Washington University

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY A major challenge for engineered T cells to combat cancer is their limited persistence. The goal of this project is to enhance the persistence of T cells by harnessing 4-1BB using structure-informed protein design. As a co- stimulatory molecule, 4-1BB improves T cell long-term immune responses. Incorporating the 4-1BB signaling domain into chimeric antigen receptors (4-1BB CAR) significantly enhances the therapeutic effects in treating hematological malignancies. However, the scope and the long-term efficacy of 4-1BB CAR-T treatment remains limited, especially for patients with solid tumors, highlighting the need for a fundamental shift in the 4-1BB CAR protein design. 4-1BB (CD137/TNFRSF9) belongs to the tumor necrosis factor receptor (TNFR) superfamily. Protein structural studies have shown that effective TNFR signaling relies on specific high-order receptor clustering. However, current protein engineering approaches for harnessing 4-1BB have overlooked this crucial requirement. Thus, the 4-1BB signals initiated by current 4-1BB CAR design does not involve TNFR like-receptor clustering. Based on the importance of receptor clustering in native TNFR signaling amplification, we postulated that the absence of this critical arrangement of 4-1BB CAR results in inefficient 4-1BB signaling. Our preliminary experiment showed that the efficacy of CAR-resident 4-1BB is limited compared to endogenous 4-1BB activation in supporting CAR-T persistence. We hypothesize that high-order receptor clustering is needed for a synthetic 4-1BB to effectively support CAR-T cell activity. We have two specific aims. Aim 1 is to identify novel key factors required for robust 4-1BB signaling by cross-examining how these two pathways diverge (CAR-resident 4-1BB v.s. endogenous 4-1BB). Aim 2 is to employ structure-guided protein design to create a potent synthetic 4-1BB capable of forming TNFR-like clustering without the need for 4-1BB ligand. Cancer cells express limited 41BB ligand to evade immune surveillance. This engineered approach will deliver potent 4-1BB signals to enhance the persistence of engineered T cells when 4-1BB ligand is lacking. The success of the project will fundamentally advance our knowledge for modulating 4-1BB pathways to improve T cell persistence and open the door to enhance the durability of various types of engineered T cells beyond CARs. Collectively, our project will mark a leap forward in protein engineering for immunotherapy.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY Bacteroides fragilis is a gram-negative intestinal commensal that comprises up to 1% of total gut bacteria. While often considered harmless, this microbe remains a leading cause of anaerobic sepsis and abdominopelvic infections. The rise of antimicrobial resistance (AMR) in B. fragilis clinical isolates has complicated treatment of these infections. In particular, a distinct clade of B. fragilis strains (clade II) that represent 10% of B. fragilis from human clinical isolates were found to harbor cfiA, a genomically encoded metallo-β-lactamase. Carbapenem resistance in these strains is concerning not only due to the lack of available treatment options but also the risk of transmission to other microbes. Recent studies have also identified differences in average nucleotide identity between clade II and clade I—the remaining 90% of B. fragilis. This suggests that these clades might actually represent two different species, and thus, biology of clade II cannot simply be assumed from clade I. As all B. fragilis niche acquisition studies to date have utilized clade I strains, the mechanisms by which a clade II strain comes to dominate and persist within the colonic niche remain unknown. Previous microbiome studies in adults have revealed predominance of a single B. fragilis strain within an individual microbiota. Interestingly, no studies have identified a single host to harbor both clade I and clade II simultaneously, suggesting that intraspecific competition between clade I and clade II may influence niche establishment and strain dominance. Further studies are thus necessary to identify factors unique to clade II biology, as long-term colonization by these strains may engender a reservoir for carbapenem resistance within the host. Recent work has identified Bcf1 as a novel secreted toxin that is utilized by clade II in the context of interbacterial competition against clade I. This method of antagonism is dependent on an interaction with Bat1—a confirmed functional target of Bcf1 and a putative TonB-dependent transporter. Orthologs of Bcf1 can be found throughout Bacteroides spp., suggesting that this may be a novel family of toxins. However, the mechanism of Bcf1 activity remains unknown and all functional studies to date have only been conducted in vitro. The proposed studies will provide mechanistic insight into Bcf1 function (Aim 1) and its role in interbacterial competition in vivo (Aim 2). Aim 1 will test the hypothesis that Bcf1 binds to Bat1 and blocks translocation of an unidentified nutrient through Bat1 via a form of nutritional competition. Aim 2 will leverage various animal models to test the hypothesis that Bcf-mediated competition allows clade II to dominate its niche and achieve long-term colonization. These studies will enable us to consider functional links between interbacterial competition and high-level drug resistance from an ecologic perspective. Understanding the inherent differences in intraspecies biology and strain-level competition dynamics may inform the development of microbiome-targeted therapies to prevent colonization of pathogenic B. fragilis strains or translate to future consideration of AMR risk during clinical treatment of B. fragilis infections.

NIH Research Projects · FY 2026 · 2026-04

Mechanical forces drive tissue function and pathophysiology, yet current high-throughput systems for drug development rarely incorporate mechanical forces, and those that do typically do not allow dynamic, feedback- based control over the forces acting on cells and/or engineered tissues. We propose to integrate key technologies developed by our team members: 1) rapid algorithms for directly estimating contractility of excitable tissues; 2) GPU-acceleration approaches for rapid computing; 3) externally triggered smart materials that can change their mechanical properties in response to magnetic fields; and 4) high-throughput engineered tissue platforms. This integration will allow us to create a high-throughput system that allows for real-time control over tissue mechanical loading based on the mechanical forces produced by the tissue. For this technology-development application, we propose milestone-driven efforts to optimize, validate, and integrate these technologies into a user-friendly, graphical-user-interface (GUI) supported platform. The approach we propose is unique in that the software-to-hardware interfacing, driven by imaging, can readily be adapted in the future by the research community, without requiring costly, user-dependent, one-time-use pure hardware-based approaches. The ability to parallelize the algorithm for computing tissue deformation, direct deformation estimation (DDE), will allow for dramatic acceleration of computing deformation, to the point that it can be computed in real-time, thereby allowing for magnetically-responsive biomaterials to be triggered in response to image-based data on contractility. We will demonstrate integration of our software-hardware interfacing based feedback approach by performing mechano-pharmacologic screens in skeletal muscle engineered from murine myoblasts and cardiac muscle engineered from human induced pluripotent stem cells. We will apply diverse loading regimes to the tissues, in combination with drugs known to have differential effects in mechanically loaded skeletal and heart muscle. We will also create tools for mining the resulting mechano-pharmacologic data. We envision that this technology will be broadly enabling for studies in mechanobiology and for improving translation of drug screens.

