the study of inflammasome activation in PWH difficult. Importantly, cannabis use is highly prevalent among PWH, and increasing evidence suggests cannabis has important immunomodulatory effects. However, the strength and directionality of cannabinoid modulation of the inflammasome in PWH remains and its impact on HIV-associated comorbidities, such as NCI, remain largely unknown. To address this challenge, this proposal will determine the impact of recreational cannabis use on the NLRP3 inflammasome in PWH leveraging biospecimens from a unique cohort of clinically and neurologically phenotyped HIV-infected CB users and non-users. We have recently found that, compared to non-users and cannabis/tobacco dual users, cannabis users exhibit distinct miRNA profiles within the CSF and plasma. Additionally, preliminary work by our group has shown that ex vivo exposure of monocytes from PWH to 9- tetrahydrocannabinol (THC) potently suppresses expression of pivotal inflammasome associated genes. To confirm these observations and further elucidate the impact of cannabis on NLRP3 inflammasome activation, this project will first determine the impact of CB use on systemic inflammation, NLRP3 inflammasome activation, and immune cell function in PWH on ART via immunophenotyping. We will then examine the impact of cannabis use on the “gut-brain immune axis, via assessing markers of intestinal integrity, blood-brain barrier (BBB) permeability and CNS activation. Lastly, we will conduct mechanistic ex vivo single cell transcriptomic experiments aimed at determining the mechanisms by which THC, CBD, and other cannabinoids modulate inflammasome activation in monocytes from our well characterized cohort of cannabis using PWH. This project will elucidate the impact of cannabis use on NLRP3 inflammasome activation in PWH to develop specific therapeutic targets for modulation to reduce inflammation associated HIV comorbidities such as NCI.

NIDA - National Institute on Drug Abuse

Cannabis use is high in PLWH, ~25-35% of PLWH report cannabis consumption. Cannabinoids have been shown to have anti-inflammatory properties in humans and animal models. Heavy cannabis use in PLWH on ART is associated with decreased levels of activated T cells in peripheral blood. The two main cannabinoids present in cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC, the psychoactive component of cannabis, is a partial agonist for cannabinoid receptors CB1 and CB2. CB1 is primarily expressed in the brain while CB2 is reportedly expressed in immune cells, including CD4+ T cells. THC exposure modulates human CD4+ T cell gene expression and in animal models skews immune responses towards a Th2 phenotype and increases CD4+ T cell production of anti-inflammatory and immunosuppressive cytokines. CBD has anti-inflammatory and antioxidant effects without the psychoactive properties of THC. CBD exposure has been shown to suppress T cell proliferation, T cell production of IL-2 and IFN-γ production in T cells. Cannabinoid treatment has also been shown to suppress neuroinflammation in animal models of Alzheimer’s disease and multiple sclerosis. Importantly, in non-human primates (NHP) cannabinoid treatment reduced SIV-induced neuroinflammation. Based on these immunomodulatory properties of cannabinoids, we hypothesize that cannabinoids impact HIV infection and persistence in the CNS. Currently, it is not known how cannabis use impacts the phenotype of CD4+ T cells in the brain and their relative abundance and distribution. Therefore, as part of this project we will evaluate the effect of cannabinoids on the natural homeostasis of human T cells in the brain. We will then assess the impact of cannabinoids on the seeding of HIV infection in CD4+ T cells in the brain, the establishment of HIV latency in brain CD4+ T cells, and the efficacy of latency reversal agents to induce HIV expression in CD4+ T cells in the brain under ART-suppression.

NIGMS - National Institute of General Medical Sciences

The Institute for Biomolecular Targeting (bioMT) infuses mechanistic investigations with a sophisticated awareness of therapeutic potential and engages translational approaches to complex diseases such as cancer and infection. In Phases I and II, our first three cohorts of research project leaders (RPL) have all received R01- equivalent funding or major venture investments, and our newest cohort is on track for similar success. Our cores provide unique protein biochemistry resources and ‘navigators’ to access microscopes campus-wide. Our core training sessions and our research seminars create a dynamic community. In Phase II, we have developed comprehensive charge-back structures and advanced educational programs. Phase III COBRE and institutional programmatic funds will enable us to deploy these new funding sources effectively over the coming five years. Phase III funding will also allow us to continue to expand the bioMT research community and launch new and collaborative research initiatives. Aim 1 is to maintain our commitment to research excellence and to refresh our intellectual community and ensure a strong foundation of investigator-initiated awards through targeted faculty development funds and new pilot projects aligned with the bioMT mission. Dartmouth recently identified our domain as a strategic priority for institutional investment, and we expect to once again exceed our target of one co-recruitment per year in Phase III. Pilot funds enable faculty to parlay emerging high-risk/high-reward research directions into long-term extramurally funded programs, and we will maintain career mentoring for our graduated RPLs and for newly hired junior faculty through promotion. Our Administrative Core will also continue to nurture a vibrant, curious, and interconnected research community. Aim 2 proposes to enhance our state-of-the-art core facilities, which will continue to serve a large group of bioMT investigators. All member laboratories will continue to benefit from our high-quality, responsive research cores. New equipment grants and additional training of core staff will continue in areas of critical need identified by our advisory councils. Core staff will continue to provide expert training. Aim 3 will focus on implementation of a multi-pronged, resilient strategy for the continued availability of core resources after the end of COBRE funding. Having developed comprehensive cost-recovery mechanisms, we will implement them step wise to enable faculty to budget accordingly, while covering a growing share of costs. We will also launch learner-centered workforce development programs that have been extensively prepared during Phase II. These programs will leverage core resources and hire dedicated staff to equip trainees for industry or PhD placements. bioMT-coordinated internships will lead them to high-value jobs, while building lasting partnerships with local and regional biotechnology partners and offsetting core costs. Taken together, these aims will expand and elevate our scholarly impact and provide financial resources to establish bioMT as a nationally visible academic enterprise central to the intellectual landscape at Dartmouth.

