University Of Connecticut Sch Of Med/Dnt

NIH Research Projects · FY 2026 · 2026-06

Project Summary/Abstract The innate immune system employs germline-encoded pattern recognition receptors (PRRs) to survey the host extra- and intra-cellular milieu for the presence of pathogen-associated molecular patterns or other danger signals and mount appropriate defense responses. These responses are typically mediated by proinflammatory cytokines and type I interferons (IFNs). Type I IFNs are potent and pleiotropic, orchestrating an effective innate immune defense against microbial pathogens. While the anti-viral functions of type I IFNs are established, their role in defense against bacterial pathogens is beginning to be understood. Previous studies from our lab showed that type I IFNs play a protective role against infection with a clinically significant enteric pathogen, enterohemorrhagic Escherichia coli (EHEC). EHEC causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. Treatment of EHEC infection is limited because antibiotics enhance the severity of HUS. Given the protective role of IFNs in EHEC disease, modulation of the type I IFN response could be a potential therapeutic strategy for EHEC disease. However, the host factors critical for initiating type I IFN response against EHEC, and bacterial pathogens in general, are not well understood. Our preliminary studies addressing this critical knowledge gap identified a crucial role for MiT family transcription factors, TFE3 and TFEB, in directing type I IFN responses against EHEC. While MiT family proteins are well characterized for their role in starvation responses and lysosomal biogenesis, their role in type I IFN induction is previously unknown. Further preliminary studies also demonstrated that the TFE transcription factor- mediated IFN regulation extends well beyond EHEC infection and that they play a significant role in promoting multiple PRR-mediated type I IFN pathways. These observations warrant further investigations into this novel role of MiT family transcription factors. In this context, the studies proposed in this project will systematically elucidate the upstream mechanism of activation of MiT transcription factors, the mechanisms by which TFEs promote type I IFN expression, and the physiological relevance of TFE-mediated anti-bacterial defense. In summary, findings from this study will assign a new function to MiT family transcription factors and provide critical insights into the host determinants of type I IFN expression.

NIH Research Projects · FY 2026 · 2026-06

Project Summary/Abstract: Regulatory T cells (Tregs) play key roles in immune homeostasis, tumor development, and cancer therapy through suppression of inflammation. How the inflammatory tumor environment shapes the behavior of Tregs in turn, is less known. This knowledge gap precludes our ability to selectively block the activity of tumor infiltrating Tregs, while leaving systemic Treg function intact. One of the prominent inflammatory pathways involves interleukin-17 (IL-17), which has been shown to promote tumor development in multiple tissues including the gut. IL-17 family cytokines promote inflammation that drives the development of colorectal neoplasia, which eventually lead to colorectal cancer (CRC). Thus far, the underlying mechanism has largely been attributed to IL-17’s impact on tumor cells and myeloid cells. Our preliminary studies show that targeted ablation of IL-17 signaling on Treg cells increased colonic tumor development in mice, demonstrating a previously unknown protective role of IL-17 in CRC. We found that IL-17 directly signals to Tregs to promote their immune suppressive signature and more importantly, alternative splicing of RNAs in these cells. We also found that IL-17 promotes the expression of the RNA binding protein (RBP), Ybx1, and enhances the splicing of RNAs whose protein products are critical for Treg function. These genes (Foxp3, IL-27RA, and Stat3) also contain RNA motifs that can be recognized by Ybx1, whose role in Treg is unknown. Ablation of Ybx1 in both human and mouse Tregs resulted in reduced Treg immune suppression function, and reduced expression of immune suppressive protein Galactin-1. We demonstrate that proper splicing of Galactin-1 mRNA depends on Ybx1. These findings lead to our hypothesis that IL-17 promotes Treg function and suppresses CRC development through Ybx1 mediated alternative RNA splicing, and that a similar IL-17-Ybx1 pathway promotes Treg activity in established tumors and impedes cancer immunotherapy. Given the critical roles of both Tregs and Th17 cells in tumor development and cancer therapy, along with the knowledge gap relating to the impact of RBPs on Treg biology, we propose the following studies: 1) delineate the mechanism by which the IL-17-Ybx1 pathway regulates alternative RNA splicing and function of Tregs; 2) test if the IL-17-Ybx1 pathway in CRC can be manipulated to prevent gut tumorigenesis; and 3) test the feasibility of targeting IL-17-Ybx1 pathway for the enhancement of cancer immunotherapy. These investigations will provide new insights into the mechanisms by which the inflammatory tumor microenvironment (TME) controls Treg function, and guide the invention and use of novel therapies for the treatment of CRC in humans. For example, based on a role for Ybx1 in promoting immune suppressive function of Tregs in CRC, we may employ targeted therapy by degrading Ybx1 in tumor infiltrating Tregs to foster an immunologically “hot” environment for enhanced cancer immunotherapy. Justification for the use of animal models We propose to use multiple animal models of cancers in this project. These models closely mimic human colorectal cancers by simulating tumor cell mutation process, microbiota interaction, and tumor environment evolution, allowing us to interrogate the mechanism of Ybx1-controlled Treg cell function in cancers. These models cannot be replaced with non-animal studies such as in vitro cell culture or organs-on-a-chip systems.