NIH Research Projects · FY 2026 · 2026-04

ABSTRACT Lung cancer is the leading cause of cancer death in both men and women, with non-small cell lung cancer (NSCLC) comprising about 85% of cases. Standard-of-care for locally advanced, non-metastatic NSCLC includes cytotoxic chemotherapy, external beam radiation therapy (XRT), and immunotherapy. Despite noted benefits, many patients do not respond favorably, and a majority who initially respond experience progression, underscoring the urgent need for improved treatment strategies. We propose a transformative approach using antibody-drug conjugates (ADCs) for targeted delivery of cytotoxic drugs that act as radiosensitizers, leveraging our breakthrough discovery of radiation-inducible antigens. Specifically, we identified Tax-interacting protein 1 (TIP1) as significantly upregulated on NSCLC cells post-XRT, positioning TIP1 as a prime target for ADCs. Preliminary data demonstrate that anti-TIP1 antibodies, which undergo endocytosis and deliver payloads specifically to tumor cells, dramatically enhance XRT efficacy. Our second-generation anti-human TIP1 ADCs feature a human antibody, an advanced drug-linking strategy to minimize premature drug release, and a highly potent payload, ensuring a robust therapeutic index. We hypothesize that these ADCs will markedly enhance the therapeutic index of XRT over chemotherapy and significantly improve the durability of immune checkpoint blockade responses, potentially transforming NSCLC treatment. Aim 1 will test the hypothesis that radiosensitization by anti-TIP1 ADCs in NSCLC is independent of mutational status. We will assess TIP1 upregulation, enhanced drug delivery, and therapeutic efficacy across NSCLC cell lines and patient-derived xenografts (PDXs), stratifying patients to identify those most benefit from ADC therapy. Aim 2 will investigate how anti-human TIP1 ADCs improve XRT-induced tumor immunity and response to immunotherapy. We will examine the impact of the ADC+XRT combination on anti-tumor immunity and response to immune checkpoint blockade in immunocompetent mice and genetically engineered mouse models (GEMMs), providing a direct comparison to chemotherapy + XRT. Aim 3 will assess the pharmacokinetics (PK), maximum tolerated dose (MTD), and payload release of anti-human TIP1 ADCs in normal tissues. We will conduct these evaluations in immunocompetent mice, leveraging the near-identical similarity between mouse and human TIP1. This research can potentially revolutionize the integration of ADCs with XRT in NSCLC, significantly improving the therapeutic index and efficacy of XRT. It will also provide critical insights into how ADC/XRT combinations modulate the tumor microenvironment and enhance anti-tumor immunity, offering new avenues to bolster checkpoint blockade responses. Our preclinical evaluation of human TIP1-ADCs will pave the way for clinical development, presenting a groundbreaking strategy for NSCLC treatment that could extend survival and improve the quality of life for patients.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY ADP-ribosylation is a modification used across domains of life to mediate biological conflicts. The covalent attachment of ADP-ribose to diverse substrates ranging from proteins and small molecules to nucleic acids can render the target inaccessible or inactive. DNA targeting ADP-ribosylation has received less attention than the modification of other substrates, but is increasingly thought to be a widespread strategy in both interbacterial conflicts and in anti-phage defense mechanisms. DNA ADP-ribosylation was discovered in bacteria less than ten years ago, and therefore despite the prevalence of this modification, little is known about its biological function. My postdoctoral research provided a major advance for the field by revealing that a widely distributed bacterial DNA targeting ADP-ribosyltransferase (ART) toxin is the effector of a family of phage defense systems, thus ascribing a clear biological function to these enzymes. DNA targeted ADP-ribosylation blocks DNA replication and is accordingly highly toxic in bacterial cells but also potently anti-viral. These bacterial DNA targeting ARTs, a family termed DarT, are normally kept inactive by a cognate, neutralizing antitoxin, DarG, which is a DNA targeting ADP-ribosylglycohydrolase (ARG). Many fundamental questions remain about the biology of DarTG systems, including how the DarT toxin becomes active after phage infection. Phages, a co-evolving biological entity, are also a rich source of anti-DNA ART mechanisms. We recently discovered that some phages have co-opted DarG-like proteins and related DNA ARGs on multiple occasions, and that these “orphan antitoxins” protect these phages from DarTG-mediated defense. Thus, as with DarT, we were able to ascribe a biological function to a widespread family of previously mysterious phage enzymes. The distribution of DNA ARGs across the tree of life further suggests that DNA ADP-ribosylation is almost certainly more widespread than currently appreciated. The major goals of this study are both to investigate the underlying biology of DarTG systems in their biologically relevant context of phage infection, as well as to develop and apply cutting edge bioinformatic approaches to identify novel DNA ART and ARG families. To this end, we will pursue the following aims: 1) elucidate the molecular mechanism by which phage infection activates DarTG1 using genome-wide, single-cell, and in vitro approaches; 2) investigate the specificity and diversity of phage- encoded DNA ARGs and other phage anti-DNA ART counter-defenses, and 3) mechanistically dissect a third, unstudied DarTG family and develop methods for discovery of additional DarT- and non-DarT-related DNA ARTs. The discoveries we make in this bacterial-phage system, with its powerful experimental and genetic tools, will reveal fundamental facets of DNA ART and ARG biology relevant to the bacterial immune system and lay the groundwork for future studies of anti-viral DNA ADP-ribosylation in eukaryotes.

NSF Awards · FY 2026 · 2026-04

Non-technical Abstract: This project offers an integrated research and education effort centered on programmable quantum materials engineered through the controlled stacking of atomically thin layers. By arranging layers in designed patterns, the project reveals how structure at the nanoscale shapes the material behavior. The research addresses fundamental questions in quantum matter by using a flexible platform to explore tunable quantum states, introducing new ways to program quantum interactions and access dynamic phase transitions that do not occur in natural materials. The educational activities translate these ideas into accessible, hands-on learning experiences through an industry-facing undergraduate experimental course and inclusive K-12 outreach efforts, including interactive demonstrations and classroom-ready kits focused on layered materials and moiré physics. Together, these activities broaden participation, strengthen pathways into STEM for students from diverse backgrounds, and support national priorities in education, workforce development, and innovation. Technical Abstract: The research effort builds on the ability to vertically stack atomically thin two-dimensional layers to engineer heterostructures with lattice mismatch or small relative twist angles. These structures give rise to long-wavelength moiré superlattices that create periodic potentials for controlling electronic and excitonic interactions at the nanoscale. This project introduces a new paradigm that exploits interference between distinct moiré length scales to generate bichromatic supermoiré lattices in van der Waals heterostructures with independently controlled orientations and compositions. The central scientific problem is how engineered interference between multiple periodic length scales enables programmable quantum interactions and collective quantum states. The research goals are to discover and control interacting quasiparticles, including electrons, excitons, and trions, and to establish broadly applicable design principles linking lattice geometry, interaction strength, and emergent quantum behavior. The research team integrates advanced optical spectroscopy and scanning probe microscopy to probe correlated quantum phases, tunable excitonic complexes, and excitonic dynamics, while systematically tuning electric field, carrier density, magnetic field, and optical excitation in situ. By enabling controlled manipulation of hopping processes and Coulomb interactions within a single, reconfigurable platform, the project establishes a new experimental framework for studying dynamic phase transitions and correlated quantum states in engineered two-dimensional materials. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2026-04