Magnolia Chamber of Commerce

This grant will fund Phase 2 of the Magnolia Chamber of Commerce mural project that was developed during 2017 with a 2017 NMF award. The mural project will enhance Magnolia Village's downtown core by painting a vibrant, interactive mural integrating art designs from neighbor school children, with a culminating community celebration event. The content of the mural is based on representing groups of three different species birds flocking together: A visual metaphor of innovative collaboration and interaction that many parts make a whole.

It Takes a Village

The Middle Passage Healing Project will foster unity among African American and East Africans communities through events such as Culture Fest, outreach sessions and cultural immersion healing circles. The community events will include digital storytelling, dance performances, visual art, musical entertainment, jewelry making, and sharing cultural recipes.

NCI - National Cancer Institute

Project Summary Somatic mutations of PHF6 are common in diverse hematologic malignancies, including T-cell acute lymphoblastic leukemia (T-ALL, 15%), mixed phenotype acute leukemia (MPAL, 23%), acute myeloid leukemia (AML, 3%), chronic myelomonocytic leukemia (CMML, 5%), and myelodysplastic syndrome (MDS, 3%). PHF6 mutations confer worse overall survival in AML and MPAL. Despite the clinical significance, the molecular mechanisms underlying PHF6 mutation mediated leukemogenesis remain to be explored. The PHF6 genetic lesions in hematological malignancies are largely frameshift and nonsense. The PHF6 gene is located on the X chromosome, and its mutation was thought to be a loss-of-function mutation. However, Phf6 D/D mice do not develop spontaneous hematologic malignancies. A recent study (Ahmed et al. Hum Mol Genet 2023) and our preliminary data showed that truncated PHF6 proteins were detectable in blood cells from three BFLS patients and an HPB ALL cell line with PHF6 truncation mutations. Furthermore, while PHF6 mutations were originally reported predominantly in male T ALL patients, subsequent studies from multiple independent groups have failed to show such a biological sex preference. All these data led us to hypothesize that the truncated PHF6 may exert gain-of-function, in addition to a loss-of-function, in leukemogenesis. We thus generated patient derived mutant Phf6 transgenic (Phf6R274XTg and Phf6R342XTg) mouse models by expressing truncated FLAG-PHF6aa1-273 or -PHF6aa1-342 protein in the hematopoietic lineages. Unlike Phf6 D/D mice, Phf6R274XTg mice and Phf6R342XTg mice developed a spectrum of spontaneous hematologic malignancies, recapitulating the characteristics of PHF6-mutated hematologic malignancy patients. Our goal is to decipher the molecular mechanisms through which the PHF6aa1- 273 leads to leukemogenesis. Three specific aims are proposed. Aim 1: Characterize the hematologic malignancies driven by truncation of PHF6 expression in vivo; Aim 2: Decipher the molecular mechanisms by which truncation of PHF6 expression leads to leukemogenesis; and Aim 3: Explore the therapeutic potential of targeting the truncation of PHF6-enhanced KAT6A/B activity in hematologic malignancies. Our newly generated mouse models, state-of-the-art epigenetic assays, and our collaborative team with longstanding expertise in HSPC biology and hematologic malignancies offer us cutting-edge platforms to successfully carry out the proposed studies. Successful completion of the proposed studies will fill a knowledge gap of the molecular mechanisms underlying PHF6 mutation-mediated hematologic malignancies, which will be pivotal for identifying novel therapeutic targets (s).

The Montana Community Foundation

The Montana Community Foundation ($150M in assets) supports nonprofits in MT through grants focused on education, environment, community development, arts. Awards range from $1,000 to $50,000.