NIH Research Projects · FY 2026 · 2026-06

Project Summary Colorectal cancer (CRC) is the third-leading cause of cancer related deaths in the US. Recent advances in cancer immunotherapies, such as immune checkpoint blockade (ICB) targeting PD-1 and other pathways, have gained remarkable progress in the treatment of human cancers. However, PD-1 blockade only works in a fraction of CRCs that harbor microsatellite instability (MSI). Therapeutic options are limited for most CRCs that are microsatellite stable (MSS). However, oncolytic viral therapies have shown promise as a cancer therapy and may prove instrumental for MSS CRCs, with viruses that selectively infect and lyse cancer cells, and simultaneously deliver immune modulating and/or cancer killing payloads. However, major hurdles in the development of oncolytic viral therapies exist. These include non-specific infection of normal tissues by viral vectors, and their inefficient accumulation in tumors upon systemic administration. These limitations restrict the use of oncolytic viruses carrying potent anti-cancer payloads (such as IL-12) to intratumoral injection, precluding their use in cancers that are not surgically reachable. We aim to resolve this limitation and fill the gap of medical need by developing and validating a “cap-linker” strategy to modify the vesicular stomatitis virus (VSV). Our preliminary data show a complete inhibition of viral activity when the VSV Indiana glycoprotein (VSV-GIN) is linked to the human LDL-R cysteine rich (CR)-2 domain (the “cap”) by a flexible, protease cleavable linker. Incorporation of the CR-2 cap into VSV-GIN results in a 700- fold activation of viral infection following linker cleavage. VSVIN harboring the modified VSV-GIN (Pro-VSVIN) with an MMP-cleavable linker showed marked increase in the safety profile compared to its WT counterpart. When injected i.v. into CACO2 human CRC bearing immune deficient mice, Pro-VSVIN achieved tumor-specific distribution and replication, resulting in more than 400-fold higher viral payload expression in the tumor compared to all normal tissues analyzed. When armed with a single chain, biologically active IL-12, i.v. injected Pro-VSVIN eradicates grafted colon tumors, inhibits lung metastatic growth of breast cancer, and enhances the efficacy of anti-PD-1 therapy. With these findings, we hypothesize that following systemic injection, the novel “cap- linker” modification of VSV-G specifically targets VSV to MMP overexpressing tumors, and when armed with immune activating payloads, such as IL-12, exhibits superior efficacy and safety compared to conventional oncolytic viral therapies. We will carry out the following studies to test this hypothesis: 1): Validate the specificity of Pro-VSVIN in MSS CRC; 2) Test the efficacy of Pro-VSVIN-IL-12 for the treatment of CRC; and 3) Optimize the treatment scheme for combined Pro-VSVIN-IL-12 and immune checkpoint blockade in MSS CRC. These investigations will confirm the cancer-specific targeting and efficacy of Pro-VSVIN, and pave the way for its use in human CRC treatments. As overexpression of MMP is common among solid tumors in humans, the impact of this study extends beyond MSS CRC to cancers of diverse origins.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Biopolymers of life fall into four broad categories: proteins, nucleic acids, lipids, and carbohydrates, also called glycans. Hybrid forms of these biopolymers are common, as is the case with glycoproteins and glycolipids on the cell surface. Nearly 80% of secreted or cell surface proteins are glycosylated; some contain up to 50% by mass carbohydrates. Recently, we discovered a new hybrid macromolecule, glycosylated RNA, expressed broadly across many cell and tissue types and is made up of N-glycans modifying small noncoding RNAs. Interestingly, these glycoRNAs are mainly found on the cell surface. While the observation that glycosylation can occur on RNA is transformative, a key question arising from this discovery is what the functional significance of RNA glycosylation is. Nucleic acid sensing is a critical immune surveillance program that monitors for invading pathogens. Innate immune sensors monitor the endosomes and the cytosol for foreign nucleic acids, with TLR3, 7, 8, and 9 sensing nucleic acids that end up in the endosome—these nucleic acids can come from pathogens and dead cells during efferocytosis. Because cells exist in a homeostatic state with glycoRNAs that can access the spaces where TLR sensors are deployed, we hypothesize that glycosylation of mammalian small RNAs prevents immune sensing of glycoRNAs. We plan to test our hypothesis through the innovative approaches proposed in three specific aims. In Aims 1 and 2, we will characterize the features of glycans that enable the “shielding” of glycoRNAs from the endosomal TLR sensing machinery and the mechanism involved. In Aim 3, we will define how pathogenetic bacteria remodel the features of cell surface glycoRNAs and connect this to the functional role of glycoRNAs during the infection of mammalian cells. Altogether, we anticipate that the results generated by this proposal will address a fundamental knowledge gap by establishing the first direct function of glycosylated RNA in the context of immune surveillance and cellular homeostasis.

NIH Research Projects · FY 2026 · 2026-05

Project Summary Almost 6 million individuals in the US have an opioid use disorder (OUD). Medications to treat OUD (MOUD) are considered the gold standard; however, most people with OUD do not receive them. Integrated care settings, such as Certified Community Behavioral Health Clinics (CCBHC), are promising settings to focus provision of MOUD. However, MOUD, particularly newer long-acting injectable formulations that may significantly improve patient outcomes, remain highly underutilized in CCBHCs. Implementation strategies to address barriers at the patient, provider, and organizational levels need to be developed and tested to improve uptake of long-acting injectable MOUD in CCBHC settings. Adopting measurement-based care (MBC) holds promise as an implementation strategy to optimize treatment delivery in patients with OUD, especially for medications. Doing so may create a learning health system model that improves clinical practice and patient outcomes. However, this proposition remains largely untested, particularly in CCBHCs. More research is needed to understand the potential benefits of implementing MBC systems to address implementation outcomes for MOUD. For example, it is not known whether adding MBC to other common implementation strategies (e.g., practice facilitation) confers additional implementation and patient outcome benefits. This two- phased R34 project proposes to develop and test implementation strategies to support long-acting injectable MOUD implementation in CCBHCs. It brings together an interdisciplinary team of implementation science researchers and CCBHC providers. During the first phase, we will develop an MBC-based multi-level implementation approach to support long-acting injectable buprenorphine implementation in CCBHCs using an iterative user-centered, participatory, co-design process with CCBHC patients and staff/providers. We will create MBC protocols and EHR systems, educational/shared decision-making tools for long-acting buprenorphine, and quality-improvement driven tailored implementation supports for long-acting buprenorphine and MBC (e.g., practice facilitation). In the second phase, a mixed-methods pilot cluster-RCT in 6 CCBHC sites serving approximately 1,000 OUD patients per year will test the multi-level MBC implementation approach vs. non-MBC based approach on implementation and patient clinical outcomes. Specifically, staff/provider implementation (adoption, acceptability, and feasibility), patient implementation (patient penetration, acceptability), and patient clinical (retention in treatment and on long-acting injectable MOUD) and functional outcomes will be compared between the two conditions. We will also examine implementation context. Results of this R34 will provide an initial test of MBC-based implementation strategies for long-acting injectable MOUD in CCBHCs and will set the stage for a larger R01-level hybrid implementation-effectiveness trial. This work will address critical gaps in knowledge about how to efficiently implement long-acting injectable MOUD in CCBHCs in the context of a learning health system model with the goal of improving treatment quality and outcomes.

NIH Research Projects · FY 2026 · 2026-05

Abstract Lung cancer remains the leading cause of cancer-related mortality worldwide, with early detection being critical for improving patient outcomes. Current diagnostic methods, including imaging and tissue biopsy, are often invasive, costly, and require specialized expertise. Liquid biopsy offers a promising alternative for noninvasive lung cancer screening by detecting circulating tumor biomarkers in blood. The detection of cell-free DNA (cfDNA) mutations enables early identification of lung cancer and assessment of treatment response. Especially, as highly effective targeted therapies become more accessible, there is a clear need for affordable and accurate cancer mutation diagnostics. Digital droplet polymerase chain reaction (ddPCR) and next-generation sequencing have been widely used to detect and quantify cfDNA mutations due to their high sensitivity, accuracy, and robustness. However, they remain limited by the complexity, high cost, and time-intensive workflow. Here, we propose to develop and validate a simple, rapid, and affordable Programmable Enzyme-Assisted Mutation Sequencing (PEAM-Seq) diagnostic platform for the quantitative detection of multiple cfDNA mutations in blood samples, enabling noninvasive, accurate, and early detection of lung cancer. In our preliminary study, we developed and validated a PCR-based PEAM-Seq Version 1 (PEAM-Seq V1.0) assay to quantitatively detect cfDNA mutations with single-nucleotide resolution. To simplify the assay, we will further develop and optimize a simple, rapid, and sensitive PEAM-Seq V2.0 assay by combining isothermal amplification, enzymatic cleavage, and CRISPR quantitative detection. To enable point-of-care diagnostics, we will incorporate the PEAM-Seq V2.0 assay into microfluidics to develop a simple and integrated PEAM-Seq chip. To eliminate the need for costly and bulky equipment, we will build a portable, smartphone-based platform to pair with the disposable PEAM-Seq chip. We will assess the clinical utility of our PEAM-Seq diagnostic platform by analyzing blood samples from lung cancer patients to validate its effectiveness in cancer early detection. While lung cancer detection serves as the initial proof of principle, this project will ultimately provide a transformative platform technology for rapid, cost-effective, and accessible diagnostics of a wide range of cancers.