Glaucoma is a leading cause of blindness, with primary open-angle glaucoma (POAG) affecting over three million Americans. The Ocular Hypertension Treatment Study (OHTS) provides a unique, high-quality longitudinal dataset to investigate visual field (VF) progression and improve early detection of POAG-related deterioration. Current diagnostic methods often fail to identify early-stage disease transitions, leading to delays in treatment and missed opportunities for intervention. Additionally, conventional metrics such as mean deviation (MD) overlook localized changes critical for assessing VF loss progression. This project aims to develop and implement novel statistical methodologies to enhance the detection and characterization of POAG progression. Specifically, we propose: (1) a change-point model for MD progression that integrates longitudinal MD measures with the time to detectable change, addressing variability in VF deterioration and identifying early disease transitions; (2) spatial-temporal modeling of pointwise VF progression to improve sensitivity in detecting localized changes, accounting for spatial correlations across retinal locations; and (3) development of a user-friendly R package to disseminate these methodologies for use by researchers and clinicians. By leveraging advanced statistical models and the extensive OHTS dataset, this research will refine estimates of differential progression rates, facilitate early identification of high-risk individuals, and optimize treatment strategies. Beyond glaucoma, these methods will have broad applicability in analyzing spatial-temporal data in other medical domains.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY Biochemistry always requires an exact buffering environment (e.g., salt condition, pH). Considering the diversity of biochemical functions and spatial complexity of cellular processes in living cells, our lab is extremely curious about how living systems orchestrate the right buffering systems to satisfy diverse cellular functions in a spatial temporal manner and how these fundamental principles of life can be implemented to understand diseases and provide new tools for bioengineering. To address these questions, our lab works at the interface of physical chemistry and molecular and cell biology to 1) explore the fundamental physicochemical and electrochemical features of living systems, 2) establish a new theoretical framework to explore whether and to what extent these fundamental electrochemical features (e.g., proton motive force) can define passive chemical functions, 3) correlate the chemical and electrochemical environments with global cellular physiology to understand the developments of diseases and 4) design new fundamental capabilities for synthetic biology to study cellular behaviors and engineer cellular functions. Our recent works in biomolecular condensates have unveiled that macromolecular condensation can influence the electrochemical environment of the cytoplasm by modulating the distribution of solvent molecules between the dilute and the dense phases. The same process sets up an interfacial electric potential that defines an interfacial electric field at the surface of condensates, which can drive diverse chemical reactions. These recent discoveries shed light onto the longstanding mystery of intracellular buffering mechanism, the regulation processes of intracellular electrochemistry and the non-enzymatic power source of biochemistry, which are critical prerequisites for cellular functions and homeostasis. However, our understandings on the underlying molecular rules and their potential impacts on cellular functions are limited. In the next five years, we will uncover the molecular mechanisms by which intrinsically disordered proteins and biomolecular condensation can modulate local and spatial chemical environments (e.g., salt and water abundance) in the cytoplasm and how these environmental features can define the electrochemical equilibria of living cells (e.g., membrane potential, pH gradient). We will study how the phase transition process can encode interfacial electrical properties at the liquid-liquid interface of condensates and explore whether the various surface charging mechanisms studied in the physical chemistry of liquid-solid and liquid-air interfaces can be manifested on the condensate surface. Lastly, with these newly uncovered functioning principles of living cells, we will design new fundamental capabilities for synthetic biology to program cellular chemical and electrochemical features for diverse bioengineering applications (e.g., metabolic engineering). Considering the critical importance of cellular environments on biochemical functions and the ubiquitous nature of disordered proteins and condensates, the proposed research will introduce a novel framework for understanding the functioning principles of cell biology, expanding our knowledge on the machineries defining cellular physiology.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY/ABSTRACT Kidney allografts are a life-saving but limited resource. Nearly ~5000 deaths each year can be attributed to the disproportionate number of patients who need an allograft compared to the number of available kidneys. The majority of available kidneys are from deceased donors. Astonishingly, ~2500 procured kidneys are discarded, often labeled as “poor quality” by a single biopsy or by a high kidney donor profile index (KDPI) score. However, measurements of kidney health from a biopsy are limited as they may not represent the variability in pathology within the kidney, and the KDPI lacks individual specificity. This proposal focuses on developing and validating imaging biomarkers to improve the accuracy and precision of the assessment of the discarded kidneys from deceased donors in order to address the critical need to increase the number of available allografts. Using discarded human kidneys, we will determine which imaging features derived by MRI can accurately detect whole kidney pathology. Then we will model those high-risk kidneys using a pig model to determine which features accurately predict outcomes. In Aim 1, we will use site-directed tissue biopsy to validate the individual image features to histopathologic scores for interstitial fibrosis, glomerulosclerosis, vascular sclerosis, and tubular atrophy. Then we will determine whether three-dimensional, multiparametric magnetic resonance imaging features in the donor kidneys can sensitively detect whole kidney pathology. We will apply statistical classification and machine learning to identify predictive imaging features. We will perform this analysis in discarded human kidneys from donors with a KDPI>85 or age>60 years. This population was chosen as it represents the largest population of discarded kidneys. We will also compare these to kidneys of lower KDPI. In Aim 2, we will model high-risk donor kidneys by inducing bilateral ischemia reperfusion injury in a pig. Once the transition to CKD is complete, the kidneys will mirror the pathology observed in human kidneys with a KDPI>85. These high-risk kidneys will undergo ex vivo imaging followed by an autotransplant of a single kidney. Glomerular filtration rate (GFR) will be measured weekly until euthanasia eight weeks after transplant. We will determine which individual or combination of imaging and histopathologic features predict kidney function after transplant. We will generate foundational evidence for the use of imaging features to overcome sampling bias and predict outcomes in high-risk kidney with the primary goal of increasing the number of kidneys for transplantation. This work directly addresses the urgent, recognized need for novel strategies to increase pool of available allografts. The long-term impact of this work will be to reduce inappropriate discards and provide biomarkers to evaluate the suitability of kidneys to expand the donor pool.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY/ABSTRACT The purpose of this career development award is to prepare Max Petersen, M.D., Ph.D., for a career in mechanistic clinical investigation of ketone and lipid metabolism in cardiometabolic health and disease. The three central objectives of the career development plan are: i) to acquire the experience and training needed to become an independent clinical investigator; ii) to develop specific expertise in clinical investigation of the physiology of ketosis and ketogenic diets; and iii) to develop specific expertise in lipid and lipoprotein kinetics research. The research strategy and career development plan will be carried out in the world-class clinical and basic research facilities of Washington University School of Medicine in St. Louis, MO. The objective of the research project is to conduct a clinical physiology study evaluating the mechanism of ketogenic diet-induced hypercholesterolemia (KDHC) in susceptible normal-weight adults. KDHC is a significant safety concern that limits the use and acceptance of ketogenic diet interventions in medicine. The Specific Aims will systematically test several proposed mechanisms for KDHC. In Aim 1, we will test the hypothesis that KDHC is primarily caused by an increased VLDL-ApoB production rate (primary outcome) and is associated with increased whole-body lipolysis and plasma LPL activity. In Aim 2, we will evaluate the alternative hypotheses that KDHC is caused by decreased LDL clearance, increased LDL production rate, and/or increased cholesterol synthesis. We will also evaluate the impact of endocrine factors (adipokines, thyroid hormones, insulin sensitivity) and cholesterol absorption on individual susceptibility to KDHC in this aim. These outcomes will be measured in a cohort of healthy, young, normal-weight adults (n = 24) pre- identified as KDHC Responders who experience KDHC after adopting a three-week screening KD. KDHC Responders will complete a randomized crossover trial with isotope tracer studies of lipoprotein and cholesterol kinetics after consuming an isocaloric ketogenic diet and an isocaloric control diet. The results will yield fundamental insights into the nutritional physiology of lipoprotein and cholesterol metabolism in people. The primary and secondary mentors for this project are Samuel Klein, M.D., and Victor Dávila-Román, M.D., who have a distinguished track record of mentoring physician-scientists to independence. In addition, a Scientific Mentoring Team of experts will provide specific training in: i) analytical procedures for lipoprotein kinetic studies; ii) compartmental modeling techniques; iii) determination of LPL activity; iv) advanced biostatistics; v) mass spectrometry; and vi) interaction of dietary composition with the circulating lipoprotein profile. A career development advisory committee of three senior physician-scientists who are experienced in mentoring junior faculty will supervise and aid Dr. Petersen’s development into an independent clinical investigator studying ketone and lipid metabolism in cardiometabolic health and disease.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY CD8+ resident memory T cells (TRM) play a crucial role in the defense against infection and cancer. Following antigenic challenge, TRM develop and surveil non-lymphoid tissues (NLTs) such as the liver and small intestine to provide long-lasting durable immunity. Within these NLTs, TRM progenitors initiate a universal residency program that is dependent upon induction of the homologous transcription factors BLIMP-1 and Hobit (encoded by Zfp683). These transcription factors are essential for TRM development, as they suppress the expression of egress-supporting molecules like S1PR1 to enforce residency. Although BLIMP-1 is expressed in circulating, conventional effector CD8+ T cells, Hobit expression is restricted to resident immune cells in NLTs including TRM. This suggests that Hobit uniquely facilitates TRM adaptation or survival within peripheral tissues. Despite the critical role of Hobit in TRM differentiation, the mechanism by which Hobit is induced is poorly defined. It is well known that cell-extrinsic factors—including IL-15, TGF-β, and retinoic acid—support the TRM fate; however, how these factors regulate Hobit expression remains unknown. Preliminary data demonstrate that several elements within the Zfp683 locus are accessible and bound by transcription factors downstream of these signals including T-bet, STAT5, and RUNX3. Therefore, I hypothesize that cell-extrinsic tissue cues cooperate with cis-regulatory elements in the Zfp683 locus to induce the expression of Hobit to support the TRM fate. In a preliminary experiment, ablation of one of these candidate cis-regulatory elements resulted in significantly reduced Zfp683 mRNA expression level in liver-resident CD8 T cells and led to a competitive disadvantage in generating TRM cells. Besides cell-extrinsic tissue factors, the presence of antigen and inflammation is also known to regulate TRM development. Previous studies using acute infection models such as LCMV Armstrong have demonstrated that TRM cells persist and expand in rechallenge contexts. However, little is known how persistent stimulation contexts such as chronic infection and autoimmunity influence TRM development and maintenance. Since BLIMP- 1 remains highly expressed during chronic infection but not acute infection, I hypothesize that contexts of persistent TRM activation alter the transcriptional requirement for Hobit during TRM development and persistence, since BLIMP-1 and Hobit play semi-redundant roles in TRM differentiation. Furthermore, I will dissect how antigen presence and inflammation differentially impact TRM responses using a novel model of neoantigen expression in the liver. This research proposal seeks to address critical gaps in our understanding of TRM biology by investigating (1) how cis-regulatory elements integrate tissue-specific cues to drive Hobit expression and (2) how chronic stimulation contexts impact the development and maintenance of TRM identity. By better understanding how TRM development is programmed at the molecular level, therapeutic interventions and vaccination strategies relevant to infection, cancer, and autoimmunity may be improved to further alleviate human disease.