National Institutes of Health

The National Institute of Diabetes and Digestive Diseases and Nutrition (NIDDK) seeks to advance its mission by continuing the work of the NIDDK Gastroparesis Consortium (GpCRC) but also to expand its scope. The collaborative efforts of the GpCRC provided a large database, the Gastroparesis Registry, which is located in the NIDDK central repository and contains information on patients with symptoms of either delayed or normal gastric emptying. It also houses the first U.S. registry of children and adolescents with gastroparesis. The GpCRC provided clarity and insight that set the stage for transforming our understanding of gastroparesis and laid out a road map for approaching other disorders of gastrointestinal (GI) motility. The findings from clinical studies and trials clearly demonstrated that the clinical burden of gastroparesis is significantly greater than previously realized and involves much more than the stomach. Importantly, the underlying mechanisms remain unclear. Interoception is the ability of the nervous system to sense, interpret and coordinate signals from various bodily systems including the GI tract. Many functional GI disorders are associated with a spectrum of overlapping symptoms including nausea, vomiting, and altered bowel habits all of which involve altered interoceptive signaling. This initiative would broaden the scope beyond gastroparesis to include other adult and pediatric GI conditions associated with impaired interoceptive processing to form a Disorders of Gastrointestinal Interoception Consortium (DGIC). The consortium may include up to 6 Clinical Research Centers (described in a companion notice) and a Scientific Data Research Center (SDRC). There would be an emphasis on multidisciplinary approaches that would reveal the underlying mechanisms that connect GI function more directly to symptoms, identify disease or response biomarkers that assess treatment efficacy, and leverage state-of-the-art technologies to identify novel therapeutic targets that could be assessed in future clinical trials. The SDRC will coordinate collaboration among the Clinical Research Centers, participant enrollment, biospecimen collections and processing, and manage the submission of data and samples to central databases and repositories.

National Institutes of Health

<p style="margin-left:0px;">The National Institute of Diabetes and Digestive Diseases and Nutrition (NIDDK) seeks to advance its mission by continuing the work of the NIDDK Gastroparesis Consortium (GpCRC) but also to expand its scope. The collaborative efforts of the GpCRC provided a large database, the Gastroparesis Registry, which is located in the NIDDK central repository and contains information on patients with symptoms of either delayed or normal gastric emptying. It also houses the first U.S. registry of children and adolescents with gastroparesis.&nbsp; The GpCRC provided clarity and insight that set the stage for transforming our understanding of gastroparesis and laid out a road map for approaching other disorders of gastrointestinal (GI) motility. The findings from clinical studies and trials clearly demonstrated that the clinical burden of gastroparesis is significantly greater than previously realized and involves much more than the stomach. Importantly, the underlying mechanisms remain unclear.&nbsp;&nbsp;</p><p style="margin-left:0px;">&nbsp;</p><p style="margin-left:0px;">Interoception is the ability of the nervous system to sense, interpret and coordinate signals from various bodily systems including the gastrointestinal tract. Many functional GI disorders are associated with a spectrum of overlapping symptoms including nausea, vomiting, and altered bowel habits all of which involve altered interoceptive signaling. This initiative would broaden the scope beyond gastroparesis to include other adult and pediatric GI conditions associated with impaired interoceptive processing to form a Disorders of Gastrointestinal Interoception Consortium (DGIC).&nbsp; The consortium may include up to 6 Clinical Research Centers and a Scientific Data Research Center (SDRC, described in a companion notice). There would be an emphasis on multidisciplinary approaches that would reveal the underlying mechanisms that connect GI function (e.g motility) more directly to symptoms, identify disease or response biomarkers that assess treatment efficacy, and leverage state-of-the-art technologies to identify novel therapeutic targets that could be assessed in future clinical trials.&nbsp;&nbsp;&nbsp;</p>

National Institutes of Health

<p style="margin-left:0px;">The National Institute of Diabetes and Digestive Diseases and Nutrition (NIDDK) seeks to advance its mission by continuing the work of the NIDDK Gastroparesis Consortium (GpCRC) but also to expand its scope. The collaborative efforts of the GpCRC provided a large database, the Gastroparesis Registry, which is located in the NIDDK central repository and contains information on patients with symptoms of either delayed or normal gastric emptying. It also houses the first U.S. registry of children and adolescents with gastroparesis. The GpCRC provided clarity and insight that set the stage for transforming our understanding of gastroparesis and laid out a road map for approaching other disorders of gastrointestinal (GI) motility. The findings from clinical studies and trials clearly demonstrated that the clinical burden of gastroparesis is significantly greater than previously realized and involves much more than the stomach. Importantly, the underlying mechanisms remain unclear.&nbsp;&nbsp;</p><p style="margin-left:0px;">&nbsp;</p><p style="margin-left:0px;">Interoception is the ability of the nervous system to sense, interpret and coordinate signals from various bodily systems including the GI tract. Many functional GI disorders are associated with a spectrum of overlapping symptoms including nausea, vomiting, and altered bowel habits all of which involve altered interoceptive signaling. This initiative would broaden the scope beyond gastroparesis to include other adult and pediatric GI conditions associated with impaired interoceptive processing to form a Disorders of Gastrointestinal Interoception Consortium (DGIC). The consortium may include up to 6 Clinical Research Centers (described in a companion notice) and a Scientific Data Research Center (SDRC).&nbsp; &nbsp;There would be an emphasis on multidisciplinary approaches that would reveal the underlying mechanisms that connect GI function more directly to symptoms, identify disease or response biomarkers that assess treatment efficacy, and leverage state-of-the-art technologies to identify novel therapeutic targets that could be assessed in future clinical trials. The SDRC will coordinate collaboration among the Clinical Research Centers, participant enrollment, biospecimen collections and processing, and manage the submission of data and samples to central databases and repositories.&nbsp; &nbsp;</p>

