Univ Of Massachusetts Med Sch Worcester

NIH Research Projects · FY 2025 · 2025-08

Project Summary Gene expression is tightly regulated by cis-regulatory elements (CREs), which interact with transcription factors and other regulatory proteins to control when and where genes are expressed in different cell types. While millions of CREs have been annotated across the human genome, key questions remain about how these elements work together, how their function varies across cell types, and how they influence alternative isoform usage. Addressing these gaps is essential to understanding the complexity of gene regulation and its role in human health and disease. This project seeks to answer three critical questions: (1) How do cCREs interact in a combinatorial manner to regulate gene transcription? (2) How do cCREs exhibit context-dependent activity in different cell types? (3) How do cCREs contribute to the regulation of transcript isoform usage, beyond simply controlling gene expression? We will answer these questions by leveraging deep learning models, such as convolutional neural networks (CNNs), to analyze large-scale genomic and epigenomic datasets. Deep learning will allow us to uncover non-linear patterns of cCRE interactions and predict their regulatory effects across different cellular contexts. To validate our computational predictions, we will employ high-throughput experimental techniques such as STARR-seq and CRISPR-based perturbation assays. These experiments will assess the regulatory activity of cCREs in their native chromatin environments, allowing for direct testing of combinatorial effects, context-dependent functions, and their impact on transcript isoform expression. Ultimately, this research will provide new insights into the regulatory mechanisms underlying gene expression, with implications for understanding complex diseases and developing targeted therapeutic strategies.

NIH Research Projects · FY 2025 · 2025-08

Project Summary. Mycobacterium tuberculosis (Mtb) is a leading cause of human deaths around the world. Despite eliciting a vigorous immune response, Mtb evolved to adapt to its host and evade clearance. While this idea was originally based on its ability to survive in the phagosome and avoid antibody immunity, we now know that Mtb also avoids T cell immunity. Mtb elicits strong CD8 T cell responses in people and CD8 T cell responses contribute to protection against Mtb in animal models. My research program has discovered that (1) not all Mtb- specific T cells recognize Mtb-infected macrophages; (2) CD8 T cells require CD4 help to maintain effector function and avoid exhaustion; and (3) memory CD8 T cells are not always as fit as naïve CD8 T cells. These factors contribute to the difficulty in observing a role for CD8 T cells in murine TB. By better characterizing how CD8 T cells recognize infected macrophages and control Mtb infection, we expect to gain insight into the role that CD8 T cells serve in immunity to Mtb. My lab has developed an innovative and productive research program that seeks to understand how CD8 T cells restrict bacterial growth and how Mtb defeats CD8 immunity. Our goal is to find new ways to elicit protective CD8 T cell responses that can inform vaccine strategies to prevent or treat TB. In Aim 1, we will combine the use of a BCG prime/ChAd.TB boost vaccine strategy that induces protective CD8 T cell responses, t-bet fate reporter mice to track memory CD8 T cells, and a novel model of latent TB infection (LTBI). This approach will permit us to characterize how vaccine-elicited CD8 T cells are recalled following Mtb challenge, identify protective antigens recognized by CD8 T cells, discover correlates of protection, and determine the role of CD8 T cells during LTBI. The focus of Aim 2 is disentangle how the lack of CD4 help and high Mtb CFU lead to dysfunctional CD8 T cells. The molecular differences between helped and unhelped CD8 T cells will be used to determine the molecular basis of protection mediated by CD8 T cells. We previously showed that CD8 T cells specific for the immunodominant TB10.4 antigen poorly recognize Mtb-infected macrophages. However, other CD8 T cells recognize Mtb-infected macrophages and efficiently inhibit Mtb replication. We have developed an experimental pipeline to identify Mtb antigens presented by infected macrophages, which we hypothesize will be ideal vaccine targets which will be the focus of Aim 3. Finally, Aim 4 will leverage the differences between protective vs. nonprotective vaccine-elicited CD8 T cell responses (Aim 1), and helped vs. exhausted CD8 T cells (Aim 2), to identify the pathways that license effector CD8 T cell activity, and perform mechanistic studies that ascertain the pathways that inhibit intracellular Mtb growth. By expanding our understanding of how effector and memory CD8 T cells participate in immunity to TB, our research program will provide actionable concepts to incorporate into novel TB vaccine strategies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Invasive pulmonary fungal infections carry unacceptably high mortality rates and contribute to over $2.4 billion in direct healthcare costs in the US alone. The most prominent fungal pathogens causing lung disease include Aspergillus fumigatus (Af). Although it is well demonstrated that the immune system is critical to a proper response to invading fungi, the airway epithelium remains a vastly understudied component of antifungal immunity. We previously demonstrated that the recognition of Aspergillus lacking the outer layer of 1,8-dihydroxy naphthalene (DHN) melanin by airway epithelial cells leads to higher transmigration of neutrophils. Our preliminary data demonstrate that purified Af melanin (termed melanin ghosts; MG) blocked the secretion of IL-8 and GROα by the epithelium, muted calcium fluxing, reduced phosphorylation of key signaling molecules, induced distinct changes in the transcriptional signatures differentially in airway epithelial cell subtypes, altered actin filamentation, and dampened the production of reactive oxygen species (ROS). These data argue that fungal melanin actively triggers distinct immunomodulatory responses in primary human epithelial cells. Thus, our overall testable hypothesis is that Af melanin blocks calcium fluxing, which silences inflammatory circuits (i.e., exocytosis pathways, TLR and CLR signaling pathways, and ROS production) in airway epithelial cells to subvert the innate immune response. To address this hypothesis, we propose the following three specific aims: [1] dissect the mechanism of how Af fungal melanin inhibits chemokine secretion, [2] determine the TLR and CLR signaling pathways affected by Af melanin; and [3] reveal the mechanism by which melanin blocks ROS production in lung epithelial cells. This work will identify the mechanisms by which fungal melanin halts chemokine secretion by airway epithelial cells and define the pathway(s) altered by this suppression through the lens of calcium signaling. Completion of this proposal will provide novel therapeutic targets for invasive fungal infections caused by melanized fungi. Furthermore, lung diseases with the hallmark of a hyper- inflammatory state, such as asthma, could be treated with synthesized melanin to dampen exacerbated inflammation.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Understanding mechanisms of drug action is important for identifying settings in which a drug will be effective, and for interpreting – even predicting – potential mechanisms of drug resistance. For anti-cancer drugs, a key aspect of their mechanism of action is the mechanism by which these drugs promote cell death. The first identified death pathway was apoptosis, a form of death that is now reasonably understood. In addition, the community has now identified at least ten distinct forms of regulated necrosis. Our recent studies highlight that these non-apoptotic forms of cell death contribute to the efficacy of many anti-cancer therapies, with each form of non-apoptotic death activated by at least one clinically used compound. A current limitation in the use of drugs that activate non-apoptotic forms of death is that we lack a detailed understanding of how most of these pathways function. To address this issue, we recently developed MEDUSA (Method for Evaluating Death Using a Simulation-assisted Approach). MEDUSA is an analytical method used to evaluate genome-wide CRISPR screens of drug efficacy (which are sometimes referred to as “chemo-genetic profiles”). Our recent publications demonstrate that MEDUSA is uniquely effective at revealing a drug’s mechanism of action and mechanisms of drug-induced lethality, including the signaling and regulatory pathways that facilitate activation of cell death. Furthermore, while many methods exist for evaluating apoptotic death – due to the stability of the apoptotic corpse and the existence of measurable biomarkers that are specific to apoptotic cells – MEDUSA is likely the only method that will be effective at identifying the genetic dependencies and mechanisms of resistance for drugs that activate non-apoptotic forms of cell death. Although MEDUSA is effective, it requires: 1) a detailed understanding of drug function to parameterize a drug-specific simulation, 2) specialized experimental requirements, such as time-resolved data, which are not common in most experiments, and 3) programming proficiency to run the MEDUSA simulations. The central goal of this proposal is to extend the MEDUSA method, to enable easier use and broad utility, particularly in the evaluation of publicly available data. We call our new version MEDUSA: Generalized Method (MEDUSA(GM)). This new method: 1) can be used without any drug-specific information, 2) does not require any specialized data, and 3) will be available on a web portal and can be used without any programming. In this proposal, we will add new functions to our MEDUSA(GM) method, stress test the MEDUSA(GM) method with therapeutically relevant drug scenarios that have not been previously evaluated, and develop the MEDUSA(GM) web portal, which will enable open access use of MEDUSA(GM).