NIH Research Projects · FY 2026 · 2026-05

Abstract Oropharyngeal Candidiasis (OPC) is a common fungal infection afflicting populations with immature or weakened immune systems. Like other oral diseases in which commensal microbes become pathogenic, OPC is associated with a dysbiotic state, an alteration in the composition and abundance of the oral health- associated bacteria, contributing to a permissive environment for Candida spp. infection. An aging population, the rise in the use of immune-compromising treatments and the increasing prevalence of drug- resistant Candida strains make it imperative to find alternative treatments for OPC. While microbiome manipulation therapies have been effective in intestinal dysbiotic diseases, none have yet been established for oral microbiome-related diseases. Microbiome directed therapies include reconstitution with probiotic bacterial consortia, that can promote oral mucosal homeostasis. It remains extremely important not only to discover new probiotic organisms but also to unravel their mechanism of action and understand their strain- specific effect. Work in our previous funding cycle showed that C. albicans causes a severe dysbiotic shift in the oral mucosal microbiota with significant reduction in bacterial alpha diversity. We also demonstrated pathogenic synergy of indigenous dysbiotic bacteria with C. albicans and identified cellular and molecular mechanisms of this synergy. Importantly, using two different types of dietary interventions in the murine OPC model we found that the oral bacterial dysbiotic shift is reversible and that diet-induced increased abundance of the commensal species Lactobacillus johnsonii is associated with less severe infection. In the next funding cycle, we propose to demonstrate that L. johnsonii induces changes in the oral bacterial microbiome consistent with mucosal homeostasis and examine mechanisms of this activity (aim 1). We also propose to demonstrate a role for this organism and its associated symbiotic bacteria in limiting the risk of C. albicans infection in immunosuppressed hosts (aim 2). In aim 3 we will dive deeper in the mechanism of protection of the oral mucosal barrier by evaluating the ability of L. johnsonii to inhibit oral epithelial adhesion, invasion and damage by C. albicans; and to inhibit Candida-induced mucosal inflammation and barrier breach. Our innovative studies will explore probiotic bacterial mechanisms that leverage the interactions between resident host bacterial microbiota, oral epithelial cells and opportunistic pathogenic fungi, providing a foundational step towards personalized, microbiome-centered treatments beyond traditional antifungal medications in oropharyngeal candidiasis. This research proposal is significant because it advances the search for alternative microbiome-targeted therapies in oral fungal infections.

NIH Research Projects · FY 2026 · 2026-04

ABSTRACT Genital herpes, a lifelong sexually transmitted infection caused by either Herpes Simplex Virus 1 or 2 (HSV1/2), is one of the most common STDs in the US with (846 million people infected worldwide). HSV1/2 can cause oral and genital lesions and establish lifelong latent infections which can undergo periodic reactivation. Reactivation can cause painful ulcers and blisters; however, many reactivations are asymptomatic, causing an increased risk of partner transmission. HSV reactivation is particularly problematic in immunocompromised patients and can lead to disseminated life-threatening infections. HSV can also be transmitted to newborns either during or after birth, potentially leading to serious neurological disorders. The severity of this public health epidemic is intensified by the lack of protective vaccines and the inability to cure infections with existing antiviral therapies. Furthermore, it is now recognized that HSV infection can increase the risk of HIV infection. Existing therapies have focused on the HSV DNA polymerase as a target, and most HSV polymerase inhibitors are nucleoside/tides, which are often associated with drug resistance, narrow spectrum and dose-limiting toxicities. New agents with different modes of action are needed not only for the treatment of resistant viruses but also for use in combination therapy to reduce dose-limiting toxicities. New therapies used prophylactically would prevent pathological sequelae of reactivation, as well as viral shedding and transmission to new hosts. We have identified the HSV alkaline nuclease (AN) as a promising and novel HSV target for anti-herpesvirus drug discovery efforts. HSV1 and HSV2 AN are encoded by the UL12 gene, which is highly conserved between HSV1 and 2. We have had a long- standing interest in understanding the role of AN in viral replication primarily relying on genetic methods. A null mutant in AN and a point mutation in the nuclease active site (D340E) are unable to produce infectious virus in cell culture. AN is a member of a large class of two-metal ion-dependent (TMID) enzymes, whose active sites coordinate divalent cations essential for catalysis. Many viruses utilize TMID enzymes for processing and manipulation of viral nucleic acids. The successful development of selective and safe inhibitors of two viral TMID enzymes (HIV integrase and influenza endonuclease, PA) shows that it is possible to block TMID nuclease activity by metal-coordinating enzyme inhibitors. We have shown that the HSV alkaline nuclease is essential for productive HSV infection as nuclease dead HSV mutants are defective in viral spread and virus production. Furthermore, we have identified several lead compounds that block AN activity and produce strong antiviral responses in cell culture and animal models. In this proposal we will continue our efforts to develop antiviral agents that inhibit HSV1/2 alkaline nuclease, UL12.

NIH Research Projects · FY 2026 · 2026-04

Project Summary The molecular mechanisms controlling the growth of axonal projections in the central nervous system (CNS) are still poorly understood. Similarly, the molecular basis of the failure of axon regeneration in the CNS are still elusive, and no regenerative therapies exist to date that could help patients with axonal injuries. Retinal ganglion cells (RGCs) are prototypical CNS projection neurons that do not spontaneously regenerate axons disrupted in optic neuropathies caused by trauma, ischemia, or glaucoma, resulting in irreversible loss of vision. Our goal is to investigate the molecular mechanisms through which the Nfe2l3 protein, whose neuroprotective functions we recently identified, regulates the regeneration process of the damaged optic nerve axons, and to test their potential for restoring simple visual functions after optic nerve injury. We will utilize established animal models and state-of-the-art technologies for investigating through which molecular mechanism the Nfe2l3 protein activates the retinal ganglion cells’ intrinsic capacity for regenerating long-distance axons through the optic nerve. We will also determine which retinal ganglion cells respond to the Nfe2l3 protein by regenerating injured axons. We expect that these studies will lead to the development of a novel approach for restoring simple visual functions after optic nerve injury, with the potential to treat different types of optic neuropathies.