ADEFUA Cultural Education Workshop

The ODUNDE Festival, hosted by ADEFUA in collaboration with the community group `Friends of ADEFUA,` is a vibrant celebration of African culture and heritage. This project encompasses a rich array of the social Odunde-Odunde Music and Dancical presented by local and special guest artists to celebrate in community with the hosted African music and dance inter-active showcases, and an African Marketplace featuring arts, crafts, and food from various West African countries. The festival includes a silent auction, an Artist Meet and Greet Reception, serving an array of Desserts and a Wine Corner that highlights local African-owned wineries and Bakeries. Through these activities, the festival aims to educate, inspire, and provide a space for meaningful cultural engagement. Odunde Festival 2024, Kicks off ADEFUA'S 40th year of being. Several cultural artists and groups are a part of the annual production, ''Evolution of Culture in Community''.

NIMH - National Institute of Mental Health

ABSTRACT HIV treatment is an essential pillar of the End the HIV Epidemic (EHE) strategy. Yet, in 2021, of the 1.2 million people living with HIV (PLWH) in the US, only 50% were retained in care, 58% achieved viral suppression and significant racial inequities persisted for Black Americans. HIV treatment outcomes are driven by poor access to HIV clinics; and for those who are on treatment, many forget to take their daily oral antiretroviral therapy (ART). Long acting injectable (LAI) ART, approved for every 1- or 2-month administration, can combat memory barriers related to taking a pill every day, but HIV clinic access remains a challenge, especially for Black Americans who are less likely to live near an HIV clinic. To increase equitable LAI ART access and viral suppression, we propose integrating LAI ART in pharmacies. Despite promising evidence that pharmacies with specialized HIV training can increase ART adherence and viral suppression, pharmacies with specialized HIV training are uncommon, likely because no free, widely available HIV treatment pharmacy trainings exist. Our work has shown that expanding HIV services in pharmacies could drastically increase access, but training is critically needed to develop a model for integrating LAI ART specifically into pharmacy workflow. Thus, we propose the Pharmacy- based LAI ART versus Clinic Effectiveness (PLACE) study to evaluate administration of LAI ART within pharmacies versus HIV clinics. We aim to 1) Examine the policy- and pharmacy staff-level barriers and facilitators of adopting LAI ART services in pharmacies among key stakeholders (policy members, pharmacy staff, HIV- clinic staff, and PLWH), 2) Develop and evaluate a pharmacy-based LAI ART training, and 3) Test the integration of pharmacy-based LAI ART delivery in pharmacies compared to HIV clinics on effectiveness, implementation, and clinical outcomes. Guided by the Exploration, Preparation, Implementation, Sustainment framework, this study will include three phases. An exploration phase of 300 online pharmacy staff surveys, and in-depth interviews of 10 Board of pharmacy members who shape pharmacy policy, 40 pharmacy staff, 10 HIV clinic staff (clinicians and nurses) and 40 diverse PLWH that examine the barriers and facilitators to pharmacy-based LAI ART provision. These data will inform the preparation phase which will develop a hybrid (virtual/in-person) LAI ART pharmacy training that will be tested among 100 community pharmacy staff. Finally, an implementation and sustainment phase, will evaluate the acceptability, feasibility, reach by race and ethnicity, uptake, costs, viral suppression and visit adherence of LAI ART service integration in 5 community pharmacies compared with 2 Ryan White funded HIV clinics among 222 LAI ART patients. Findings will inform a model that could be implemented across Ryan White funded clinics to scale pharmacy-based LAI ART services in the US Southeast where efforts to improve equitable HIV treatment and reduce HIV transmission are desperately needed to EHE.

The Pittsburgh Foundation

The Pittsburgh Foundation ($1.5B in assets) supports nonprofits in PA through grants focused on education, community development, health, arts, environment. Awards range from $5,000 to $200,000.