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Our MD-PhD Program was founded in 1983 and first obtained NIH MSTP funding in 2013. We currently have a steady enrollment of about 80 students at all stages at any given time. Our Institutional leadership is highly supportive of the Program, as is amply demonstrated by its investment in the Program. Our Program leadership changed in December 2021, interrupting T32 funding but enabling substantial positive changes. The Mission of the Program is to train a diverse group of physician-scientists who will excel in their chosen research fields through innovative and high-impact research. We prepare them to become leaders in academic medicine and other health-related enterprises. Our graduates will combine their investigative skills with their clinical experience and expertise to produce new knowledge that will improve the health of both individuals and populations. Our Objectives are that, throughout their professional careers, our trainees will 1) think critically and innovatively; 2) inform their research through clinical observation and, conversely, apply a hypothesis-driven approach to their care of patients; 3) conduct rigorous, reproducible, and transparent science as a fundamental focus of their professional activities; 4) collaborate effectively with other researchers in academia, government, and the private sector. Other programmatic objectives include 5) increasing faculty and student diversity, both scientific and sociodemographic; and 6) enhancing our mentoring and evaluation structures. Historically, MD-PhD programs have focused on the basic sciences, and UMass Chan has world-renowned basic scientists. However, our faculty also includes thought leaders in translational science, and in population health sciences, including epidemiology, biostatistics, health informatics, data science, and health equity research. We believe that physician-scientists trained in the population health will make significant contributions to improving the health of both individuals and populations, and that the scarcity of MD-PhD graduates with a focus on the population sciences is a missed opportunity for our country. We intend to maintain the size of our MD-PhD trainee group, but we aim to increase the proportion of trainees in the population sciences from about 10% to about 25%. Also, we have begun and will continue to restructure our Program with 1) markedly enhanced and longitudinal mentoring with Individual Development Plans (IDPs) playing a key role and centrally supported effort for mentors; 2) an intense focus on reducing time to dual degree (TTD) by eliminating redundancies in the medical and graduate school curricula, a structured and mentored approach to choosing thesis advisors, and requiring mentor training and attention to reducing TTD by thesis advisors ; 3) enhanced and early emphasis on research-intensive residency selection; and 4) an entirely revamped evaluation system, with dynamic data-driven feedback loops for Program improvement. We request 20 MSTP T32 slots each year, while institutional resources will directly defray over 80% of the cost of our MD-PhD Program. Our Vision is that all of our trainees will pursue careers as physician-scientists and contribute toward improving the health of individuals and populations through medicine-inspired scientific discovery.

NIH Research Projects · FY 2025 · 2025-08

AAV-Delivery of Broadly Neutralizing Antibodies to Augment ART-Free Post-Treatment Control of HIV-1 in Children Initiation of antiretroviral therapy (ART) to infants with HIV-1 within 3 months of birth can durably suppress HIV-1 replication, preserve immune function, limit the establishment and persistence of viral reservoirs, and reduce HIV-related morbidity and mortality. However, rates of viral suppression in early childhood are low due to the many challenges inherent in delivering lifetime, daily ART. There is thus a critical need for new therapeutic approaches to achieve durable HIV-1 control off ART in young children. This clinical trial planning grant, submitted in response to PAR-23-206, addresses key barriers to the efficient translation of promising pre- clinical data from infant macaque studies into a proof-of-concept POC Phase I clinical trial that will evaluate whether a single administration of two AAV9-vectored broadly-neutralizing antibodies (AAV9-VRC07-523LS; AAV9-10-1074LS) to children on ART will be safe, tolerable, and lead to sustained production of bNAbs at levels that will allow control of HIV-1 replication after ART discontinuation. Intramuscular AAV9 administration will selectively target long-lived muscle cells using a dose likely to provide sustained in vivo production of antibodies with minimal off-target effects and immunogenicity. Administration to young children capitalizes on the relative tolerogenic immune milieu in early life to circumvent ADA, a major barrier to this approach in adults. Primary trial outcomes include safety, pharmacokinetics (bNAb levels), and the proportion of children who maintain HIV control (plasma HIV-1 RNA < LOD) off ART. To enable efficient POC clinical trial development and implementation, we will coordinate multiple partners to: 1) develop a full regulatory strategy; 2) complete a full clinical trial protocol and associated documents; and 3) engage communities and ethics committees to inform clinical trial development and implementation. Enabling the timely design and completion of the POC trial will be an important step to advancing broadly accessible, safe, durable, and effective therapies to achieve control of HIV-1 replication off ART in children.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Despite their potent innate cytotoxic activity, NK cells are unable to completely control HIV, however, their depletion enhances acute viremia, and their proliferation during hyperacute infection is associated with better control of infection, suggesting they do apply immune pressure. In vitro, infected cells that survive NK cell exposure have higher levels of MHC-I, suggesting that the MHC-Ilow cells are preferentially eliminated due to “missing self”. However, our preliminary data demonstrates that while genetic ablation of the HIV accessory protein, Nef, rescues MHC-I surface expression on infected cells, these cells are not completely protected from killing, suggesting mechanisms beyond “missing self” are driving NK cell stimulation. Indeed, we have discovered several new NK cell ligands that are differentially regulated with infection. Unexpectedly, we find that deletion of Vif from HIV enhances MHC-I downregulation and augments NK cell killing in co-culture assays, suggesting an additional role for Vif in controlling NK cell function. Finally, humanized mice harboring intact NK cell responses exhibit an enrichment of mutations in Nef’s acidic cluster (the dominant factor controlling MHC-I surface expression), resulting in enhanced surface expression of MHC-I. This suggests a new mechanism of viral escape from NK cell responses via changes to accessory protein regulatory elements. The overall objective of this proposal is to uncover new mechanisms of HIV accessory protein-mediated control of NK cell function. We hypothesize that while MHC-I remains the dominant regulator of NK cell activity, the virus employs additional mechanisms to fine tune this “missing self” trigger. Specific Aim #1 will identify new HIV-regulated NK cell ligands. This includes the testing of HIV clinical isolates from different clades, which exhibit accessory protein sequence diversity, and characterizing how the NK ligands CEACAM1, ICAM1, HEVM, CD43, CD44, and CD58 affect innate and ADCC-mediated NK killing. In addition, we will use mass spec to identify natural cytotoxicity receptor ligands on infected cells that trigger NK cells, and develop a custom CITE-Seq panel with an expanded repertoire of NK ligand antibodies for use in future clinical studies. Specific Aim #2 will define how HIV Vif regulates MHC-I and NK cell activation. Using mutated viruses, mRNA, and siRNA, we propose to assess how the effects of Vif on Nef may contribute to MHC-I regulation. Finally, Specific Aim #3 will characterize the effects of NK cell pressure on Nef, Vpu, Vpr, and Vif sequence evolution in vivo using humanized mice with an enhanced NK cell compartment. Mutants will be reconstructed in vitro to characterize the effects on MHC-I and other NK cell ligands, and NK cell killing. Finally, enriched mutated sequences from the animal studies will be compared to sequences in the Los Alamos HIV Database to assess their clinical relevance. Together, this work will provide mechanistic insights into viral immunoevasion mechanisms that directly affect NK cell function, which may aid the development of NK cell-based therapies for cure strategies, such as blocking accessory protein function with small molecules.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY Opioid use and deaths from overdoses have skyrocketed in the United States over recent years. Most people with opioid use disorder (OUD) relapse within weeks to months despite treatment. Relapse vulnerability is strongly associated with severe and persistent sleep and circadian rhythm disruptions, suggesting therapeutics that mitigate these alterations during withdrawal and abstinence may reduce craving and risk for relapse. However, our understanding of the mechanisms across cellular and molecular levels in the brains of people with OUD is extremely limited. We recently demonstrated alterations in several pathways related to dopamine, opioid, and glutamate signaling in human brain associated with OUD. We also reported significant disruptions in molecular rhythms associated with OUD in human striatum, encompassing major neural substrates for reward and motivation, as well as arousal, sleep, and circadian rhythms. We and others developed a novel and innovative approach for using time-of-death (TOD) of the subject to measure molecular rhythms in human postmortem brain associated with various psychiatric disorders, gaining insights into disease-specific neurobiological mechanisms and pathways in human brain. In this proposal, we will use TOD computational tools combined with single nuclei RNA-sequencing (snRNA-seq) to investigate the relationship between molecular rhythm disruption in specific cell types of the striatum (nucleus accumbens (NAc), caudate, putamen) in OUD using postmortem brains from unaffected subjects and subjects with OUD, followed by mechanistic studies using brain region- and cell type-specific ablation of molecular rhythms in mouse models of opioid self- administration. In Aim 1, we will use snRNA-seq and TOD analyses to create a cell type specific map of molecular rhythms in subregions of human striatum. In Aim 2, we will compare molecular rhythms in specific cell types of the striatum from unaffected subjects to molecular rhythms in cell types from subjects with OUD to determine the extent of molecular rhythm disruptions associated with OUD at the cellular level. Results will be integrated with human GWAS sleep and opioid traits. snRNA-seq findings will be validated using in situ RNAscope hybridization of the top rhythmic transcripts and cell type specific markers. We then directly investigate the functional relevance of molecular rhythm ablation in specific striatal cell types during opioid self- administration and sleep physiology and assess the impact of opioids on cell type-specific molecular rhythms in mice in Aim 3. The findings from our proposal will resolve molecular rhythm alterations at the cell type-specific level in the brains of subjects with OUD to begin to identify the mechanisms underlying the relationships between circadian rhythms and OUD in the hopes of progressing towards strategies that target the circadian system in the treatment of OUD.