NIH Research Projects · FY 2026 · 2026-04

Abstract The quantitative detection of nucleic acids plays an increasingly important role in the early detection and screening of cancers. Although the real-time quantitative PCR method has become the gold standard for nucleic acid quantification, its reliance on expensive equipment and trained personnel limits its use in resource-limited settings. Recently, CRISPR technology has emerged as a simple and powerful tool for highly sensitive and specific nucleic acid detection when combined with nucleic acid pre-amplification. However, due to the incorporation of the pre-amplification step, CRISPR-based detection methods require multiple operations and lack the quantitative detection capabilities needed for nucleic acid targets. Therefore, developing a simple and sensitive CRISPR approach for the quantitative detection of nucleic acid biomarkers (e.g., microRNAs) remains a challenge. Recent studies have demonstrated that exosomal microRNAs (exo-miRNAs) are promising liquid biopsy biomarkers for detecting cancer progression and assessing therapy efficacy with high sensitivity and specificity. However, current methods for exo-miRNA detection often require expensive equipment and specialized expertise, hindering their widespread clinical application. Here, we propose to study an asymmetric CRISPR approach for the quantitative detection of nucleic acids (e.g., exo-miRNAs). Furthermore, we will integrate the asymmetric CRISPR assay into a microfluidic chip to develop an asymmetric CRISPR diagnostic platform for exo-miRNA profiling in clinical blood samples. As an example application, we will adapt our asymmetric CRISPR diagnostic platform for the non-invasive early detection of breast cancer—the most commonly diagnosed cancer in the world—and rigorously validate its clinical application. Ultimately, the success of our proposed project will enable clinical translation, facilitating the development of a simple, sensitive, nucleic acid-based molecular detection method for early cancer detection and personalized medicine.

NIH Research Projects · FY 2026 · 2026-04

Project Abstract Abdominal aortic aneurysm (AAA) affects 1–2% of the elderly population and, when ruptured, often causes fatal hemorrhage. As AAA enlarges, the rupture risk increases, yet no pharmacological therapy exists to slow its progression, posing a major challenge, especially for patients ineligible for surgery. A key feature of AAA is vascular smooth muscle cell (VSMC) reprogramming from a contractile to a synthetic state, driven primarily by Kruppel-like factor 4 (KLF4). While Ca2+-dependent pathways induce KLF4 expression, the precise mechanism of its aberrant activation in AAA remains unclear. TRPM7 is a unique chanzyme (ion channel with kinase activity) abundantly expressed in VSMCs. TRPM7 is negatively regulated by ADP-ribosylation factor-like protein 15 (ARL15), but its biological significance is completely unknown. TRPM7-mediated ionic signaling and TRPM7 kinase play a critical role in enhancing inflammatory responses. Considering AAA is featured by the chronic inflammatory conditions of the aortic wall, I propose to investigate the contribution of TRPM7 activity in VSMCs reprogramming and AAA pathogenesis. In Aim 1, the expression and regulation of TRPM7 and ARL15 in human VSMCs and human AAA samples will be examined. The pathogenesis of human AAA will be investigated using the latest snRNA-seq technology, which offers more than twice the resolution of previous methods. Additionally, the role of VSMC’s TRPM7 in aneurysmal remodeling will be comprehensively evaluated through scRNA-seq and CITE-seq using my HA- TRPM7 transgenic mouse strain, and VSMC-specific TRPM7 knockout mouse strain with lineage tracing experiments. To elucidate the role of ARL15, the endogenous inhibitor of TRPM7, in AAA development, I will modulate its expression in vivo through overexpression or knockdown using adenoviral or AAV delivery systems. In Aim 2, the molecular mechanisms underlying TRPM7-induced AAA pathogenesis will be investigated. To determine whether TRPM7’s channel or kinase function is responsible for AAA progression, I have generated an inducible channel-dead TRPM7 (TRPM7E1047K) knock-in mice, which will be paired with kinase-dead TRPM7 (TRPM7K1646R) knock-in mice. The involvement of TRPM7-mediated ionic signaling pathways, including Ca2+, Mg2+, or Zn2+, will also be explored. Finally, I am generating inducible gain-of-function TRPM7N1098Q knock-in mice to directly assess whether TRPM7 overactivation serves as a primary driver of AAA pathogenesis or merely contributes secondarily to AAA related vascular remodeling. In summary, my goal is to elucidate the mechanisms underlying AAA and establish TRPM7 overactivation as a key driver of vascular remodeling and AAA pathogenesis, providing a foundation for potential future therapeutic strategies to limit AAA progression. Additionally, the development of inducible gain-of-function and channel-dead TRPM7 knock-in mouse strains will facilitate broader investigations into TRPM7’s role in other physiological and pathological processes, enabling future collaborations with other research groups.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY/ABSTRACT Cell death and the effective clearance of dead or dying cells are essential processes for maintaining homeostasis. Cells die in various physiological or pathological contexts, including tissue injury, infection, anti- tumor chemotherapy, or radiation therapy. Dead or dying cell corpses, along with the released intracellular contents, are potent activators of cellular responses such as wound healing and inflammation. These processes must be tightly regulated to prevent triggering autoimmune, fibrotic, or other diseases. Moreover, cell death can occur through distinct pathways, such as non-lytic apoptosis, or lytic necroptosis and pyroptosis, each having different effects on the surrounding milieu. Therefore, manipulating the mode of cell death or its subsequent biological effects holds great potential for developing more effective therapies against diseases like cancer. Efferocytosis, the process by which phagocytes internalize dead cells, removes and processes dead cell remnants to prevent harmful accumulation. Dead cell-containing phagosomes formed during efferocytosis are filled with entities that bind and recognize dead cells, serving as a platform for decoding death-associated information via processes such as antigen presentation. Recently, we have established a proximity-labeling- based approach, dubbed PhagoPL, to profile the protein compositions of microbe-containing phagosomes, systematically exploring microbe-sensing mechanisms in macrophages (Li et al., Nature 2024). This proof-of- concept application of the PhagoPL technique demonstrates that the composition of the phagosome is adaptive to the biochemical nature of its cargo and proves that PhagoPL holds the potential to uncover novel players in cell recognition. In this proposal, the cutting-edge PhagoPL approach will be used to investigate the protein compositions of various dead cell-containing phagosomes. By doing this, we seek to address two significant gaps in the literature related to how phagocytes interact with dead cells: (1) What are the different sets of proteins used by phagocytes to uptake, process and respond to distinct types of dead cells, specifically apoptotic, necroptotic, and pyroptotic cells? (2) How does phagosome composition dictate the ability of phagocytes, especially different types of dendritic cells, to perform antigen cross-presentation? By answering these unexplored but fundamentally important questions, this research program will provide guidance and potentially novel targets for the development of new therapeutics for autoimmune diseases, cancer, and infections.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Oligodendrocyte precursor cells (OPCs) are best known as precursor cells that generate myelinating oligodendrocytes. In demyelinating disorders such as multiple sclerosis, OPCs are an important source of remyelinating cells. OPCs exist abundantly not only during development when myelinating cells are generated but also in the mature brain in the gray matter as well as white matter. This suggests that OPCs may be involved in cellular functions other than generating myelinating oligodendrocytes. Unlike other non-neuronal cell types, they receive synapses from neurons and depolarize upon stimulation. While past studies have focused on the role of neuron-to- OPC synapses in activity-dependent myelin plasticity, emerging evidence suggests that OPCs play an active role in the neural network, possibly through a mechanism that is independent of myelin. A fundamental question that has not been resolved is how OPCs modulate neuronal network function. Accumulating reports that many of the synaptic proteins previously considered to be unique to neurons are also expressed by OPCs and supports their role in synapse formation or reorganization. As a first step toward elucidating the role of OPCs in neuronal synapses, this project will develop improved techniques and test the hypothesis that OPCs in an activity-dependent manner dynamically modulate nearby neuron-neuron synapses. This will be examined after chemogenetic OPC activation (Aim 1) and in which the gene encoding the synaptic organizer protein C1QL1 is selectively deleted from OPCs (Aim 2). A current challenge in studying neuron-OPC synapses is an inability to quantify them free of confounding signals from nearby neuron-neuron synapses. To solve this problem, while simultaneously monitoring neuron-neuron synapses, we will develop a transposon-mediated conditional in utero electroporation approach optimized to deliver nanobodies that specifically mark excitatory and inhibitory postsynaptic proteins in OPCs. The outcome of the proposed study will likely lead to novel principles regarding the role of OPCs in regulating neuronal excitation or inhibition and have profound impact on the regulatory mechanism and development of therapeutic strategies to ameliorate pathological conditions of altered excitation/inhibition imbalance such as epilepsy, schizophrenia, and autism spectrum disorders.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Splicing factors (SFs) are RNA-binding proteins that regulate alternative splicing (AS), enabling a single gene to produce a variety of mRNA transcripts and corresponding proteins. AS plays an integral role in development, cancer, and aging, and many SFs are essential for embryonic development. Therefore, SF levels must be tightly controlled to maintain proper gene expression, which can be achieved through the AS of poison exons (PEs) within their own transcripts. PEs within SF transcripts, or SF-PEs, introduce premature termination codons, triggering nonsense-mediated decay (NMD) to reduce SF protein levels, a process known as AS- NMD. Conversely, PE skipping increases SF abundance. Prior studies highlight SF-PEs as critical for cancer cell survival, but their role in non-cancerous cells remains unclear. The goal of this proposal is to determine how SF-PEs maintain SF homeostasis to modulate transcriptomes that sustain pluripotency and differentiation. Our preliminary data suggest that PEs in Srsf3 and Tra2b, two SFs linked to cancer and developmental disease, are essential for pluripotent stem cell survival and embryonic viability. However, the morphological, functional, and transcriptomic effects of PE knockout remain unclear, as does the broader role of SF-PEs in pluripotent stem cell survival. We hypothesize that SF PEs fine-tune pluripotency by buffering SF gene expression and modulating AS of target genes critical for maintaining pluripotent cell viability. Aim 1 will utilize an in vivo reverse genetics approach and long-read RNA sequencing (LR-seq) to characterize how Srsf3- and Tra2b-PEs shape mouse embryonic development. Aim 2 will investigate SF AS-NMD dynamics in vitro using a high-throughput CRISPR-based exon deletion screen to identify SF-PEs essential for iPSC viability. Conditional knockout iPSC models will be engineered to assess effects of SF-PE knockout on transcriptomes using LR-seq, SF target binding using eCLIP, and differentiation phenotypes using functional assays. Successful completion of these Aims will elucidate how SF-PEs modulate transcriptomes, safeguard cell pluripotency, and drive differentiation. This Fellowship will provide me essential training in RNA splicing, stem cell biology, genomics, and scientific communication—critical for my future career as a physician-scientist translating basic research into clinical applications.