Power of Rage

A one-day event highlighting art, entertainment and food, including an indoor street fair will be held on Sunday March 8, 2020 at historic Washington Hall, 153 14th Avenue from 10:00 am - 6:00 pm. The program will feature local performers, visual artists, artisans, informational tabling from community organizations, and culinary delights provided by local pop-up restaurant chefs. This event is intended to promote acceptance of cultural, social, physical and racial differences while promoting civic participation.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Heart failure (HF) is a leading cause of mortality worldwide. The metabolism of the failing heart is commonly characterized by increased glucose uptake, glycolytic dependence, and reduced oxidative phosphorylation. We previously demonstrated that blocking glucose oxidation is sufficient to cause hypertrophy and subsequent HF. Additionally, our preliminary data shows that an altered pyruvate-lactate metabolic axis may be pivotal in human HF. Research investigating both the mechanistic regulation and biological roles of the pyruvate-lactate metabolic axis in cardiac metabolism during HF and cardiac recovery is warranted and also has the potential to identify novel druggable pathways to target for future pharmacological approaches. The overall objective of this application is to test the hypothesis that impaired pyruvate oxidation is a cardinal feature of HF in humans and animal models and that myocardial recovery is tightly coupled to normalization of the pyruvate-lactate metabolic axis. We will quantify the pyruvate-lactate metabolic axis in human HF and myocardial recovery (Aim 1). Next, we will determine the essentiality of the pyruvate-lactate metabolic axis for HF and cardiac recovery (Aim 2). Lastly, we will define cell-autonomous mechanisms that regulate the pyruvate-lactate axis in HF and recovery (Aim 3). These experiments will allow us to identify patterns of metabolic alteration in the pyruvate-lactate axis and molecular pathways during HF and myocardial recovery. Understanding the role of pyruvate and lactate metabolism in HF and myocardial recovery is cutting-edge research. Our unique access to human HF myocardium from patients administered stable isotope-labeled glucose or lactate to quantitate pyruvate metabolism in HF and recovery is state-of-the-art and will likely help us reveal new fundamental mechanisms of cardiac metabolism and expedite the successful translation of therapeutics being validated in various models of HF and recovery.

NIGMS - National Institute of General Medical Sciences

Project Summary Modern genome sequencing has shown that many species exchange genes with their close relatives through a process known as hybridization. As a result, the genomes of modern species are a mosaic of regions derived from past hybridization. Because of this, many species including our own must contend with the potentially negative consequences that can arise from mixing two divergent genomes. One of these negative consequences is the exposure of “hybrid incompatibilities” or genes that do not interact properly in hybrids. Uncovering the evolutionary forces that drive the formation of these hybrid incompatibilities is crucial to understanding how the genomes of modern species function. Although this is an important question, we have rarely been able to identify the genetic architecture of hybrid incompatibilities in vertebrates and lack the empirical data needed to understand what predisposes certain genes or genomic regions to negative interactions in hybrids. My postdoctoral research will investigate the evolution of a repeatedly evolved hybrid incompatibility in fish species where hybrid offspring from multiple crosses develop melanoma. I will combine classical genetic crosses, population genomics, and state-of-the-art functional genomic techniques to generate a comprehensive model of how hybrid incompatibilities evolve. In Aim 1, I will perform multiple genetic mapping crosses to identify genomic regions that drive hybrid melanoma. In Aim 2, I will characterize structural variation in the genome and its functional consequences on pigmentation genes involved in hybrid melanoma. Finally, in Aim 3, I will complement this work with a comparative genomic and transcriptomic approach to investigate how genes controlling pigmentation function within gene regulatory networks and become disrupted in hybrids with melanoma. Together, these approaches will give us unprecedented insights into how hybridization has shaped our genomes and the repeated origin of an evolutionarily and biomedically important phenotype. My primary goal under this NRSA F32 fellowship is to receive the scientific and professional training I need to establish my own independent research lab that unites molecular and computational biology with cutting-edge genomic approaches to establish models for how evolutionary processes shape genome content and function. As a postdoctoral fellow in the Schumer and Petrov labs at Stanford University, I will receive training in cutting-edge genomic techniques and analytical approaches. In addition to my scientific training, I will strengthen the professional skills needed to establish my future lab including grantsmanship, network building, and mentorship. In sum, with the training I will receive under this fellowship, I will be poised to lead a research program with great power to link molecular mechanisms to evolutionary outcomes and connect genotypes to phenotypes at the molecular and organismal level.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract: HIV reservoirs are present in several tissues, including the bloodstream, lymphoid, and CNS. However, the nature and mechanisms by which these viral reservoirs are generated and maintained under ART are unknown. The recent development of several techniques to detect, quantify, and characterize viral reservoirs in situ has provided a unique tool to examine these reservoirs. Our laboratory has become a major contributor to detecting rare viral reservoirs in tissues and bloodstream and the effects of drug use on viral reservoir formation and stability. Our imaging systems correspond to correlative microscopy (live-cell, confocal, and electron microscopy), with mass spectrometry imaging and spatial proteomic/mRNA. The combination of all these technologies is unique for infectious diseases. Our data clearly indicates that despite long- term ART and full systemic suppression, synthesis of viral proteins and defective pro-viruses is still occurring even after 26 years in ART. Viral proteins such as tat, gp120, and nef are produced by viral reservoirs and taken up for surrounding uninfected cells, triggering chronic neuro and immune system compromise. Interestingly, sex and drug are critical determinants of residual viral expression and the radius of diffusion of these viral factors. Our proposal is designed in response to the PAR-25-330 with a focus on defective pro-viruses in HIV persistence and pathogenesis during ART.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary/Abstract: This NRSA proposal, tailored to Ms. Hibbard, provides high-quality predoctoral research training and career development centered upon her future goals. The sponsor’s excellent mentoring record, collaborations with leading biomedical researchers, and the outstanding environment at the IUSM and Indiana Center for Musculoskeletal Health (ICMH) will foster the successful completion of this project. Additionally, participation in the Preparing Future Faculty and Professionals program for ethics and grant writing courses, manuscript preparation, departmental seminars and journal clubs, as well as national meetings will enhance Ms. Hibbard’s career development towards becoming a well-rounded, independent investigator. Patients with chronic kidney disease (CKD) develop renal fibrosis, which is a common pathological manifestation of virtually all etiologies of CKD, and one of the major causes of end-stage renal failure. Currently, there are no direct therapies for this manifestation. My preliminary data demonstrate that fibroblast activation protein (FAP) is highly expressed in CKD kidney, and our initial RNAseq datasets support that FAP is associated with altered myofibroblast motility and matrix composition. Thus, our models of the interactions between fibrosis and the effects of FAP on this patient phenotype remain incompletely understood. The primary goal of the present application is to test new hypotheses regarding drivers of CKD fibrosis, including FAP as well as its direct targeting in pre-clinical studies. Although my initial results show increased Fap mRNA and protein in the kidney of mice with CKD, the effects of targeting FAP to reduce renal fibrosis, are unstudied. Thus, the central hypothesis is: FAP increases renal fibrosis onset and progression in CKD through enhancing matrix secretion and cell migration, and CD5/LNP-FAPCAR will target FAP+ activated fibroblasts to reduce pathologic CKD outcomes. In Aim 1, the role of Fap in progression of CKD fibrosis will be tested using FAP-KO mice, and in isolated cell culture studies. Aim 2 will test the translational, pre-clinical rescue of renal fibrosis during CKD using a novel targeted anti-fibrotic therapy and scRNAseq. By performing these studies, Ms. Hibbard will gain new research skills in utilizing state-of-the-art translational mouse models, bioinformatic skills, and CKD treatments. Collectively, this proposal will also provide excellent research, ethics, and written and oral presentation training to Ms. Hibbard, as well as test important disease mechanisms that result in kidney fibrosis, and its potential resolution.