NIH Research Projects · FY 2026 · 2025-08

Project summary Maple Syrup Urine Disease (MSUD) is an autosomal recessive amino acid metabolism disorder that is regarded as the most severe amino acidopathy and is invariably fatal if left untreated. MSUD is caused by buildup of the branched chain amino acids (BCAAs) leucine, isoleucine and valine. The current standard of care for MSUD is either BCAA-free formula diet or liver transplantation. MSUD liver transplantation does do not reverse prior damage to the CNS, does not prevent the low IQ, ADHD or the profound psychiatric disease, which is unresponsive to antidepressants. MSUD also naturally occurs in calves, and the disease is strikingly similar to humans both in disease course, biochemistry and post-mortem analyses. In proof-of-concept data we treated an MSUD calf by intravenous adeno associated viral (AAV) gene therapy and our therapeutic response was equivalent to that of the standard of care (liver transplant or formula diet) in MSUD patients. However, the calf exhibited white matter abnormalities on MRI like that of MSUD patients, low glutamate on MR spectroscopy and in cerebrospinal fluid, also consistent with human MSUD. Therefore, this proposal focuses on addressing the neurological aspects of MSUD while maintaining the biochemical correction in the periphery. Our team consists of Dr. Dan Wang, an AAV gene therapy expert that tested this therapy in MSUD mice and showed profound efficacy, Dr. Guangping Gao the director of the Horae Gene Therapy Center who has subsidized the cost to make sufficient amounts of AAV to treat MSUD calves and Dr. Kevin Strauss, the physician who cares for all the MSUD patients and has pioneered all the therapeutic approaches for MSUD including liver transplant and developed the MSUD formula. Dr. Gray-Edwards (PI) is an expert in large animal AAV gene therapy and has taken several AAV gene therapies to the clinic through testing in large animal models of human genetic diseases. The experimental aims of the proposal is as follows: Aim 1: Determine if high dose intravenous or combined intravenous and CSF delivery of AAV9-A-BiP-B can address the neurological components of MSUD. Aim 2: Long term assessment of the best AAV gene therapy approach Aim 3: Underpinning transcriptomic and metabolomic changes in the MSUD brain receiving the standard of care and determining the effect of gene therapy.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The germline contains the genetic information that will be passed to future generations. Therefore, maintaining the germline genome is essential for fertility and species survival. One mechanism of germline genome maintenance is that of the PIWI/piRNA pathway, in which small RNAs known as piRNAs interact with a PIWI Argonaute protein to form what is known as a piRNA-Induced Silencing Complex (piRISC). Through its endonuclease activity, piRISC silences repetitive elements (i.e., transposons) to protect the genome for future generations and regulates gene expression to ensure proper germ cell development and function. Loss of PIWI function leads to infertility in at least one sex in many animals, including human males. Recent work has revealed that the small zinc-finger protein, gametocyte-specific factor 1 (GTSF1), accelerates piRISC target cleavage. Loss of GTSF1 function in mice and human males leads to infertility. Preliminary kinetic evidence suggests that GTSF1 is not required for target binding or target release. However, the piRISC catalytic states associated with GTSF1 binding have not been explored. Seven GTSF1 residues have been identified as key for target cleavage, but most of these amino acids are not conserved in the other mouse GTSF paralogs, GTSF1L and GTSF2, even though they also accelerate piRISC target cleavage. This proposal seeks to test the hypothesis that mammalian GTSF proteins stabilize a catalytically active state of piRISC via key contacts with both the piRNA-target RNA duplex and PIWI protein. Aim 1 will use single-molecule FRET to probe piRISC conformational changes in the absence and presence of GTSF proteins to determine which, if any, catalytic state is stabilized in the presence of GTSF. Aim 2 will employ a high-throughput screening method to identify all amino acid positions across GTSF paralogs which are required to interact with piRISC. This study will provide insights into how GTSF1 accelerates piRISC target cleavage and determine which GTSF residues are key for its function, providing insight into why some human GTSF1 mutations lead to infertility. The proposed research will provide training in microscopy, in vitro biochemistry, and bioinformatics to prepare the fellow for a postdoc studying epigenetic inheritance and a future career as an independent investigator.