NIH Research Projects · FY 2026 · 2026-02

Project summary Directional cell migration is crucial for many physiological processes, including angiogenesis, wound healing, tissue remodeling, and immune responses. Dysregulated cell migration is associated with various diseases, such as fibrosis and metastatic cancer. Understanding the molecular mechanisms underlying cell migration has broad implications for human health. Cells employ different modes of migration—collective, mesenchymal, amoeboid, etc.—depending on their intracellular state and extracellular environment. Regardless of the mode, cell migration fundamentally requires coordination of cytoskeletal components (e.g., actin, microtubules, intermediate filaments), extracellular environment sensing, and the generation of physical forces for locomotion. Previous studies have highlighted the critical role of microtubules in various aspects of cell migration. While microtubules undergo several post-translational modifications, how these modifications influence cell migration remains largely unexplored. Microtubule acetylation (acetylation of Lysine-40 on α- tubulin) and detyrosination (removal of the carboxy-terminal tyrosine residue on α-tubulin) have been implicated in metastatic cancer, underscoring the need for a mechanistic understanding of their effects on cell migration and cytoskeletal dynamics. However, elucidating these molecular pathways is technically challenging due to the lack of tools to acutely and specifically manipulate these modifications in living cells. To address this gap, I have developed genetically encoded, light-inducible molecular actuators—optoTAT and optoVASH—to specifically and reversibly induce microtubule acetylation and detyrosination, respectively, in living cells. Over the next five years, we will employ these optogenetic tools alongside genetic and pharmacological perturbations to investigate several critical, yet currently intractable, questions: (1) What are the effects of microtubule acetylation and detyrosination on actin and vimentin dynamics, and what molecular signaling pathways mediate these cytoskeletal interactions? (2) How do microtubule acetylation and detyrosination influence cell contractility, mechanosensing, and engagement with the extracellular matrix? (3) What are the effects of these modifications on the mode of migration in two-dimensional environments (flat surfaces) and three-dimensional matrices? Results from this study will illuminate an underexplored area of cytoskeletal biology—microtubule post-translational modifications—and identify key factors that could serve as diagnostic markers or therapeutic targets.

NIH Research Projects · FY 2026 · 2026-02

Abstract The proposed K99/R00 award aims to guide the applicant to research independence, specifically in mechanisms of action (MoA) research of recovery support services for substance use disorders. The opioid epidemic is an ongoing public health crisis with opioid-related overdose deaths continuing to rise. Although there are effective treatments for opioid use disorder (OUD; e.g., medication-assisted treatment), many patients drop out of treatment too early to realize the effects of these interventions. Peer recovery support (PRS) is a promising tool to improve treatment retention for individuals with OUD; however, the mechanisms underlying PRS remain unknown. This study seeks to partner with individuals impacted by the opioid epidemic to develop: 1) a client- centered assessment battery of MoA underlying PRS; and 2) an MoA-enhanced PRS program designed to enhance mechanisms responsible for increasing retention in OUD treatment. During the K99 phase, the PI, in collaboration with two community boards of 1) individuals with lived experience with OUD and 2) certified PRS specialists (certified “peers”) who support people in treatment for OUD, will identify potential MoA and compile appropriate assessments of those MoA (Aim 1a). Next, using iterative mixed methodology, the assessment battery will be iteratively refined (Aim 1b) through two waves of qualitative interviews informed by human- centered design principles (n = 10) and think-aloud protocols (n = 10). Following refinement of the battery, these assessments will be incorporated into an ongoing clinical trial to examine effects of a PRS program on these MoA (N = 82; Aim 2). MoA with the largest effects will then be incorporated into an MoA-enhanced PRS program co-developed with community boards (Aim 3). During the R00 phase, feasibility and acceptability of the MoA- enhanced PRS program will be evaluated in a randomized controlled trial (RCT) comparing this program plus standard care to standard care only (Aim 4) in a sample of patients initiating treatment for OUD (N = 70). Preliminary efficacy signals related to the impact of the PRS program on treatment retention and opioid use will be examined as an exploratory aim. The training and career development plan includes courses, workshops, seminars, readings, and hands-on research in areas critical to the PI’s independence: mixed methods design and data analysis including human-centered design and qualitative interviewing, principles and methods of community-based participatory research, skills to develop novel peer recovery support services in community settings, examining feasibility, acceptability, early signs of efficacy, and MoA in RCTs, and transition to independence in research through training in grants management, grant writing, scholarly writing, and scientific presentations. The R00 project will provide pilot data to support future R01 applications for fully-powered efficacy trials of the MoA-enhanced PRS program. This K99/R00 award will set the foundation of the PI’s independent research career, specializing in MoA for behavioral interventions.