NIA - National Institute on Aging

PROJECT SUMMMARY/ABSTRACT Background: Aging is associated with reduced muscle mass and physical function, which may be exacerbated by age-associated diseases including peripheral artery disease (PAD). PAD affects about 8-12 million individuals in the United States and an estimated 10-15% of the population age ≥65. In PAD, skeletal muscle (SkM) metabolic dysfunction contributes to physical limitation and mobility disability. Few therapies have been identified that improve walking impairment in people with PAD; thus, therapeutic interventions targeting the pathophysiology of the SkM metabolic myopathy are promising. MicroRNAs (miRs) are powerful regulators of gene expression, specifically in the context of energy metabolism. MiRs have great potential as therapeutic agents due to the ability of a single miR to regulate entire pathways. In my preliminary studies, I found that miR-1, the most abundant miR in SkM, appears to play a critical role in hindlimb ischemia models and in human PAD. I developed a genetically modified mouse model for inducible, SkM-specific knockout of miR-1 and found that loss of miR-1 results in metabolic inflexibility and compromised running performance. Utilizing state-of-the-art experimental approaches (Argonaute (AGO) enhanced crosslinking and immunoprecipitation, coupled with high-throughput sequencing (eCLIP-seq)), I identified dysregulation of the pyruvate metabolic pathway as a mechanism for reduced SkM oxidative metabolism with miR-1 loss. Proposed Research: The purpose of this proposal is to define the miR-1 regulated transcriptome and investigate how miR-1 and miR-1 target genes contribute to SkM metabolic myopathy and mobility limitation in experimental PAD as well as in the clinical disease. Aim 1 will identify whether rescuing SkM miR-1 expression will ameliorate ischemic pathology. Aim 2 will determine the role of miR-1 in exercise training adaptations in hindlimb ischemia. Aim 3 will assess miR:target binding in PAD samples to determine pathophysiologically relevant mechanistic targets. Together, these Aims will define the miR-regulated transcriptomic response in PAD and will provide a foundation for the development of miR-based therapeutics aimed at SkM metabolism in PAD. Candidate: I have led projects that investigated several aspects of PAD pathophysiology. I have also led studies that explored miRs in SkM through various novel approaches and technologies. This expertise and my established track record in working with transgenic mouse models will ensure the successful completion of these aims. I will follow up this work and the associated publications with an R01 proposal focused on the role of post-transcriptional regulatory mechanisms in the exercise response heterogeneity in older participants with PAD. The K22 award will be fundamental as I launch my independent investigator career, offering management and grant writing training, helping to hone my skills as a mentor/PI and establish a long-term funded research program.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