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract Invasive fungal infections cause millions of deaths annually. Despite the obvious need, there are no licensed human fungal vaccines. Delays in diagnosing fungal infections are common due in part to suboptimal diagnostic tests. A major impediment to the development of vaccines and diagnostics is the paucity of publicly available, defined, and validated immunogenic fungal antigens. To address this resource deficiency, teams from three institutions (UMass Chan, UTSA, and UWM) will produce, validate, and provide relevant fungal antigens to the scientific community. Crude antigenic preparations from Cryptococcus neoformans, C. gattii, Candida albicans, C. auris, Aspergillus fumigatus, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, and C. posadasii will be manufactured. These include heat-killed whole fungi, Coccidioides formalin-fixed spherules, fractions from bead-beaten fungi, and fungal extracts. The biochemical composition and immunogenicity of the crude antigens will be determined. Purified fungal antigens will also be made. The UMass Chan, UTSA, and UWM teams selected 36 vaccine-candidate proteins (12 each from Cryptococcus, Coccidioides, and Blastomyces) for study. These 36 fungal proteins will be recombinantly produced in E. coli, formulated into vaccines, and tested for their ability to protect DR4 mice (which express a human MHC Class II allele) from a lethal fungal challenge. Based on the data obtained and aided by immunoinformatic analyses, four proteins/team will be down-selected for in-depth studies. Vaccine-mediated protection will be extended to include humanized DR3 mice (which express a different MHC Class II allele from DR4 mice). Overlapping peptide libraries will be synthesized and used to map the human MHC Class II epitopes that stimulate CD4+ T cell responses in vaccinated DR4 and DR3 mice. Proteins will be recombinantly expressed in yeast strains identical to or closely related to their strain of origin. A comparison of immune responses following vaccination with proteins recombinantly expressed in E. coli and yeast will enable determination of how epitope recognition is affected by yeast post-translational modifications. Finally, the ability of the recombinant proteins to stimulate CD4+ T cell responses in PBMCs from human subjects exposed to or infected with Cryptococcus, Coccidioides, Blastomyces, or Histoplasma will be determined. The fungal antigens manufactured and validated will be provided as resources to the scientific community. 1) Validated crude antigen preps will be deposited in the Biodefense and Emerging Infections Research Resources Repository (BEI) repository. 2) The E. coli and yeast recombinant antigens, along with the strains expressing these recombinant proteins, will be deposited to BEI. 3) The peptide epitopes and the corresponding MHC Class II alleles stimulated will be uploaded to the Immune Epitope Database (IEDB). These resources will be valuable to investigators studying fungal immunology, vaccinology, and diagnostics, particularly newcomers to the field and those who do not have the biosafety facilities or approval needed to study pathogenic fungi.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Pancreatic cancer is highly metastatic, and patients are often not diagnosed until metastases have already formed, with the vast majority (>75%) presenting with oligometastatic disease. Current genetically engineered mouse models of pancreatic cancer are metastatic in 30-40% of animals and harbor only small focal metastases, complicating the study of metastatic drivers in mice. Preliminary experiments identified Calcium/calmodulin- dependent protein kinase II beta, Camk2b, as a gene whose loss enhances tumor metastasis and creates a highly immunosuppressive tumor microenvironment (TME). Genetically engineered mice with tumor cell-specific Camk2b knockout form metastases in 80% of animals and present with >25 metastatic lesions per mouse. Metastatic burden is so profound in this model that the animals succumb to disease twice as fast as control tumor bearing mice. Preliminary results, show that deletion of Camk2b results in tumors that are more metastatic and express higher levels of Lysyl Oxidase (Lox). Aim 1 will focus on exploring tumor cell intrinsic mechanisms through which Camk2b-deletion promotes tumor cell metastasis. The metastatic cascade typically begins by the modulation of tumor cell epithelial identity through programs such as epithelial-to-mesenchymal (EMT). Thus, Subaim 1.1 will investigate the effect of Camk2b loss on tumor cell epithelial cell identity using isogenic cell lines, genetically engineered mouse model, and patient-derived organoid systems. Subaim 1.2 will investigate the functional contribution of LOX on metastatic competency and the ability of LOX-targeting to block metastatic outgrowth. Preliminary experiments indicate that deletion of Camk2b in tumor cells shifts the immune milieu towards a pro- tumor state that is more permissive to tumor metastasis. In this regard, Camk2b-null tumor cells alter their local microenvironment and confer an immune desert phenotype that may facilitate tumor growth and metastasis. To explore mechanisms of this immunosuppressive phenotype, Aim 2 will test the contribution of the immunosuppressive microenvironment in Camk2b-null tumors on metastasis. Subaim 2.1 will selectively deplete macrophages in Camk2b-deletion tumors and evaluate the impact on the metastatic niche and gross tumor cell metastasis. Subaim 2.2 will investigate the contribution of Tbc1d9, a calcium-responsive gene activated in Camk2b deleted tumor cells, to suppression of the NK and T cell response. Collectively, our work will demonstrate that Camk2b is a metastasis suppressing gene whose loss activates pro- metastatic signaling networks. Our functional and mechanistic work will define targetable downstream signaling nodes, within tumor cells and in the microenvironment, to block immunosuppression and metastatic outgrowth.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY The goal of this project is to determine the mechanisms by which interferon gamma (IFNg) signaling promotes tumor growth and immune suppression to drive neuroendocrine prostate cancer (NEPC) progression and resistance to immune checkpoint blockade (ICB) therapies. Following hormone therapy failure, some patients develop an aggressive subtype of prostate cancer known as NEPC that is lacking in effective treatment options. Though most prostate cancers are defined by a “cold” tumor microenvironment (TME) lacking T cells that mediate anti-tumor immunity and are necessary for immunotherapy responses, it was recently shown that a subset of NEPC patient tumors are “inflamed” with T cells and have heightened expression of interferon gamma (IFNg) signatures. As IFNg is associated with activation of adaptive immunity and better ICB outcomes, this suggests that NEPC patients may potentially benefit from immunotherapy. To determine the impact of IFNg on tumor- immune interactions in situ, I developed a novel mouse model of NEPC through in vivo electroporation of CRISPR constructs targeting three tumor suppressor genes, Pten, p53, and Rb1 (PtPRb), commonly disrupted in NEPC patients. Despite PtPRb NEPC tumors recapitulating the inflamed TME of human NEPC with enhanced IFNg signaling and a significant influx of CD8+ T cells, paradoxically, mice failed to respond and even had worse survival following anti-PD-1 ICB. Inhibiting Nuclear Factor kappa B (NFkB) signaling downstream of IFNg in NEPC cell lines in vitro or blocking the IFNg receptor (IFNGR1) in NEPC tumors in vivo not only inhibited tumor growth and prolonged survival, but also reduced the numbers of macrophages that highly infiltrate NEPC tumors and can suppress T cell and immunotherapy responses. Based on these preliminary results, our central hypothesis is that IFNg signaling contributes directly to tumor growth and macrophage dysfunction, and that targeting it will activate immunotherapy responses in NEPC. In Aim 1, we will test whether IFNg signaling directly contributes to NEPC viability and growth signaling. Genetic and pharmacological approaches will be used inhibit different upstream and downstream IFNg signaling regulators in murine NEPC tumor cells and human NEPC organoids to investigate their role in driving growth factor signaling and growth phenotypes in vitro and in vivo. In Aim 2, we will test whether IFNg drives macrophage-mediated immune suppression and immunotherapy resistance. The impact of IFNGR1 KO on macrophage polarization, phenotypes, and T cell interactions will be assessed in vitro by tumor-immune co-culture assays and in vivo in murine NEPC tumor models by multiplexed error-robust fluorescence in situ hybridization (MERFISH) spatial transcriptomics. Finally, we will evaluate the effects of macrophage or IFNGR1 blockade on tumor and immune responses and anti-PD-1 ICB outcomes in our preclinical NEPC animal models. Ultimately, this work will not only unveil new tumor intrinsic and extrinsic mechanisms by which IFNg signaling drives NEPC progression, but also advance novel strategies targeting IFNg regulators to enhance immunotherapy outcomes in this aggressive disease.