NIH Research Projects · FY 2026 · 2026-02

ABSTRACT Regulation of mRNA translation is vital for cells to acclimate and respond to changing environments and stress. A paradoxical mechanism of protein synthesis regulation is the modulation of translational fidelity. Mounting evidence shows that organisms, from bacteria to humans, regulate errors during translation to resist and acclimate to cellular stresses. Despite recent progress and exciting discoveries, our understanding of how trans- lational fidelity is regulated remains incomplete. The long-term objective of my research program is to determine how and why changes in translational fidelity occur and the molecular mechanisms underlying its regulation. In this MIRA proposal, we focus on the function of two families of translation factors: a rare tryptophanyl-tRNA synthetase (TrpRS) and an aminoacyl-tRNA deacylase (CtdA). Our preliminary and published results indicate that these protein factors coordinate translational fidelity in response to environmental and physiological changes. TrpRS is a universal and essential enzyme that ligates tryptophan to tRNAs during protein synthesis. TrpRS’s substrate specificity is critical in translational fidelity as ligation of non-cognate amino acids causes translation errors. Our preliminary data show that some organisms, including pathogens of public health interest, such as Salmonella enterica and Klebsiella pneumoniae, encode an unconventional TrpRS (named TrpRS-B2) with relaxed specificity that confers resistance against oxidative stress. TrpRS-B2 is predominantly found in organisms encoding an additional TrpRS (TrpRS-B1) that lacks the characteristics of TrpRS-B2. CtdA is a newly discovered family of aminoacyl-tRNA deacylases that maintain translational fidelity of Arg codons. CtdA hydrolyzes canavanyl-tRNAArg resulting from the ligation of canavanine, a toxic non-standard amino acid, to tRNAArg by arginyl-tRNA synthetase, which confuses canavanine with Arg. However, little else is known about the potential involvement of CtdA in other cellular functions and its diversification during evolution. The emphasis of our proposed research is to establish how TrpRS-B2 and CtdA contribute to the biology of several human-associated bacteria by regulating translation. In the next five years, we will address several fundamental questions using Salmonella enterica as a model organism because it offers a facile and established system to interrogate diverse environmental, physiological, and host-interaction conditions. We will investigate the molecular and biochemical differences between TrpRS-B2 and TrpRS-B1 and how TrpRS-B2 confers toler- ance to oxidative stress and promotes virulence. Moreover, we will examine the role(s) of CtdA beyond canavanine detoxification and the function of newly identified CtdA paralogs. These studies will shed light on the diverse regulatory mechanisms of translational fidelity and expand our understanding of the survival strategies of bacteria. Ultimately, this knowledge can help develop new approaches to treating human bacterial diseases.

NIH Research Projects · FY 2026 · 2026-02

Abstract Borrelia burgdorferi (Bb), the Lyme disease (LD) spirochete, cycles between an Ixodes spp. tick vector and a reservoir host, typically small rodents. To sustain its dual-host lifestyle, Bb employs sophisticated, yet parsimonious, regulatory mechanisms to reshape its transcriptome in response to tick and mammalian host signals. BosR–Bb's sole member of the FUR (ferric uptake regulator) family of regulators was initially described for its putative role in regulation of genes involved in Bb's oxidative stress response in vitro. It subsequently gained prominence from the discovery that it binds to the rpoS promoter and is required for RpoN-dependent expression of rpoS as well as RNAP-RpoS function in mammals. For the past two decades, the LD field has focused almost exclusively on BosR's role in the expression of rpoS and RpoS-regulated genes. BosR's Fur-like regulatory functions outside the RpoN/RpoS pathway remain a major knowledge gap. Our preliminary studies with our dialysis membrane chamber (DMCs) system for generating mammalian host-adapted Bb revealed that BosR regulates 82 genes independently of RpoS in mammals. We also found that BosR regulates 90 genes in vitro independently of RpoS, but only three of these are regulated by BosR in DMCs. Notably, none of the oxidative stress genes previously linked to BosR were dysregulated in ΔbosR under either condition. We further engineered an otherwise WT Bb strain expressing a BosR point mutant (R39A) with impaired DNA-binding activity and found that DNA-binding by BosR is essential for transcription of rpoS as well as its RpoS- independent function in DMCs. Collectively, these findings support our central hypothesis: Bb expresses distinct BosR regulons in ticks and mammals as a product of its Fur-like function that (i) is unrelated to the RpoN/RpoS pathway and (ii) involves differential promoter recognition in response to environmental signals. In our first Aim, we will define the BosR regulon in ticks using TBDCapSeq, a Bb-specific transcript enrichment methodology that we recently developed to investigate Bb gene expression in feeding ticks. Experiments in larvae are particularly relevant as they could prove that BosR has gene regulatory functions during a phase of the enzootic cycle when RpoS is OFF. Studies in this Aim will also establish whether BosR regulates distinct regulons in ticks and mammals. In our second Aim, we will examine the mechanism used by BosR to regulate gene expression independently of RpoS. Accordingly, we seek to establish that bind of BosR to a degenerate DNA motif gives rise to the BosR regulon(s). Unlike other Furs, BosR lacks a clearly defined regulatory metal binding site. Therefore, we will also investigate whether allosteric regulation of BosR, either by heme binding as suggested by AlphaFold3 structural modelling or interactions of BosR with c-di-GMP-liganded PlzA dictate distinct functions of BosR in ticks and mammals.