ABSTRACT Alcohol use disorder (AUD) is a highly prevalent, chronic, relapsing disorder for which pharmacological treatments remain few. People affected by AUD show heavy, compulsive alcohol drinking, a negative emotional state when abstaining from alcohol, and an inability to reduce or stop intake. Key neuroadaptations induced by chronic alcohol include the recruitment of stress neurotransmitter systems in the bed nucleus of the stria terminalis (BNST). The BNST is a brain region that plays a key role in both excessive drinking and anxiety-like behavior. This project concerns pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide particularly abundant in the BNST, which has recently emerged as a master regulator of the stress response. The central hypothesis of this application is that the central PACAP system is a key mediator of heavy alcohol drinking and associated anxiety-like behavior and heightened pain sensitivity. Furthermore, we hypothesize that PACAP acts in the BNST via the stimulation of corticotropin-releasing factor (CRF) neurons. Finally, we hypothesize that PACAP projections from the lateral parabrachial nucleus to the BNST are those mediating the effects. These hypotheses will be tested using well-established animal models of heavy alcohol drinking and affective behavior, combined with state of the art pharmacological, molecular, and viral approaches. This highly translational and mechanistic research will shed light on the role of a key neuropeptide system in heavy drinking and anxiety-like behavior. A deeper understanding of the molecular mechanisms underlying heavy drinking and the neuroadaptations occurring in the extended amygdala neurocircuitry may lead to the discovery of novel therapeutic agents for AUD.

NHLBI - National Heart Lung and Blood Institute

Abstract As of today, lung transplantation continues to be the only treatment option for patients suffering from end-stage respiratory failure. Ischemia reperfusion injury (IRI)-mediated primary graft dysfunction (PGD) is a very common complication of lung transplantation affecting more than 50% of recipients. PGD is the main risk factor for short- term mortality and any degree of PGD is associated with chronic lung allograft dysfunction (CLAD) which remains the main barrier for long term survival. Therefore, it is of critical importance to better understand and characterize the mechanisms responsible for the development of IRI for clinical translation into improved outcomes. Currently, induction and maintenance therapies after lung transplantation are centered around the reduction of the robust T cell immune response against the allograft, including T cell help to B cell activation. However, it is known that T-cell independent signals can activate B cells to develop strong immune responses which are not targeted in contemporary treatment regimens. We have recently reported on the role of lung-infiltrating B cells in the pathogenesis of IRI. We found that the synergistic activation of the BCR/TLR4 receptors on these B cells is necessary for the production of the monocyte chemokine CCL7 in a TRIF-dependent fashion, a critical step contributing to classical monocyte (CM) recruitment and subsequent neutrophil extravasation, resulting in worse lung function. Also, we have generated new data suggesting that IRI produces a specific IRI-derived lung effector B cell subpopulation that upregulates activation and costimulatory markers (CXCR5+ CD25+ CD30+ CD80+ CD86+) which are essential for the development of B cell immune responses that could be important for donor specific antibody (DSA) production and development of AMR and CLAD after lung transplantation. The overarching goals of this application are to identify the key pathways associated with B cell recruitment after IRI; the mechanisms of early activation of recipient-derived B cells in the lung and which role these early events have on the development of subsequent deleterious immune responses by B cells. In this proposal, we will use state-of-the-art techniques including, murine lung transplantation, intravital microscopy, single cell RNA sequencing, proteomics and transcriptomic techniques to elucidate how lung macrophages direct B cells recruitment and colocalization to the lung after transplantation (SA1), if T-cell independent signals are capable of activate recipient-derived B cells early after reperfusion (SA2.1), If intravascular BCR activation on graft-infiltrating recipient B cells is necessary for their extravasation (SA 2.2) and to examine mechanisms that drive the development of deleterious B cell-mediated immune responses after lung transplantation (SA3). As B cells can be depleted with currently available agents, this project has the potential to change current paradigms of induction and maintenance immunosuppression after lung transplantation by identifying immunological targets for B cell-centered therapies which are not currently used in contemporary treatment regimens, therefore, improving the survival of lung transplant recipients.