NIH Research Projects · FY 2026 · 2026-04

Project Summary/Abstract This proposal describes a 5-year plan for Dr. Portales-Castillo to become an independent investigator with expertise in parathyroid hormone /parathyroid hormone related peptide receptor (PTH1R) signaling in patients with skeletal dysplasias. Dr. Portales completed his clinical nephrology training at the Massachusetts General Hospital (MGH). During his time as a clinical fellow and following his interest in renal osteodystrophy, he learned basic research at the Endocrine Unit in the Laboratories of Drs Jüppner and Gardella, leaders in PTH1R research. Dr. Portales work at MGH included the functional characterization of homozygous PTH1R mutants found in patients with Eiken syndrome. Patients with Eiken syndrome have delayed ossification, i.e gain of function effects on PTHrP pathways at the growth plate, but on the other hand, exhibit resistance to PTH actions in proximal renal tubule (PT). Thus, Eiken syndrome exemplifies a paradox in PTH1R signaling: How can the same receptor lead to gain-of-function effects for the function of one of the ligands (PTHrP) but loss-of-function for the other ligand (PTH)? By performing detailed in vitro analyses, Dr. Portales found that three PTH1R mutants found in patients with Eiken syndrome fail to desensitize when treated with PTHrP but not with PTH. Of special interest for this project, is the PTH1R mutant R485X. The PTH1R-R485X mutation introduces a premature stop codon that truncates the receptor C- , a key molecule for receptor desensitization. Derived from the insights gained from the in vitro findings, a novel humanized mouse model of Eiken syndrome was generated. In his role, as an Assistant Professor at Washington University of St. Louis, Dr. work revealed that delayed ossification in the novel humanized Eiken mice is caused by enhanced response to PTHrP in vivo and that like humans, Eiken mice exhibit resistance to PTH calcemic actions . In separate experiments, Dr. Portales also observed that the mammary fat pad (MFP) of mice with Eiken syndrome is smaller due to enhanced PTHrP actions in adipose tissue. The overall hypothesis of this proposal is that the PTH1R C-tail interactions are required to restrain PTHrP responses at the growth plate and adipose tissue but on the other hand promote PTH response in the PT. To test this hypothesis, the proposal has 3 aims: Aim 1 Aim 2: Aim 3: . Trough the work over the next 5 years with his mentorship team comprising of leaders in bone (Drs. Civitelli, Nickolas, Jüppner and Gardella), adipose tissue (Dr. Brian Finck) and PT (Dr. Humphreys) fields, Dr. Portales can leverage this K08 award to become and independent investigator with expertise in PTH1R signaling.