NIH Research Projects · FY 2026 · 2025-07

ABSTRACT Opioid use disorder (OUD) is a significant public health threat that affects millions of people and is associated with high rates of co-occurring conditions such as HIV. Approximately 17% of the 15 million people who inject drugs globally are HIV-positive. Conversely, HIV-positive individuals often experience chronic pain, which leads to increased opioid use and a higher risk of addiction. OUD causes persistent molecular changes in the brain, potentially further altering or exacerbating HIV-related brain impairment. However, transcriptional alterations by alternative mechanisms remain understudied in the human brain and may be a major factor in OUD and HIV. Constituting over 70% of the human genome, long non-coding RNAs (lncRNAs) are increasingly recognized for their significant roles in gene regulation and disease pathogenesis, including OUD and HIV-related neurocognitive disorders. However, lncRNAs have not yet been well studied in either OUD or HIV, let alone in the coexistence of OUD and HIV. Our pilot study suggests that lncRNAs are especially abundant in the brain. Some of these lncRNAs are also conserved between humans and mice. We also found that conserved OUD- dysregulated lncRNAs exhibit cell type-specific expression in neurons and non-neurons in the brain. Therefore, we hypothesize that lncRNAs may regulate brain functions in a cell type-specific manner, and these lncRNAs might also potentially modulate brain functions in individuals with OUD, HIV, or co-occurring OUD and HIV. Several NIH programs, such as SCORCH and BICAN, have generated large amounts of single-cell (sc) RNA sequencing (scRNA-seq) data in human and rodent brains with or without OUD or HIV exposure. These data offer excellent opportunities to test our hypothesis. However, current approaches cannot comprehensively study cell type-specific lncRNAs at single-cell resolution because most disease-, tissue-, or cell type-specific lncRNAs have not yet been discovered. Therefore, to test our hypothesis, (1) we will expand our current lncRNA study methods to systematically discover and characterize lncRNAs in OUD and HIV at the single-cell level by analyzing large-scale omics data of human and mouse brains obtained from SCORCH, BRAIN, and other public data sources; (2) we will functionally characterize conserved lncRNAs in mouse models to uncover lncRNAs as potential new molecular mechanisms in OUD and HIV, as well as in their co-occurrence. This study will discover novel lncRNAs as potential molecular factors at the single-cell level in key brain regions involved in OUD, HIV, and their co-occurrence. Understanding lncRNAs in key brain regions affected by OUD and HIV is likely to reveal novel drug targets for treating patients who suffer from one or both conditions. Additionally, this study will provide a new computational framework broadly applicable to existing and future scRNA-seq datasets for understanding lncRNAs in other substance use disorders at single-cell resolution, and consequently, it will improve the utility of datasets from NIH SCORCH, HuBMAP, and BICAN programs.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Mutations in genes encoding proteins that are essential for cell processes such as DNA replication can lead to cellular dysfunction and disease. Protein defects can perturb these cell processes by removing or surpassing cell cycle checkpoints, leading to errant growth and proliferation of cells, defining characteristics of cancer. As such, it is essential to investigate Proliferating Cell Nuclear Antigen (PCNA), the central player that coordinates DNA replication, DNA repair, and cell-cycle regulation. PCNA, also known as the sliding clamp, is a homotrimeric ring that slides along DNA to facilitate interactions of over 100 known proteins, many involved in cancer development and other important cellular processes. The sliding clamp is conserved across all life forms, providing insight into the evolution of DNA replication and cell-cycle machinery. Thus, PCNA is an ideal target to investigate mutational effects on protein function and the long-term impacts on the cell. In Aim 1, we will address an interesting paradox related to PCNA. Point mutations in PCNA that result in subtle biochemical effects cause severe disruption of organism fitness, suggesting that PCNA is especially sensitive to mutations. Conversely, PCNA genes across evolution are widely varying in sequence suggesting that PCNA is actually accepting of mutations. To investigate this contradiction, we will perform a mutational scan of all potential point mutations in the yeast PCNA protein. These mutants will then be exposed to DNA-damaging agents to assess the effects of the PCNA mutants on various PCNA functions. I predict that a mutational screen of PCNA will show mutational effects on cell viability and DNA damage response based on residue location in PCNA providing insight into the acceptability of point mutations in PCNA. This data will also provide insights into potential disease mutations that could impact human PCNA. The Kelch lab has previously investigated two disease-associated germline mutations in PCNA. These mutations lead to PCNA-associated DNA repair disorder (PARD), characterized by UV sensitivity, neurodegeneration, premature aging, and, most notably, the development of skin cancer. In Aim 2, we will investigate how patient-associated mutations in PCNA affect biochemical and cellular function. I selected variants based on association with cancer or PARD. I will establish mutant human retinal pigment epithelial (RPE1) cell lines using CRISPR/Cas9 techniques. Once these cell lines are established, I will assess the cellular impacts by using flow cytometry and DNA-damage assays. I will compare these results with tests of the biochemical functions using isothermal titration calorimetry and thermal shift assays. I predict that the mutations will exhibit defects in thermostability, cell regulation, and DNA repair. The impact of this study is two- fold. First, the study will enhance our understanding of how PCNA function and evolution are intertwined. Second, the study will investigate select human PCNA mutants that can inform cancer diagnosis and provide a framework for investigating other proteins.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Osteogenesis imperfecta (OI) is the most common bone fragility disease, with an incidence of approximately 1 in 25,000–50,000 individuals in the US. OI is an inherited genetic disorder caused by mutations in the genes involved in collagen production, processing, and cross-linking. Genetic mutations and phenotypes in OI patients are highly variable, so current treatments mainly focus on symptomatic improvements, such as increase bone mass and strength to reduce bone fragility, fracture, and pain, using the drugs initially developed to treat osteoporosis. One innovative approach to treat OI patients who require life-long treatments is a long-lasting, bone-targeted gene therapy that improves skeletal health using recombinant adeno- associated virus (rAAV). To avoid potential off-target adverse effects of AAV gene therapy in non-skeletal tissues, we improved rAAV9 serotype’s bone-specific tropism and expression by implementing a novel bone- targeted capsid and incorporating miRNA-mediated repression of transgene expression in the liver, muscle, and heart into the vector design. Using this bone-tropic rAAV, we enhanced bone-forming WNT signaling in osteoblasts (OBs) by silencing the expression of two WNT inhibitors, Sclerostin (Sost) and Schnurri3 (Shn3). Here, we propose that with a single treatment, systemic delivery of the bone-tropic rAAV conferring single or dual silencing of Shn3 and/or Sost improves bone mass and strength, and increases grip strength, promotes the healing of fracture, and improves mobility in OI mice. We will determine the molecular mechanisms of how these processes occur using mouse and human OI skeletal organoids. First, we will examine whether bone-tropic AAV:WNT modulators improve skeletal health in OI mice (Aim 1). Given that bone-tropic rAAV-mediated silencing of Shn3 and/or Sost promoted bone formation by augmenting WNT/b-catenin signaling and OB function in osteoporosis, this aim will examine their ability to ameliorate skeletal deformities and increase bone mass and strength throughout the skeleton, promote fracture healing, and enhance grip strength and mobility in mice with three different subtypes of OI forms. OI skeletal phenotypes continuously develop throughout lifetime, so we will examine rAAV’s effectiveness and durability at multiple intervention timepoints. Next, we will determine molecular mechanisms of bone-tropic AAV:WNT modulators using mouse (Aim 2) and human (Aim 3) OI skeletal organoids. The 3D-skeletal organoids seeded with mouse or human OI-OBs will be developed for in vitro culture or implantation into a xenograft mouse model. These organoids will allow us to study the molecular actions of the bone-tropic rAAV reversing abnormalities in WNT/b-catenin signaling, osteoblast and osteocyte development, mineralization activity, and collagen biochemistry. We will perform transcriptome profiling of AAV-transduced OI-OBs to identify the downstream regulators of the WNT pathway contributing to collagen production and processing. Successful completion of these aims will provide proof-of-concept evidence for bone-tropic AAV gene therapy as an alternative long-lasting treatment option for OI patients and improve our understanding of its mechanisms of action in the treatment of OI.