NIH Research Projects · FY 2026 · 2026-02

Inflammasomes are large cytosolic multiprotein complexes formed in response to infections and cellular stresses that drive auto-activation of inflammatory caspases, production of inflammatory mediators, and pyroptosis. Inflammasomes are important for controlling bacterial infections; however, if hyperactive and persistent, are damaging to tissues due to their nature of inflammation. Notably, inflammasomes are involved in the pathogenesis of sepsis, a life-threatening illness due to the human body’s extreme inflammatory response to infections, usually bacterial infections. There are >1.5 million cases of sepsis and >250,000 sepsis-related deaths each year in the United States. However, there are no effective therapies. Most inflammasomes are assembled by a pathogen- or danger-associated pattern recognition receptor (PRR) and an adaptor (optional), which serve as a platform for the recruitment and auto-processing caspase-1. However, human caspase-4/5 and murine caspase-11 can be directly bound and activated by a bacterial endotoxin, lipopolysaccharide (LPS); therefore, they are recognized as the non-canonical inflammasomes. Although much has been learned about the regulatory mechanisms for the canonical inflammasomes, little is known for the non-canonical inflammasomes. To address this gap, we employed an unbiased manner to identify caspase-4 interactors and found that ubiquitin regulatory X (UBX) domain-containing protein 1 (UBXN1) was a top hit. Preliminary studies showed that UBXN1 was positive regulator of the non-canonical inflammasome signaling and facilitated the pathogenesis of LPS and polymicrobial sepsis. Mechanistically, our initial results suggest that UBXN1 enriches and tethers lysine (K) 63- and K48-linked unanchored ubiquitin chains to caspase-4/11, thus promoting the assembly and activation of caspase-4/11. In contrast to the conventional ubiquitination that adds a ubiquitin molecule (Ub) via the covalent bond to a substrate protein and additional Ub molecules in tandem (polyubiquitination), unanchored (free) ubiquitin chains are derived from deubiquitylation or de novo synthesis and interact with a target protein non- covalently. However, the research on free polyUb is still in its infancy. We further demonstrated that tetrameric free K63-Ub4 and K48-Ub4 enhanced caspase-4 activation. Herein, we hypothesize that unanchored K48/63-Ub chains bind and facilitate the non-canonical inflammasome activation in a UBXN1-dependent manner. We will employ multiple approaches including cell-free, biochemical, microscopic, genetic, pharmacological, in vivo tools and murine models. We hope to advance the fundamental knowledge about unanchored polyubiquitin chains and comprehensively understand the in vivo physiological functions of UBXN1 in the pathogenesis of polymicrobial and single bacterial sepsis and its molecular mechanism of action. These pieces of knowledge will lay a solid foundation for the future development of small molecular inhibitors of UBXN1 to treat sepsis and other autoinflammatory diseases.

NIH Research Projects · FY 2026 · 2025-12

Project Summary/Abstract The staggering prevalence of obesity presents major public health and economic consequences. Effective anti- obesity drugs are desperately needed to combat the obesity epidemic, as behavioral strategies offer limited success. Analogs of the endogenous satiety signal glucagon-like peptide-1 (GLP-1) suppress food intake and body weight and are FDA-approved for obesity treatment. However, GLP-1 analogs (e.g. semaglutide) are burdened by side effects, namely nausea and emesis. Therefore, increasing the therapeutic potential of GLP-1 receptor (GLP-1R) agonists requires characterization of the central mechanisms that mediate both the food intake-suppressive and nausea/emesis effects of GLP-1. Preliminary data in the rat indicate that GLP-1Rs in the locus coeruleus (LC), a source of norepinephrine (NE) output in the brain, are pharmacologically and physiologically relevant for the food intake and illness-like effects of GLP-1. However, the circuit by which endogenous GLP-1 signaling in the LC contributes to food intake suppression and nausea/emesis remains unclear. Additionally, the functional relevance of LC GLP-1Rs to the food intake suppressive and nausea/emesis effects of the semaglutide is not known. The main goal of the proposed 5- year research career development plan is to facilitate the applicant’s transition to a tenure-track Assistant Professor with independent R01 funding. To this end, the proposed research will train the applicant in a variety of approaches to identify the behavioral, cellular, and circuit-level mechanisms behind LC GLP-1R induced anorexia and illness-like behaviors. Aim I will utilize pharmacological, chemogenetic and RNAi-mediated GLP-1R knockdown strategies in the rat and musk shrew, a preclinical model that has an emetic profile similar to humans, to reveal a circuit by which endogenous GLP-1 signaling in the LC contributes to food intake suppression, nausea and emesis. Aim II will take a translational approach by determining the real-time calcium signaling dynamics of LC NE neurons to semaglutide as well as the pharmacological relevance of LC GLP-1Rs to the food intake suppression, nausea/emesis and calcium signaling evoked by systemic semaglutide. Aim II will use also cutting-edge single nucleus RNA sequencing and bioinformatic analysis to probe semaglutide-induced changes in the LC NE neuron transcriptome to reveal the fingerprint of LC neurons and regulation of LC NE neuron genes by semaglutide. Results from these experiments will inform the development of more efficacious and tolerated obesity treatments and will provide the applicant with a unique set of skills and pilot data to encourage her transition to research independence.

NIH Research Projects · FY 2025 · 2025-09

Abstract Chemical threat agents can potentially be weaponized to cause mass casualties. The warfare agent sulfur mustard and its chemical analog nitrogen mustard (NM) cause severe injury to ocular tissues with acute epithelial defects, pain and delayed onset of keratitis. As yet, a comprehensive understanding of the cellular contributors and pathophysiological molecular mechanisms of corneal pathology and sensory dysfunction is lacking. Recently, we have described remarkable changes in citrullination, a protein posttranslational modification (PTM), manifests in NM-injured murine corneas. Citrullination is catalyzed by peptidyl arginine deiminases (PADs). Our preliminary findings show that PAD4 could be driving citrullination in the corneal epithelium and stroma, but its citrullinated targets have yet to be formally identified. In addition, it is still unclear how vesicant injury affects corneal sensory function and what molecular pathways in corneal Schwann cells (cSCs) contribute to axonal degeneration and regeneration. To study such complex facets of corneal pathology- structural integrity and sensory function – in this R01 grant proposal, we will investigate the epithelium and sensory system, using citrullinomics and transcriptomics approaches, respectively. Specifically in Aim 1, we will investigate what proteins are temporally altered by PAD4- driven citrullination to illuminate biological pathways and cellular processes affected by citrullinated proteins. These protein hits will be validated and assayed for function in human corneal epithelial cell culture experiments. In Aim 2, we will focus on a newly identified druggable target in cSCs whose pharmacological inhibition in NM injury promotes cSCs and axonal network regeneration and sensory recovery, and hence understanding this SC-target’s mechanisms in cSCs would be important. We will exploit a single nuclear RNA sequence analysis paradigm to pursue temporal transcriptomic changes occurring in injured corneas to illuminate novel effectors of this regenerative paradigm. Bioinformatic approaches will be used to interrogate these data to achieve a comprehensive overview of the pathways affected by citrullination mediated by PAD4 in the cornea, and a similar decoding of transcriptomic information for understanding cSC regenerative function. Ultimately, this two-pronged approach could illuminate new biomarkers and effectors of corneal injury and unravel repair mechanisms that have not been previously established.