NIH Research Projects · FY 2026 · 2026-04

ABSTRACT Arguably one of the greatest medical breakthroughs in the 20th century was the introduction of antibiotics into clinical use. Soon afterwards several downsides to antibiotics were recognized, including antibiotic-associated colitis (now known to be due to Clostridioides difficile) and emergence of antibiotic-resistant organisms (ARO) that colonize the gut. CDI and ARO are now urgent threats to human health. The healthy human gut microbiome provides colonization resistance against C. difficile and ARO, and antibiotic-induced gut microbiome disruption leads to C. difficile and ARO colonization, infection, and transmission to other people. Unfortunately, patient populations that have benefited the most from antibiotics, such as HCT recipients, are now at greatest risk for CDI and ARO infections. SYN-004 (ribaxamase) is an orally administered recombinant class A beta-lactamase that breaks down beta-lactam antibiotics in the gut and has been found to protect against gut microbiome disruption, C. difficile and ARO colonization, and C. difficile infection (CDI) during IV beta-lactam exposures in people. Due to the high incidence of CDI and ARO infections, HCT recipients are a potential target population for phase 3 trials of SYN-004 to prevent these infections. However, HCT recipients often have significant disruption of the gut mucosal barrier, which may lead to absorption of biologically active SYN-004 and reduction in systemic antibiotic efficacy. Therefore, with this application we propose to complete a phase 1b/2a clinical trial investigating the potential for systemic absorption of biologically active SYN-004 and impact SYN-004 has on cefepime (FEP) pharmacokinetics (PK), the microbiome, and C. difficile and ARO colonization in HCT recipients with the following aims: Aim 1: Determine if there is systemic absorption of biologically active SYN-004 and/or alterations in FEP PK among allogeneic HCT recipients who receive myeloablative conditioning compared to placebo; Aim 2: Define the gut microbiome composition and metabolic function sparing effects of SYN-004 compared to placebo in allogenic HCT recipients who receive FEP; and Aim 3: Characterize the acquisition and abundance of AROs and AR genes in the guts of allogenic HCT recipients who receive SYN-004 versus placebo during cefepime administration. We hypothesize systemic absorption of biologically active SYN-004 will not be detected in HCT recipients and FEP PK will not be altered, and that SYN-004 recipients will have less microbiome disruption and C. difficile and ARO colonization/abundance compared to placebo. The rationale and motivation for this study are novel approaches to prevent CDI and ARO are urgently needed. Our proposal is significant because CDI and ARO are urgent problems. Our proposal is innovative because SYN-004 protection of the gut microbiome will change current paradigms to treat infections and prevent CDI and AROs. The proposed work is impactful because this phase 1b/2a trial will provide supporting evidence and justification for a phase 3 trial with a primary outcome of CDI prevention in HCT recipients.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY High-density lipoproteins (HDL) perform the essential function of removing excess insoluble cholesterol from cells. The process is so important that abnormal HDL levels were recently linked to all-cause mortality including for non-cardiovascular diseases such as cancer and to the severity of infectious disease. However, due to the focus on HDL in cardiovascular disease, what cell types and biological processes require homeostatic cholesterol efflux remains unknown. Humans lacking two cholesterol transporters that facilitate cholesterol efflux to HDL, present with yellowed and lipid-laden lymphoid tissues without developing premature cardiovascular disease. Preliminary data shows that specific deletion of these transporters in the macrophages causes mice to develop profound lipid accumulation in the medullary sinus of the lymph node even on a diet without excess cholesterol. Furthermore, fluorescently tagged HDL developed in the Randolph laboratory is enriched in the medullary sinus and binds to macrophages. Medullary sinus macrophages (MSMs) filter molecules draining from tissues in the lymph, including apoptotic cell products. The capture of particulate and apoptotic debris would introduce excess cholesterol to MSMs but also provide MSMs with a complete survey of antigens in the lymph. Current dogma states that antigen is acquired and presented by migratory dendritic cells (DCs) or can be directly captured by resident DCs if it is small enough to enter the T cell zone of the lymph node (LN). Studies of viral infection found that DCs resident to the lymph node were essential to produce a CD8 T cell response, but it is unclear how these resident DCs could acquire antigens that are too large to enter the T cell zone. This fellowship proposes that MSMs capture particulate antigens and transfer them to resident DCs but rely on HDL-mediated cholesterol efflux to clear accumulating cholesterol. Previous work showed that macrophages in the spleen directly transfer antigen to DCs and that LN macrophages are required for developing a tumor-protective CD8 T cell response against injected dead cell-associated antigen independent of migratory DCs. The proposed work will test whether MSMs transfer large model antigens to resident DCs in the lymph node using novel genetic approaches to target MSMs and imaging approaches to capture the interactions between MSMs and DCs. It will also define interactions between LN macrophages and HDL for the first time using a novel fluorescently tagged HDL and asses the contribution of dead cell cholesterol to MSMs. MSMs in tumor-draining LNs have an elevated signature of lipid handling, thus the proposed study will test if mice with cholesterol efflux-impaired macrophages are unable to form a cytotoxic T cell response against an immunogenic tumor. The proposed fellowship will take place under the mentorship of Gwendalyn Randolph an expert in macrophages, DCs, lymphatics, and HDL with support from a committee of experts in the exceptional environment of the Washinton University Immunology program. The proposed fellowship will train the awardee to be a skilled immunologist with an interdisciplinary twist that will aid in understanding complex biology at the root cause of disease in their future scientific career.