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Zoonotic transmission of avian Influenza A virus (IAV) to humans poses a pandemic threat. Recent transmission of avian IAV to dairy cattle, and to dairy farm employees emphasizes the importance of identifying molecular mechanisms that regulate zoonotic events. It is thought that the IAV envelope glycoprotein, hemagglutinin (HA), must adapt its receptor specificity to bind α2,6-linked SA that predominates the human upper airway to initiate infection. However, recognition of α2,6-linked SA by HA is insufficient for avian H5N1 IAVs to enter human cells, indicating that other adaptations are necessary for zoonotic transmission to occur. Although evidence suggests that the pH and temperature sensitivity of HA are important factors that govern host tropism of IAV, a molecular mechanism that explains the temperature and pH dependence is missing. To define the pH and temperature dependence of HAs pre- to post-fusion conformational change, we first focused on visualizing conformational dynamics of HA with single-molecule FRET (smFRET). Preliminary data reveal that under neutral pH and room temperature conditions, the head domains of A/Vietnam/1194/2004 (H5N1) (VN04) HA, HA1, undergo a breathing motion where the heads are either fully caged, or partially uncaged. At pH8.0 HA spends 50% of the time in the fully caged conformation and 50% of the time in the partially uncaged conformation. Decreasing the pH to 6.5 shifts the equilibrium to where HA spends 30% of the time in the fully caged conformation and 80% of the time in the partially uncaged conformation. These data allow us to establish a conformational phenotype for HA where can determine the pH and temperature dependence of a given conformational phenotype. Thus, the central hypotheses of this proposal are that (1) the regulation of the pre- to post-fusion conformational change of avian HA is differentially regulated by pH and temperature compared to human adapted HA and (2) this difference in regulation directly affects the fusogenic function of HA. Experiments performed in Aim 1 will characterize the pH and temperature dependence of HAs conformational phenotype by monitoring conformational dynamics of HA at pH 8.0, pH 6.5, and pH 5.5 as well as at room temperature and 37°C. I will implement the smFRET for three HA serotypes: A/Vietnam/1194/2004 (H5N1) (VN04), A/California/07/2009 (H1N1) (CA09) and A/Hong Kong/1/1968 (H3N2) (HK68). Furthermore, mutations that alter pH and temperature stability will be characterized to evaluate a molecular mechanism that can explain the differences in the conformational phenotype between the different HA serotypes. Aim 2 will characterize the pH and temperature dependence on HA-mediated membrane fusion by monitoring the extent and rates of fusion of HA pseudo-typed virus with the three previously mentioned HA serotypes. Additionally, the same pH and temperature stability mutations will be used to assess their impact membrane fusion. Collectively, these data will reveal a mechanism that explains the zoonotic transmission potential of IAV and help improve genetic surveillance efforts to identify variants of concern.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Skin diseases affect nearly one-third of the global population. Current dermatological drugs are inadequate for many of these diseases, and some have few or no treatments available. There is an urgent need to develop new dermatological therapies. RNA interference (RNAi)-based therapeutics—e.g., small interfering RNA (siRNA)— represent a third class of therapeutic modalities, in addition to small molecules and biologics, that enable specific and sustained mRNA silencing to prevent production of proteins driving disease. With recent advances in nucleic acid chemistries, five siRNA drugs have been approved by the FDA for treatment of liver-associated diseases. Key benefits of siRNA drugs include: (a) ease of sequence-based design; (b) high specificity; (c) a well-defined mechanism of action; and (d) long durability (up to 3-6 months from one dose). These properties of siRNAs allow rapid drug discovery and durable modulation of disease targets previously considered “undruggable”. The goal of this proposal is to expand the clinical utility of therapeutic siRNAs for the treatment of skin diseases. An siRNA that silences expression of the immune mediator, JAK1 (human and mouse), and supports functional inhibition of JAK1 in vivo, has been identified. Aim 1 (K99 phase) will systematically evaluate the safety and efficacy of selective JAK1 silencing by therapeutic siRNA in mouse models of autoimmune and inflammatory skin diseases (i.e., vitiligo, cutaneous lupus erythematosus, and skin fibrosis). Gene silencing in skin requires efficient delivery of chemically-engineered siRNA to skin cell types expressing the target gene. Aim 2 (K99 phase) will dissect the skin biodistribution and efficacy profiles of hydrophobically conjugated siRNAs targeting JAK1 following local and systemic delivery. The R00 phase will expand the scope of the siRNA delivery platform by determining skin cell biodistribution and efficacy of new siRNA conjugates and scaffolds, and siRNAs targeting immune mediators in the IFN-γ (i.e., IFNGR1, JAK2, STAT1, and CXCL9/10/11) and IL15 (i.e., IL15 and IL15RA) pathways, for which lead compounds have already been identified. Preliminary work has developed a dual-targeting siRNA scaffold supporting simultaneous silencing of two genes in vivo. Aim 3 (K99 phase) will test the efficacy of modulating two inflammatory targets in the mouse models mentioned in Aim 1. In the R00 phase, biocompatible click chemistry and nanostructure engineering strategies will be used to construct programmable unimolecular multi-targeting siRNA scaffolds to potentiate combinatorial modulation of 3 or more inflammatory targets, a crucial goal for treating skin diseases with complex pathology. This project will establish an siRNA-based platform for modulating gene expression in the skin, and foster the PI’s continued scientific and professional training. Research in the mentored K99 phase will be carried out under the guidance of an esteemed mentor committee, whose expertise range from basic RNA biology to clinical dermatology. By the R00 phase, the PI will be ready to establish an independent lab focused on developing novel siRNA therapeutic programs for complex skin diseases.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Cardiovascular disease is the leading cause of death in the United States. Despite decades of research, it is unclear how the RNase A family nuclease angiogenin stimulates blood vessel formation, and why angiogenin dysfunction is associated with heart failure and poor cardiovascular health. Recent work revealed that angiogenin’s nuclease activity, which is required for its angiogenic function, is stimulated by binding to the ribosome, but it remains unclear whether angiogenin’s ribosome-dependent mechanism is involved in angiogenesis. A bacterial nuclease named ribocin with a strikingly angiogenin-like structure and ribosome- dependent activity holds similar potential for understanding lung health. Nearly all Cystic Fibrosis patients experience Pseudomonas aeruginosa bacterial pneumonia and subsequently suffer from lung tissue inflammation and lasting damage long after the infection has cleared. Ribocin encoded by P. aeruginosa damages human ribosomes specifically at central helix 69 of the 28S rRNA and inhibits translation. We hypothesize that like other ribosome-inactivating proteins, ribocin induces a ribotoxic stress response that causes inflammation and cell death in human lung tissues. To inform future therapeutic studies aimed at treating cardiovascular disease and post-infection pulmonary damage, the mechanisms of translation control by these RNase A-family nucleases must be elucidated in the context of their cellular functions. The goal of this project is to determine the structural basis of translation control by angiogenin during angiogenesis and determine the impact of translation control by ribocin on ribotoxic stress response and cell death. With guidance from the sponsor, an expert in biochemical and structural basis of translation, and collaborators, who are experts in the RNA developmental biology and cryo-EM method development, the trainee will apply cutting edge methods for in-cell cryogenic electron microscopy (cryo-EM) complemented by cell assay and biochemical approaches to visualize structural changes to actively translating ribosomes during angiogenin- stimulated vascularization and ribocin-mediated tissue damage. Aim 1 will determine the contribution of angiogenin’s ribosome-specific activity on tube formation (angiogenesis) in human umbilical vascular endothelial cells (HUVEC). Aim 2 will elucidate the structural mechanism of translation inhibition by ribocin and investigate the impact of selective ribosome damage by ribocin on ribotoxic stress response in human lung cells (IB3). The results of this study will reveal details of mechanisms underlying fundamental cardiovascular function and novel components of pulmonary disfunction, necessary for future work in developing therapeutics for cardiovascular and lung disease.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of patient morbidity and mortality in the United States, causing 150,000 deaths, 873,000 emergency department visits, and 700,000 hospitalizations annually. COPD exacerbations diminish patient quality of life, widen health disparities, and strain acute care resources. Frequent assessment and early treatment prevent severe COPD exacerbations and emergency services encounters. However, strategies for optimizing timely, effective evaluation and intervention for acute disease symptoms are lacking. Mobile integrated health (MIH) programs, which deploy highly trained paramedics supervised remotely by physicians into the community to care for patients in their homes, may improve COPD care delivery by overcoming barriers to timely evaluation and treatment. There have been no prior investigations of the implementation or effectiveness of MIH initiatives to manage COPD exacerbation. In this proposal, I seek to implement, refine, and evaluate the Paramedic Evaluation for Acute COPD Exacerbation (PEACE) intervention. This program dispatches community paramedics into patients' homes on-demand, in collaboration with ambulatory teams to manage COPD exacerbation proactively. I have evaluated barriers and facilitators to the adoption of MIH to manage COPD exacerbation. I have also demonstrated the feasibility and safety of an MIH program to care for acutely ill, medically complex adults. Using my findings from this preliminary work, I have designed a prototype intervention for managing acute COPD exacerbation. My research plan includes three aims: Aim 1: Test and refine the prototype PEACE intervention to manage COPD exacerbation in community- dwelling adults with moderate to severe COPD. Aim 2: Complete a pilot randomized controlled trial with 50 patients to evaluate the feasibility and preliminary effect of the intervention, and Aim 3: Evaluate implementation, of the PEACE intervention using the PRISM implementation framework. My career goal is to lead hypothesis-driven research that investigates mobile community-based healthcare delivery strategies to improve the lives of patients living with COPD. To help me achieve my aims and career goals, I have developed a training plan that includes: 1) acquiring training and experiential skills in implementation and dissemination methods, 2) obtaining advanced skills in pragmatic clinical trial design and analytic techniques, and 3) enhancing leadership, grantsmanship and writing skills. I will achieve these goals through directed coursework, focused seminars, and practical experience. I am well supported by a highly experienced team of mentors and advisors. At the end of this mentored career development award, I will have an enhanced intervention, robust preliminary data, and a refined research strategy, well-positioning me to perform an R01-funded multi-center trial as an independent investigator.