NIH Research Projects · FY 2025 · 2025-09

Abstract Borrelia burgdorferi (Bb) is maintained in nature within an enzootic cycle involving a mammalian reservoir host and a tick vector. To sustain this dual-host lifestyle, Bb must remodel its transcriptome as it shuttles between these two host milieus. The RpoN/RpoS pathway is central to borrelial gene regulation. In addition to upregulating genes required for tick transmission and mammalian infection, this pathway represses tick-phase genes, hence its designation as the `gatekeeper'. Activation of the spirochete's other major pathway, Hk1/Rrp1, results in synthesis of the second messenger c-di-GMP, which is required for survival in ticks. c-di-GMP exerts its regulatory effect via the effector protein PlzA. Using a novel transcriptomic approach, TBDCapSeq, we compared the RpoS regulons during the nymphal blood meal and mammalian host-adaptation. These analyses established unequivocally that the contours of the RpoS regulon differ dramatically during the tick and mammalian phases and that RpoS-mediated repression occurs exclusively within mammals. Unexpectedly, we found that deletion of BosR in an IPTG-inducible rpoS strain diminished expression of RpoS-upregulated genes and abrogated RpoS-mediated repression. Thus, in addition to its well-recognized function as a cofactor for RpoN-dependent transcription of rpoS, BosR also plays an essential downstream role modulating promoter recognition by RNAP-RpoS. Using a Bb strain that synthesizes c-di-GMP constitutively, we made the intriguing observation that liganded-PlzA (PlzALig) exerts a `brake' effect on RNAP-RpoS, antagonizing RpoS-mediated repression and diminishing transcription of RpoS-upregulated genes. Remarkably, the effect of PlzALig on RNAP- RpoS mirrors BosR-deficiency. These findings lead to our central hypothesis that PlzALig and BosR determine the contours of the RpoS regulon by exerting reciprocal effects on RNAP-RpoS. In Aim 1, we will determine how DNA binding by BosR shapes the RpoS regulon and investigate the mechanism underlying RpoS-mediated repression. Experiments in Aim 2 will investigate two possible mechanisms to explain the PlzALig brake. Lastly, in Aim 3, we will assess the contributions of other regulatory factors to modulation of the tick- and mammalian-phase RpoS regulons. Our long-term objective is to develop a holistic understanding of how the RpoN/RpoS regulatory networks orchestrate gene expression to sustain Bb in nature.

NIH Research Projects · FY 2025 · 2025-09

The transcriptional regulation and intercellular signaling at the maternal fetal interface impacts a number of pregnancy related pathologies. Specifically, the endometrial cells, such as the endometrial stromal trophoblasts undergo transcriptional regulation of distinct mechanistic programs that affect their permissiveness to placental invasion. Dysregulation of this placental invasion can underly the etiology of invasion related maternal fetal diseases (IMFDs) such as pre-eclampisa, fetal growth restriction, and placenta previa. This project will study the evolution of transcriptional regulatory elements underlying tissue mechanical integrity in the endometrium. Using machine learning and statistical models, combined with comparative genomics and computational regulatory element prediction, we will prioritize putative elements that functionally control endometrial tissue integrity in the face of placental invasion. In addition, the mechanistic insights thus gained will be combined with a data-centric approach to develop a prognostic model to relate individual transcriptional profiles of endometrial cells to IMFD disease risk.

NIH Research Projects · FY 2025 · 2025-09

Abstract NMR is one of the most versatile analytic tools for investigating biomolecules. It can probe structure and dynamics in solution at atomic resolution, even for disordered molecules, and it can quantify the affinity and kinetics of biomolecular interactions and the sites of interaction. It is capable of quantifying individual components of complex mixtures of small molecules, with applications to metabolomics, drug discovery, and clinical diagnostics. The broad goal of this project is to develop computational methods and tools that enhance the application of NMR in these biomedical applications, by improving sensitivity, resolution, lowering the barriers to implementing complex experimental and analytic workflows, and making them more reproducible and the resulting data easier to survey and share. The approach will leverage and enhance the open, international repository for biomolecular NMR data, the Biological Magnetic Resonance Data Bank, and the NMRbox computational platform, comprised of >250 NMR-related software packages and high performance computational and storage resources. Special emphasis is given to the application of NMR for investigating dynamics and disorder in biological macromolecules, which are inaccessible to other methods, and to the quantitative analysis of mixtures of small molecules, for NMR-based screening, metabolomics, and biomarker discovery.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Of the 7 million individuals in The US with heart failure (HF), ~1 in 500 have hypertrophic cardiomyopathy (HCM), while ~1 in 200 suffer from dilated cardiomyopathy (DCM). Large-scale genetic sequencing studies have shown that both HCM and DCM can be caused by pathogenic or likely-pathogenic (P/LP) genetic variants in the TNNT2 gene. This gene encodes cardiac troponin T, a regulatory protein in the sarcomere essential for the heart’s contractile function. ~115,000 individuals in the US with HCM or DCM have been identified to carry P/LP TNNT2 variants. These genetic variants disrupt the sarcomere’s calcium (Ca2+) sensitivity, causing hypercontractility in HCM or hypocontractility for DCM. The severity of these effects depends on the ratio of mutant TNNT2 to wild- type TNNT2 protein. Despite extensive research on the impact of P/LP TNNT2 variants, no treatments currently target this gene. Attempts to use small molecule drugs to address these genetic effects have been unsuccessful. To overcome this challenge, we engineered first generation adeno-associated viruses (AAVs) capable of delivering wild-type TNNT2 to cardiomyocytes. In pilot studies conducted in mice, these AAVs successfully prevented DCM in vivo for the first time. Additionally, the simultaneous knockdown of mutant mRNA significantly improved outcomes. To move toward clinical application, we propose optimizing and screening next-generation AAVs to identify a lead therapeutic candidate suitable for an investigational new drug (IND) application. This project follows a milestone-driven approach. In the R61 Phase: 1) AAV development and validation—to identify validate and screen promising AAV candidates; 2) micro RNA (miR) targeting—to design a miR to selectively suppress mutant TNNT2 mRNA; 3) preliminary efficacy testing—conduct human cell-based assays to evaluate efficacy and establish dose-response curves, and to utilize human 3D cardiac microtissue models of HCM and DCM to simulate real-life conditions and assess therapeutic effects; and 4) in vivo mouse studies—to perform short-term efficacy and dose-response studies using a mouse DCM model, while simultaneously identifying serum biomarkers to aid in the prediction of therapeutic response in vivo. R33 Phase progression will require identifying at least one AAV that restores normal contractile function with minimal toxicity. Key R33 activities include: 1) preclinical testing in DCM mice using a prevention and treatment study design; 2) comprehensive dose-response analyses across cardiac structure and function (echocardiography), single cardiomyocyte physiology and Ca2+ analyses (IonOptix); transcriptomic profiling (RNA-seq and qPCR) and serum biomarker studies (ELISA); and 3) tissue distribution and preliminary toxicity measurements (ddPCR, ELISA and histology). These studies will support intellectual property, large-animal safety testing, IND applications, and eventually, Phase I clinical trials in collaboration with our Accelerator Partner (JAX Accelerator), the NHLBI Catalyze Coordinating Center, and outside consultants. Our ultimate goal is to create the first TNNT2-targeted gene therapy for cardiomyopathy.