NIH Research Projects · FY 2026 · 2026-04

Project Summary Transcription of a HIV-1 provirus generates a 9-kilobase full-length transcript, that either remains unspliced or undergoes extensive alternative splicing resulting in generation of over 100 transcripts. While the completely spliced viral mRNAs can easily access the host mRNA nuclear export pathway, the unspliced genomic RNA and the partially spliced mRNAs are retained in the nucleus by unknown mechanisms. Nuclear export of intron- containing HIV-1 RNAs is mediated by the viral Rev protein (translated from fully spliced mRNAs) which binds to the Rev-response element (RRE) on these RNA subsets and tethers them to the host CRM1 export protein. How intron containing HIV-1 transcripts are retained in the nucleus is a long-standing question. The majority of cellular pre-mRNAs are alternatively spliced, and recruitment of mRNA export factors is closely coupled with splicing. Accordingly, the first widely accepted model proposes that lack of splicing leads to a block in deposition of RNA export factors on the unspliced and partially spliced HIV-1 transcripts. On the other hand, it is noteworthy that ~5% of protein-coding genes in humans do not contain introns. Hence, splicing is not a strict pre-requisite for RNA export. The second model proposes that intron-containing HIV-1 transcripts are actively retained in the nucleus due to distinct features present within these RNAs. This model is supported by the finding that codon- optimization of unspliced and partially spliced viral mRNAs overcomes nuclear retention independent of splicing modulation. On the other hand, the minimal sequence features necessary and sufficient for nuclear retention and whether trans-acting host factors are involved remain unknown. A distinguishing feature of the HIV-1 genome is its unusually biased nucleotide composition, rich in adenosines (~36%) and poor in cytosines (~18%). Preliminary studies provided herein raise the possibility that this property may underlie the nuclear retention of unspliced and partially spliced HIV-1 transcripts. The central hypothesis of this application is that unspliced and partially spliced HIV-1 mRNAs are actively retained in the nucleus by host proteins that recognize adenosine- rich sequences on these transcripts. In preliminary studies, we found that altering the codon usage of multiple reporter genes (e.g. GFP, mCherry, firefly luciferase) to resemble HIV-1 codon usage, hence making them adenosine-rich, results in their nuclear retention and dependence of reporter gene expression on Rev/RRE. In Aim 1, we propose to identify the minimal features of these RNAs that result in nuclear retention and determine whether this property is conserved in other mammalian species. In Aim 2, we propose to leverage these minimal HIV-1-like reporter RNAs in genome-wide CRISPR and siRNA screens to identify putative host factors that mediate nuclear retention of HIV-1 unspliced and partially spliced transcripts. The proposed studies have the potential to significantly advance our understanding of a fundamental aspect of HIV-1 replication. Knowledge gained herein will also more broadly impact viral and eukaryotic gene regulation fields.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY/ABSTRACT. Viral triggers of autoimmune disease remain poorly understood and have been hypothesized to contribute as initial triggers of the immune disturbances that underlie clinical autoimmunity as well as promoters of disease. Human Roseoloviruses, Human Herpesvirus (HHV)-6 and 7, have been associated with several autoimmune diseases notably encephalitis, and multiple sclerosis (MS). Due to high species-specific tropism, human herpesviruses are difficult to study in a controlled environment, and their relationship with the development of these disorders remains elusive. Recently however, the murine roseolovirus (MRV) was identified and found to be a close homolog of HHV-6 and 7. MRV is a thymotropic virus that causes transient, severe thymic atrophy with the most pronounced loss occurring in CD4/CD8 double-positive (DP) and CD4 single-positive (SP) thymocytes. Single-cell RNA sequencing data shows that CD4 SPs, DPs and double negative (DN) thymocytes as well as medullary thymic epithelial cells (mTECs) can support productive infection of MRV. Neonatal infection of mice with MRV causes autoimmune gastritis (AIG) in adulthood. CD4 T cells are both required and sufficient for AIG development in this model. MRV infections also results in loss of AIRE transcripts in mTECs, and development of autoantibodies to several tissue antigens targeting almost all organ systems. Additionally, type I interferon (IFN) signaling is required for development of AIG in neonatally infected mice. The goal of this study is to understand the role of type I IFN signaling in auto-immune gastritis development in the setting of neonatal MRV infection. We hypothesize that type I IFN is a major contributor in the disruption of central tolerance, specifically leading to alterations in T cell selection and development of autoreactive T cells, as well as an aberrant T regulatory cell (Treg) response. I will investigate whether type I IFN is required for the development of autoreactive T cells and whether type I IFN leads to disruptions in Treg development. Using a unique model of virus-induced autoimmunity, our study has the potential to shed light on the infectious triggers of the immune disturbances observed sometimes years prior to clinical symptoms, providing insight into the early mechanisms underlying autoimmune disease onset. As an MD/PhD student, my long-term goal is to become a physician-scientist leading a research lab in dermatology. Completion of this project will equip me with a strong foundation at the intersection of immunology, virology, and autoimmunity, fields that often converge in skin-related clinical presentations. My robust training plan at WashU in St Louis, one of the nation's leading MD/PhD programs integrates strong mentorship, rigorous coursework, independent study, and participation in research meetings ranging from local to international. This tailored approach is designed to support the successful execution of my research proposal and to position me for long-term success in achieving my career goals.

NSF Awards · FY 2026 · 2026-04

Non-technical description: This project brings together researchers from USA and Germany to develop a new class of optical materials that can control light in unusual and highly customizable ways. These semiconductor materials, which we refer to as tunable anisotropic chalcogenides for optics have the potential to enable faster light-based communication systems, improved sensors, mixed reality displays, photon routers for quantum computing, and advanced tools for laser-based manufacturing. The team discover new materials by combining theory, computer simulations, and materials informatics, followed by the formation of single crystals and thin films using state-of-the-art synthesis techniques. The project includes extensive training for the next generation of materials scientists and engineers, international exchange opportunities for students, and community building activities such as an online photonics research forum. It includes a coordinated student exchange with the DFG partner, collaboration with Air Force Research Laboratory, and activities that cultivate entrepreneurship across the participating institutions. Technical description: The proposed research will create a new class of low-loss optical materials called tunable anisotropic chalcogenides for optics that have large optical anisotropy with controlled spatial variations and, in select cases, dynamically tunable anisotropy across the visible to mid-infrared spectral ranges. The team use first-principles density functional theory and materials informatics to identify promising low dimensional chalcogenides containing transition metal cations, followed by synthesis via vapor transport crystal growth and pulsed laser deposition. Structural and optical properties are probed using X-ray, neutron, and electron-based methods along with optical spectroscopies capable of quantifying linear and circular anisotropy. Alloying and ion bombardment are employed to systematically tune the anisotropy. The integrated closed feedback loop supports iterative optimization within a high dimensional materials space, thereby expediting the rapid discovery and developments of TACOs. The project is expected to lead to an open-access database with physical properties of optically anisotropic crystals. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2026-03

PROJECT ABSTRACT Obesity-associated metabolic diseases have roots in the maladaptation of hypothalamic circuits. Compared to neural circuits regulating energy intake, mechanisms regulating energy expenditure have been less studied and poorly understood; however, they remain appealing therapeutic targets. This is particularly true for increasing thermogenic expenditure (non-shivering thermogenesis), which may carry additional benefits from increased brown adipose tissue (BAT) activity. Further, white adipose tissue which as a central role in obesity induced disorders can undergo remodeling to become more like BAT becoming beige adipose tissue with increase metabolic activity. The long-term goal of this project is to identify and elucidate the functioning of hypothalamic neural circuits that regulate energy balance and metabolism, thereby addressing these growing disease burdens. Converging lines of study led to identification of a subpopulation of neurons in the preoptic area of the hypothalamus (POA) that express KOR (POAKOR+). Evidence indicates the kappa opioid system is important for the regulation of energy balance and metabolism, but the putative roles of KOR-expressing neurons are not yet known. Preliminary data indicate that POAKOR+ neurons regulate energy expenditure and metabolic pathways. The central hypotheses of this proposal are that POAKOR+ neurons are: (Aim 1) recruited following by circadian timing and inhibited by feeding; (Aim 2) are key regulators of energy expenditure; and (Aim 3) can be targeted to drive negative energy balance, cause weight loss due to decreased adipose tissue, improve markers of metabolic health, and remodel white adipose tissue. To test these hypotheses, a range of methods, including fiber photometry, indirect calorimetry, metabolic profiling, home cage monitoring, thermal imaging, and MS metabolomics will be utilized. Results from the proposed studies are expected to inform our understanding of hypothalamic circuits regulating energy expenditure and metabolic activity, by demonstrating that POAKOR+ neurons are previously unappreciated important regulators of energy expenditure and metabolism. Supporting the translational relevance of POAKOR+ neurons, data from the Allen database show KOR expression is enriched in the human preoptic hypothalamus. The studies will determine if POAKOR+ neurons are potential therapeutic targets to modulate energy balance (weight loss) and metabolic dysfunction.