NIH Research Projects · FY 2025 · 2025-06

Abstract Pneumonia is a leading cause of morbidity and mortality worldwide. Microbes that cause respiratory infections are encountered by virtually everyone, but effective immunity in healthy individuals typically prevents such exposures from progressing to pneumonia. Yet, host signals guiding lung defense constitute a major knowledge gap, limiting opportunities for clinical intervention in patients at risk for pneumonia. A goal of our research is to delineate when, where, and how prior experience with respiratory pathogens influences pneumonia susceptibility thereafter. Recovery from lung infections elicits strong and rapid protection in response to a subsequent challenge with related but distinct organisms (e.g., different strains/serotypes). We and others have demonstrated that this heterotypic protection is location-dependent, restricted to the previously exposed region and independent of circulating factors. Moreover, accelerated immunity in experienced lungs requires lung CD4+ resident memory T (TRM) cells, which accumulate in the interstitium near bronchovascular bundles and pulmonary veins. CD4+ T cells are required for the rapid and protective neutrophil response observed in experienced lungs, and our pilot studies, using a novel crystal ribcage approach to connect experienced lungs to the circulation of naïve mice, indicate that this is specifically driven by lung-localized CD4+ TRM cells. Excitingly, this same approach revealed neutrophil recruitment as remote as subpleural alveoli in as few as 4hrs in the experienced lung, demanding a better understanding of the multi- cellular signaling network tethering CD4+ TRM cells to enhanced neutrophilic defense. The primary objectives of this proposal are to determine the spatiotemporal relationship between centrally concentrated CD4+ TRM cells and emigrated neutrophils during pneumonia, while developing a transcriptional atlas of key immune processes corresponding with enhanced defense in the experienced lung. To do so, we will test the hypotheses that: 1) Infection of the experienced lung activates CD4+ T cells in the interstitium of bronchovascular bundles and pulmonary veins, coinciding with diffusely parenchymal neutrophil recruitment; and 2) CD4+ T cells in the experienced lung trigger rapid, diffuse, and heterogeneous transcriptome changes across diverse distal lung and immune cells. This will be accomplished using state-of-the-art approaches for imaging, cytometry, and spatial transcriptomics, leveraging expertise and resources across two neighboring institutions. Results from this study will provide key insights about the cells and signals dictating host immunity. Furthermore, data from the proposed experiments will provide a foundation for future investigations by our groups and others, having a lasting impact on the field.

NIH Research Projects · FY 2025 · 2025-06

Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a progressive neurodegenerative disease caused by loss of upper and lower motor neurons in the brain and spinal cord. Frontotemporal dementia (FTD), the most common presenile dementia under the age of 60, is caused by focal degeneration of frontal and/or temporal lobes. Despite decades of intensive research, there is still no effective treatment for either disease. Pinpointing the underlying pathogenic mechanisms poses a challenge, as about 90% of ALS cases and 60% of FTD cases are sporadic. However, nuclear depletion and cytoplasmic aggregates of TDP-43 are found in about 97% of ALS cases and up to 50% of FTD cases, as well as in many patients with Alzheimer's disease. Thus, it is critically important to understand the molecular and cellular mechanisms underlying TDP-43 pathology. In this exploratory project based on some of our unexpected preliminary findings, we will use mouse models and CRISPR-edited human neurons derived from induced pluripotent stem cells to investigate how the ubiquitination/deubiquitination pathway regulates the subcellular localization of TDP-43 and TDP-43 aggregate formation. These studies will provide mechanistic insights into TDP-43 biology and may suggest novel therapeutic targets for neurodegenerative diseases characterized by TDP-43 pathology.