NIH

Significance to VA: Lung cancer (LC) is the leading cause of cancer death for US veterans (USvets). Non-small cell LC (NSCLC) represents the majority of LCs with a poor 5-yr survival rate (~23%). NSCLC patients in the VA Health Care System are increasing as USvets often acquire tobacco addiction during military service fostering a large percentage of high-risk current & former smokers. The lung cancer incidence is also higher in USvets with lower survival rates, and LC is linked to service-connected exposure to carcinogens. Current treatment options for NSCLC are palliative, but have recently evolved with the use of immune checkpoint inhibitors (ICIs). Unfortunately, the effectiveness of ICIs in NSCLC remains modest with underlying resistance mechanisms elusive. Our research will define these resistance mechanisms and identify potential molecular targets & strategies to target both the NSCLC tumor & enhance ICI efficacy to produce a more durable outcome for USvets. Innovation & Impact: STK11 mutations (mtSTK11) are common in NSCLC and associated with resistance to immune checkpoint inhibitors (ICIs). Our data connected mtSTK11 to a novel dysregulation of caspase 9 (C9) alternative RNA splicing (ARS). Specifically, mtSTK11 NSCLC preferentially expressed C9b, which induces tumorigenesis, and a tumor immunosuppressive microenvironment (TIME) that supports ICI resistance. Genetic removal of C9b sensitized mtSTK11 NSCLC to ICI therapy in a NSCLC mouse model, thus highlighting the potential utility of modulating dysregulated ARS as a therapeutic. Human mtSTK11 NSCLC tumors also presented with the dysregulation of additional ARS events, which our data also support roles in NSCLC tumorigenesis & TIMEs. These data support the hypothesis that dysregulated ARS in mtSTK11 NSCLC modulates tumorigenesis and induces a TIME that promotes ICI resistance. Specific Aims (SAs): To interrogate our innovative hypothesis, we are proposing three specific aims: SA1: Determine the role of ARS events linked to the mtSTK11 oncogenotype in NSCLC tumorigenesis; SA2: Determine the role of specific STK11-regulated ARS events in ICI responses; & SA3: Determine the signaling mechanisms driving dysregulated ARS in mtSTK11 NSCLC. Methodologies: Unbiased “splicomic” analysis in human NSCLC will identify ARS events dysregulated in the mtSTK11 oncogenotype. Once validated for STK11/LKB1-regulation (e.g., by qRT-PCR), their roles in cancer biology will be determined using cells models (e.g., clonogenic potential) and mtSTK11 NSCLC mouse models (e.g., tumorigenesis & ICI resistance). The mechanism of action for specific ARS events will be determined by interrogation of the cell composition of the TIME using multiplex immunofluorescent histology & Aurora flow cytometry. State-of-the-art molecular manipulations (e.g., CRISPR), novel molecular “tools”, and complementary biophysical studies will be employed to modulate specific ARS events in our cellular & in vivo models and determine mechanistic function. Important ARS events and mechanisms will be interrogated in human NSCLC tumors for translational outcomes (e.g., survival, ICI resistance). Path to translation/implementation: Validation of our hypothesis would lead to the identification of new chemical entities (NCEs) that specifically block cancer-related ARS to foster a new generation of therapeutics for NSCLC. These NCEs would have limited toxic side effects: the “Achilles Heel” for some global ARS inhibitors. Additionally, our laboratory has shown that ceramide induction in NSCLC cells will reverse the dysregulated C9 ARS and sensitize cells to standard of care NSCLC treatments. A new ceramide-induction therapy, ceramide nanoliposomes, recently completed a phase I clinical trial (NCT02834611) for solid tumors (e.g., NSCLC) with an excellent safety profile. Thus, our proposed studies will build the molecular, mechanistic, & pre-clinical foundation for the development of new targeted ARS therapeutics and the clinical implementation of combination therapies using both ARS inhibitors & ceramide-induction therapy with ICIs in NSCLC to improve outcomes.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Squamous cell carcinoma (SCC) is one of the most common cancers worldwide, yet the early events that drive mutated epithelial stem cells toward malignancy remain poorly understood. While cellular plasticity has emerged as a key process enabling clinically normal mutation-harboring skin to acquire oncogenic potential and undergo malignant transformation, the non-genetic variables that regulate this process and ultimately drive tumorigenesis remain largely unknown. These gaps in knowledge can largely be attributed to a dearth of tools and models that capture pre-oncogenic stem cell identity landscape in intact tissues. In this project, I will investigate how the lymphatic vascular niche, increasingly recognized as a key regulator of epithelial stem cell, controls cancer stem cell fate transitions during early tumor initiation and malignant progression in skin SCC. Using deep imaging and sequencing approaches, we discovered that lymphatic vascular insufficiency predisposes stem cells to malignant transformation, while oncogenic plasticity propelling the transition from benign to metastatic carcinoma is preceded by dynamic lymphatic remodeling. The central hypothesis is that lymphatic niches evolve during tumor progression, initially supporting tolerance to oncogenic stress and later driving epigenetic rewiring that enables malignant transformation. Aim 1 will determine how lymphatic regression influences tolerance to oncogenic stress and promotes malignant transformation using in vivo lymphatic ablation models, chromatin accessibility profiling and organotypic culture systems. Aim 2 will define the molecular and spatial interactions between tumor-initiating cells and lymphatic niches during malignant progression. Leveraging enhancer-based proximity sensors, I will identify lymphatic-derived signals that promote stem cell plasticity potential. This work is expected to reveal context-dependent lymphatic cues that regulate early tumor initiation and malignant transition, while generating new tools to study vascular–stem cell interactions in vivo. These studies aim to uncover actionable pathways that can be targeted to intercept skin cancer before it becomes invasive, offering new opportunities for early intervention in a disease that affects millions. My training will take place in the Gur-Cohen Lab at UC San Diego and the Sanford Stem Cell Institute, a highly interdisciplinary environment with access to state-of-the-art tools in imaging, genomics, and computational biology. In this fellowship, I will gain advanced expertise in 3-dimensional tumor imaging, epigenomic analysis, and the development of experimental tools to interrogate the cancer stem cell interactome in real time. Under the guidance of a dedicated mentoring team, I will also strengthen my skills in scientific communication, grant writing, and mentorship. This integrated research and training plan will prepare me for an independent academic career focused on understanding how systemic signals influence stem cell fate and oncogenic potential.