NIH Research Projects · FY 2026 · 2026-03

Over 81,000 individuals died of an opioid overdose (OD) in 2023, and >92% of those cases involved fentanyl. Fentanyl has very potent opioid-induced respiratory depression (OIRD) effects, the primary cause of opioid OD death. Key brainstem regions, the preBötzinger complex and ventral respiratory group (preBot/VRG) and parabrachial and pontine Kölliker-Fuse nuclei (PB/KFn), play critical roles in respiration, and have been implicated in OIRD. Therefore, it is likely that the cellular etiology of opioid OD, notably the sites of action of fentanyl, involves these understudied brain regions. Despite evidence for individual differences including heritable variation in opioid OD (e.g., our GWAS of opioid OD death), most biologically mechanistic studies have focused on addiction-centric brain regions. Consistent with the likelihood of differential cellular signatures across these brain regions, our preliminary data suggest pervasive bulk tissue expression differences in the preBot (i.e., genes distinct from those implicated in regions relevant to addiction). In addition, prior studies have primarily relied on bulk tissue or single omics approaches to characterize the cellular etiology of opioid OD death. New multiome methods allow us to deeply characterize how the partnership between chromatin accessibility and gene expression in these brain regions drives opioid OD involving fentanyl, thus offering insights into biology and implicating genes that could be therapeutically targeted. Against the backdrop of unabated fentanyl-driven OD deaths, our proposal will: (A) generate the first single-nucleus (sn) paired RNAseq and snATACseq (i.e., multiome) data from the preBot/VRG and PB/KFn, from diverse human fentanyl OD decedents (N=40) and matched controls (N=40). (B) Characterize cell populations, states and cell-type specific relationships between differential gene expression and chromatin accessibility in each brain region across groups. (C) Estimate enrichment of opioid GWAS variants and genes, yielding mechanistic insights to large-scale population-scale genetic discovery. (D) Combine existing opioid-related expression differences in the same and other brain regions to delineate a brain-wide network of transcriptional variability associated with OUD and opioid OD, and produce in-silico prioritization of gene-drug pairs that are viable for preclinical follow-up.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Neurodevelopmental disorders (NDDs) often result from mutations in genes essential for brain development and function. Recent advances in gene replacement therapy have shown promise for rescuing molecular and behavioral deficits in mouse models, even when gene restoration occurs postnatally. However, the context and feasibility of gene replacement for specific disorders remain unclear. This project focuses on Tatton Brown Rahman Syndrome (TBRS), a rare NDD caused by mutations in DNMT3A, a gene critical for DNA methylation and neuronal development. TBRS patients exhibit intellectual disability, overgrowth, joint hypermobility, and seizures. In mice, loss of DNMT3A leads to altered neuronal differentiation and synaptic function, emphasizing its importance in early brain development. Kim will explore the potential for restoring DNMT3A function using innovative mouse models and gene therapy approaches. In Aim 1, Kim will employ spatial transcriptomics and single-nucleus RNA sequencing to assess how DNMT3A loss impacts cell type distributions and gene expression in the cerebral cortex and whether these changes can be reversed by restoring DNMT3A expression. In Aim 2, she will evaluate the feasibility of gene replacement therapy for TBRS using adeno-associated viruses (AAVs). These studies will address timing, delivery methods, and baseline efficacy of DNMT3A reinstatement in both tamoxifen-inducible and disease-relevant mouse models. This work will determine whether postnatal DNMT3A restoration can rescue molecular, cellular, and behavioral deficits associated with TBRS and provide a foundation for gene therapy strategies targeting NDDs. The findings will contribute to understanding the therapeutic potential of gene replacement, with implications for improving outcomes and quality of life for patients and families affected by TBRS and related conditions. This project will be conducted at Washington University in St. Louis, a phenomenal research environment that integrates cutting-edge genomic technologies, advanced imaging platforms, and expertise in neurodevelopmental disorders. The lab is supported by collaborations with leading researchers in mouse behavior, epigenetics, and computational biology, ensuring access to unparalleled resources and mentorship. This environment fosters innovation, collaboration, and rigorous scientific inquiry, creating the ideal setting to achieve the goals set forth by this proposal.

NIH Research Projects · FY 2026 · 2026-03

Abstract Phosphatidylserine (PS), which constitutes only 2-10% of plasma membrane lipids, is a key phospholipid involved in intercellular communication/signaling. PS is actively sequestered to the inner leaflet of the plasma membrane and cells spend significant energy to maintain this asymmetry. PS externalization to the outer leaflet under specific contexts, such as after induction of apoptosis, allows phagocytes to recognize and engulf the dying cells via efferocytosis. Further, the engagement of PS on apoptotic cells by PS receptors on phagocytes (such as macrophages) induces anti-inflammatory signaling, which is a hallmark of efferocytosis during homeostasis. Despite our recognition of PS as a key apoptotic marker, nearly all studies consider PS as an isolated entity, and the PS-proximal proteins influencing PS recognition and signaling remain largely unexplored. Our exciting preliminary studies reveal that PS exposure on apoptotic cells occurs in patches and occurs adjacent to specific proteins, which can, in turn, influence efferocytosis. Our overarching hypothesis tested in this proposal is that proteins in the PS neighborhood on apoptotic cells critically influence/modulate the efferocytosis of dying cells, and the downstream signaling/responses elicited within phagocytes. In Aim 1, based on preliminary data identifying specific PS-proximal proteins, we test how they differ between apoptotic versus 'live’ cells engineered to expose PS (without caspase activation), the proximity of PS to specific scramblases that mediate PS exposure, and how different cell death modalities may alter PS- proximal proteins. In Aim 2, we test specific PS-proximal tetraspanin proteins in regulating efferocytosis ex vivo and in vivo, via models of tissue inflammation. The novel approaches taken here to define the PS neighborhood on apoptotic cells and its influence on efferocytosis address a long-standing key gap in our knowledge, with relevance to auto-inflammatory diseases.

NIH Research Projects · FY 2026 · 2026-03

Genetic studies in the context of Alzheimer Disease (AD) are significantly biased by information coming from the same homogeneous population: Non-Hispanics Whites (NHW). The ADSP Follow-Up Study (FUS) long term goals include to fully reveal the genetic architecture of AD in multiple ethnic groups and examine the AD genome in the context of US-representative population. As of 2016, approximately 13 million people of the total U.S. population, have Caribbean ancestry. The Caribbean Omics & Genomics Alzheimer Study (CONGAS) will contribute to ADSP-FUS mission with 5,000 samples from elderly individuals (>65 yo) across the Caribbean countries and Spain. We will generate blood biomarkers, array data, whole genome sequence, and proteomics for the CONGAS dataset. Participants will be classified according to ATN criteria that will be used as endpoint phenotype in genome, and proteome association analyses, while taking into account admixture and ancestry components. Ultimately, we will identify causal genes and druggable targets and create better prediction models, by using genetics and multi-omic data as tools.