NIH Research Projects · FY 2025 · 2025-06

Project Summary: Tuberculosis (TB), like many infectious diseases, demonstrates striking variation between individuals in its clinical manifestation. People infected with the causative agent, Mycobacterium Tuberculosis, range in disease state from completely asymptomatic infection to life-threatening disseminated disease. Even though many infected individuals do not develop symptoms and most resolve the infection with treatment, TB is perennially the leading cause of death by a single infectious agent with an annual death toll of more than one million people. A major goal of TB research is to understand the basis of the variability in infection outcomes. Such an understanding will help in identifying ways to improve treatments and tip the scale away from pathological inflammation lung damage toward healthy resolution of infection. One important factor that contributes to the outcome of TB is the genetic background of the infected individual. Many studies have addressed the role of natural genetic variants in determining TB outcomes in both humans and mice. However, relatively few studies have investigated these associations in the context of macrophages, a central cell type of interest in TB. This project will explore the relationship between genetic variants, transcriptional responses, and Mtb infection phenotypes in macrophages derived from genetically diverse Collaborative Cross (CC) mice. In preliminary studies, I associated variants in the Stat2 locus with the strength of type I interferon signaling in stimulated CC macrophages. Therefore, I hypothesize that polymorphisms, such as those in Stat2, underlying variation in transcriptional responses are responsible for heterogeneity in bacterial control in CC macrophages. I will test this hypothesis using a novel tool developed to specifically investigate the role of natural genetic variation in determining macrophage phenotypes. This hypothesis will be tested in three specific aims which will both generate novel genetic association and determine the mechanism of previously identified associations. I will use an exploratory approach in the first aim to discover what the genetic and transcriptional determinants of Mtb restriction are in this population of macrophages. This approach is likely to discover novel mechanistic insights into how macrophage respond to Mtb infection. In the second and third aims, I will delineate the mechanism by which polymorphisms in Stat2, a mediator of type I interferon signaling, alter its function. The second and third aim will test an updated model of the recognition of DNA by STAT2 which I generated by combining macrophage genetic association studies and structural modeling. In the proposed studies, I will utilize and continue to develop an innovative new tool, the CC macrophage library. I will incorporate genetic, biochemical, and systems approaches to discover and characterize associations of polymorphisms and transcriptional networks with Mtb infection outcomes. The results of these studies will provide insight into innate immune processes and will be important to consider for future studies into TB and other infectious diseases using the Collaborative Cross.

NIH Research Projects · FY 2026 · 2025-06

Project Summary Uterine smooth muscle cells (USMCs) generate uterine peristalsis that is crucial for menstruation, sperm and embryo transfer, and embryo implantation; dysfunction in uterine peristalsis is associated with adenomyosis, a uterine disease that affects millions of women with physical and mental distress. To date, the molecular basis of uterine peristalsis and its precise role in reproduction, and adenomyosis remain unclear. We recently discovered that the activation of L-type Ca2+ channels in USMCs generates intercellular Ca2+ waves that lead to uterine peristalsis. We also found in our published or preliminary studies: (1). Among the four α1 pore- forming subunits of the L-type channel, Cav1.2 and Cav1.3 are expressed in non-pregnant uteri and their levels are decreased in uteri at the peri-implantation stage. (2). Smooth muscle cell-specific Cav1.2 deletion disrupts Ca2+ waves and uterine peristalsis, causing infertility. (3). Intercellular Ca2+ waves and uterine peristalsis from tamoxifen-induced adenomyotic mice are altered, with a down regulation of Cav1.2. (4). Tamoxifen-induced adenomyotic mice show impaired embryo implantation. And (5). Myometrium from normal regions of uterine slices from patients with adenomyosis exhibits synchronized Ca2+ waves and robust shortening. In contrast, the myometrium around adenomyotic lesions from the same patients produces asynchronized Ca2+ waves with less shortening. These functional changes are associated with a decrease in Cav1.2 mRNA and an increase in Cav1.3 mRNA in the adenomyotic regions. Given these findings, we hypothesize that (1) in both mice and humans, Cav1.2 and Cav1.3 contribute to the L-type Ca2+ current in USMCs, (2) Cav1.2 and Cav1.3 play distinct roles in uterine peristalsis, uniquely influencing embryo implantation, and (3) Cav1.2 down-regulation underlies alterations in uterine peristalsis in adenomyosis and contributes to adenomyosis pathogenesis. To test these hypotheses, we will investigate the roles of Cav1.2 and Cav1.3 in Ca2+ signaling, uterine peristalsis, and embryo implantation using Cav1.3 knockout mice and smooth muscle cell-specific and inducible Cav1.2 knockout mice (Aim 1). We will also determine whether Cav1.2 down-regulation alters uterine peristalsis, leading to adenomyosis and the resultant impaired embryo implantation in mice (Aim 2). Finally, to translate our findings in mice to humans, we will use RNA interference to determine whether Cav1.2 and/or Cav1.3 comprise L-type Ca2+ channels in human USMCs, identify and quantify the signatures of L-type Ca2+ currents, Ca2+ signals, and uterine peristalsis in human adenomyotic USMCs, and uncover the underlying mechanisms for generating these signatures (Aim 3). This study is expected to provide a fundamental understanding of uterine peristalsis and its role in fertility, as well as deep insights into the mechanisms of adenomyosis pathogenesis. The outcome of this study may provide the basis for targeting the Cav1 family in USMCs for new treatments of gynecologic disorders.

NIH Research Projects · FY 2025 · 2025-06

MASSI/XU – ABSTRACT Pathological molecular alteration and the associated aggregation of the TAR-DNA-binding protein-43 (TDP-43), a protein involved in RNA processing, are hallmarks of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). They are also observed in a variety of other devastating neurodegenerative diseases, including traumatic brain injury (TBI), Alzheimer's disease (AD), Parkinson's disease (PD), and limbic-predominant age-related TDP-43 encephalopathy (LATE). Recently, we and others have observed that, in patient protein aggregates, TDP-43 is citrullinated at two specific sites. Protein citrullination (PC) is a common post-translational modification known to modulate protein function. Interestingly, altered PC has been observed in various neurodegenerative diseases including ALS, PD, AD, Creutzfeldt-Jakob disease (CJD) and multiple sclerosis (MS). The goal of this proposal is to interrogate the hypothesis that citrullination of TDP-43 impacts its RNA-binding activity and stability, resulting in cytoplasmic mislocalization and aggregation. We aim to elucidate the structural basis underlying the effect of PC on both the structure, stability and liquid-liquid phase separation (LLPS) of TDP-43 (Aim 1). We also aim to determine how citrullination impacts TDP-43 RNA-binding activity and splicing regulation (Aim 2). Finally, we will characterize the effect of citrullination on the nuclear versus cytoplasmic localization of TDP-43 to determine if mislocalization of TDP-43 to the cytoplasm leads to its aggregation (Aim 3). Collectively, the outcomes from these aims will provide novel insights into the role of PC in neurodegeneration in general and ALS in particular. The long-term goal of this research is to utilize these molecular insights to develop TDP-43-based therapeutic strategies to treat a broad class of devastating neurodegenerative diseases.