NIH Research Projects · FY 2026 · 2026-03

Project Summary: Age-related neurodegenerative diseases are a rapidly growing cause of mortality and morbidity worldwide. The overwhelming majority of neurodegenerative disease, referred to as ‘sporadic,’ is caused by poorly understood interactions between genetic and environmental risk factors. Due in part to the complex etiology of neurodegenerative disease, broadly effective therapies are lacking. While the clinical symptoms of these disorders are heterogenous, reflecting selective neuronal death in distinct brain regions, there are pathophysiological features that link some neurodegenerative diseases. One such commonly observed pathological hallmark is the aberrant nuclear clearance and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43), which, in addition to being the defining pathology of limbic-predominant age-related TDP-43 encephalopathy (LATE), is observed in 97% of patients with amyotrophic lateral sclerosis (ALS), 50% of patients suffering from frontotemporal dementia (FTD), and up to 57% of Alzheimer’s disease (AD) patients. Together, the neurodegenerative disorders characterized by TDP-43 pathology can be referred to as ‘TDP-43 proteinopathies.’ Therapies that slow or reverse TDP-43 dysfunction thus have the potential to impact a broad group of neurogenerative disease patients; however, potent modifiers of TDP-43 pathology are lacking. Moreover, the mechanism by which TDP-43 mislocalization results in neuronal death is incompletely understood, and it is likely that therapy targets that mediate this disease process remain undiscovered. We recently found that genetic variants impacting alternative polyadenylation (APA) of ATXN3 are a novel genetic risk factor for ALS. Subsequent experiments revealed that modulation of ATXN3 substantially impacts TDP-43 pathology in multiple cell types, including in human iPSC-derived neurons and in ALS/FTD patient brain tissue. In Aim 1 of this proposal, I will determine the functional significance of ATXN3 alternative polyadenylation and identify cellular pathways to modulate ATXN3 expression in human neurons. TDP-43 is a prolific RNA-binding protein, directly impacting the metabolism of over 6,000 RNAs. Recent high-profile studies have identified RNAs that are differentially expressed (e.g., stmn2) or alternatively spliced (e.g., unc13a) upon nuclear depletion of TDP-43; however, transcripts alternatively polyadenylated upon TDP- 43 nuclear loss remain comparatively unexplored, despite the fact that regulation of APA is a key function of TDP-43. We have identified hundreds of previously unknown APA genes in ALS/FTD patient neurons exhibiting nuclear clearance of TDP-43. Notably, we found significant APA of MARK3, a tau kinase associated with early tau S262 phosphorylation in AD, reflecting a possible mechanistic link between TDP-43 and tau pathology. In Aim 2, I propose to identify and characterize new TDP-43 target genes in human neurons, first by studying the function of MARK3 APA, and second, by developing a new cellular tool to precisely define the transcriptome of human neurons undergoing TDP-43 nuclear clearance in a cell-type and temporally-controllable manner.

NIH Research Projects · FY 2026 · 2026-03

Project Summary/Abstract Opioid addiction results in the death of tens of thousands of individuals in the U.S. every year. That number is only continuing to grow. A large reason for the growing number of opioid-related deaths is the introduction and mass production of synthetic opioids like fentanyl. Synthetic opioids are much more potent than traditional opioids, and therefore their usage is more often associated with addiction and subsequent death. To combat this growing opioid epidemic, we need a better understanding of the brain regions that propagate opioid and, in particular, synthetic opioid, seeking. One potential critical modulator of opioid seeking is the locus coeruleus (LC). The LC, which is the origin of the primary central noradrenergic (NE) system, is a critical modulator of the somatic symptoms of opioid withdrawal and opioid reinstatement. Additionally, the LC densely express mu opioid receptors (MOR), the target receptor for synthetic opioids. However, despite its dense expression of MOR and its established role in opioid-related behaviors, the LC’s role in opioid seeking is not well established. This proposal aims to establish the role of the LC and, in particular, LC MOR in fentanyl drug seeking. My preliminary data shows that a conditional knockout of MOR on noradrenergic cells potentiates morphine place preference and increases fentanyl consumption in a two-bottle choice paradigm. This data does not, however, implicate a specific brain region in the modulation of these behaviors. The NE system is a vast collection of ascending and descending projections across the brain, and dopamine beta hydroxylase, the rate limiting enzyme in the synthesis of norepinephrine, is also expressed in any epinephrine producing cells, as well as within the sympathetic ganglia, and adrenal glands. We therefore hypothesize that the LC is the region responsible for these findings, and a decrease in LC MOR will also increase fentanyl consumption. In Aim 1 I will determine what chronic fentanyl exposure does to LC MOR; specifically, whether chronic fentanyl exposure downregulates LC MOR. To do so I will implant mice with osmotic minipumps for reliable and consistent delivery of fentanyl over a two-week period. Following this period, brain slices containing the LC will be collected and LC MOR will be assessed both functionally, with the use of whole-cell electrophysiological recordings, and physically, with the use of quantitative polymerase chain reaction. In Aim 2 I will determine whether knockout of LC MOR drives fentanyl consumption. To do so I will first virally knockout LC MOR, after which mice will undergo a two-bottle choice paradigm of fentanyl consumption. I will then test whether our preliminary findings showing that a conditional knockout of NE MOR increases fentanyl consumption are being driven by the LC. Specifically, I will rescue LC MOR expression in these NE MOR conditional knockout mice before they undergo the same two- bottle choice fentanyl consumption paradigm. Overall, this proposal will help establish the role of the LC, and LC MOR specifically, in fentanyl consumption. Furthermore, it will support my growth as an independent scientist ultimately allowing for me to position myself well for a future career in opioid research.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Urinary tract infections (UTIs) affect approximately 150 million people per year worldwide. An estimated 50% of women report having had a UTI at some point in their lifetime. Antibiotic use, while the mainstay of UTI treatment, has contributed to the rise in antimicrobial resistance. Uropathogenic E. coli (UPEC) is implicated in about 75% of uncomplicated UTI cases, which has led to interest in understanding the mechanisms utilized by UPEC cause UTI. Previous work has shown that UPEC colonizes both the bladder and gastrointestinal (GI) tract, where UPEC is thought to establish a long-term reservoir and seeds the urinary tract. Thus, targeting UPEC in the GI tract could contribute considerably to UTI prevention by reducing UPEC shedding in the feces and subsequent colonization of the urinary tract. Previous work from our lab has shown that the susceptibility of UPEC to the host mucosal immune response in the GI tract depends on the localization of UPEC within the gut, which in turn is determined by the degree of microbial competition from other members of the gut microbiota. This competition can drive UPEC strains to occupy two distinct gut compartments depending on when they were introduced. When UPEC is introduced first in the gut, it inhabits the luminal and mucosal compartment, while a second strain, introduced after the first strain, prefers the mucosal compartment. Interested in the fitness factors and metabolic requirements that enable this differential localization, we created a barcoded transposon library in UPEC strain UTI89. Transposon libraries are an established tool for conducting genome-wide screens in bacteria. Furthermore, barcoding the library simplifies sequencing preparation, allowing users to test more experimental conditions. We tested our UTI89 pooled barcoded transposon library by growing it in different growth medias, which would allow us to both validate the library and reveal nutritional dependencies of UPEC in different environments. Aim 1 focuses on identifying UPEC fitness factors in vitro under conditions that mimic the gut environment. First, we will conduct an in vitro UTI89 pooled library screen in five different conditions. After validating the pooled library and identifying genes of interest in the mucin media condition, we will conduct another in vitro pooled library screen using the control, mucin-containing and glucose-containing media under anaerobic conditions, which we use to model the physiological gut environment. We have also assembled an arrayed version of this library which we will use to test gene candidates identified in both screens. Aim 2 will use gnotobiotic mice to perform an in vivo pooled library screen to identify fitness factors important in UPEC gut colonization when UPEC is introduced as a primary colonizer to a naïve gut and as a secondary invader in the presence of an existing gut microbiome. We will also test whether a low-fiber diet changes the landscape of UPEC gut colonization and repeat the above in vivo screen with mice on a low-fiber diet as an additional arm. Identifying UPEC fitness determinants will elucidate UPEC biogeographical niche occupancy and inform design of antibiotic-sparing interventions for UTI.