Washington University

NSF Awards · FY 2025 · 2025-08

Urbanization has remained a dominant global demographic trend since its origins nearly 6,000 years ago. This doctoral dissertation research project examines what factors are most predictive of urbanization, deurbanization, and urban de-nucleation. Much of the archaeological data has focused on sedentary societies in arable river valleys, which yield models of gradual settlement growth and decline largely based on population and agricultural surplus. A larger, more varied dataset is necessary to get a clearer archaeological understanding of more rapid trajectories toward urbanization and deurbanization among non-farming societies. A crucial component of this research involves using satellite imagery to identify and map small non-urban settlements surrounding known urban sites. In doing so, this project will help to develop U.S. geospatial research capabilities by refining and publishing open-source and reproducible methodologies on a large satellite image dataset. This effort will provide students with valuable training in geospatial methods. This project responds to research priorities in the science of artificial intelligence through the study and usage of computers and software through the development satellite imagery-based machine learning and other deep learning models. To better understand the social and environmental factors surrounding urbanization and deurbanization, it is necessary to determine functional differences between large central settlements and the smaller sites in their peripheries. The investigators use satellite image surveys coupled with systematic soil coring to 1) investigate two settlements in a period of technological development and 2) identify alternative sites in the peripheries of these two sites. Preliminary investigations suggest that metallurgic production may have played a crucial role in the formation of large, fortified pastoralist settlements in this region; therefore, the investigators analyze soil chemistry to find traces of metallurgical activity in both urban and non-urban settlements. These soil cores provide artifacts and organic materials for radiocarbon dating, allowing the investigators to establish a chronology; thereby determining if urban sites were surrounded by smaller contemporary sites that provisioned them with food or metallurgical resources, and illustrating how/when these landscapes were abandoned. These data will help the investigators explore hypotheses to explain the formation and dissolution of pastoralist urban landscapes. 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 2025 · 2025-08

PROJECT SUMMARY Brown adipose tissue (BAT) is a thermogenic fat pad that is essential for maintaining core body temperature and is associated with protection from numerous metabolic diseases. Adult patients with intact BAT activity are much less likely to have obesity, type 2 diabetes, or cardiovascular disease (CVD), and obese individuals exhibit impaired BAT activation after exposure to cold environmental temperatures. Consequently, there is significant interest in identifying novel pathways that regulate BAT function, with the goal of developing new therapeutic strategies to treat metabolic diseases such as obesity and type 2 diabetes. It is well established that sympathetic neurons are the dominant drivers of brown adipocyte activation by secreting the catecholamines epinephrine and norepinephrine (E/NE). Macrophages then degrade Epi/NE to form a negative feedback loop that is essential for maintaining BAT homeostasis. Recently, we characterized the immune cell landscape of BAT and surprisingly found that neutrophils are the most abundant immune cell type in this tissue, greatly outnumbering macrophages. However, the role of neutrophils in regulating BAT function is unknown. Neutrophils are best known for their role in phagocytosis of bacteria and foreign material and are highly glycolytic cells with few mitochondria. In new preliminary studies, we have found that neutrophils are recruited from the blood specifically into BAT following exposure to cold temperatures and undergo massive metabolic and transcriptional reprogramming to acquire a unique reliance on oxidative phosphorylation. Antibody-based depletion of neutrophils leads to decreased BAT mass, impaired adaptive thermogenesis, and hypothermia. Strikingly, mice that are born without any neutrophils die if subjected to cold stress but are rescued from this fate if they receive a single adoptive transfer of wildtype neutrophils into the BAT. In addition, we found that the recruitment of neutrophils into BAT is dependent on ATG14, and deletion of ATG14 in myeloid cells leads to decreased core body temperature, decreased BAT mass, loss of stored lipids in BAT, decreased expression of Uncoupling protein 1, and exacerbated HFD-indued obesity. Furthermore, neutrophils directly activate brown adipocyte metabolism. These findings lead to our central hypothesis that ATG14-dependent neutrophils are recruited into BAT and undergo local transcriptional and metabolic reprogramming to critically support BAT activation and adaptive thermogenesis. We will address this hypothesis in two aims. In Aim 1, we will identify the factors that drive neutrophil reprogramming upon entry into BAT and determine whether disruption of their metabolic reprograming impairs BAT thermogenesis. In Aim 2, we will determine the mechanisms by which ATG14-dependent neutrophils in BAT sustain adaptive thermogenesis to ameliorate metabolic disease pathogenesis. These studies will identify for the first time a role for neutrophils and ATG14 in regulating BAT function and adaptive thermogenesis, revealing a novel immunometabolic pathway that can be targeted to enhance BAT activity to treat multiple metabolic diseases, such as obesity, type 2 diabetes, and/or CVD.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract This proposal outlines a 5-year research and career development plan to support Dr. Amit Bery’s transition to independence as a physician-scientist studying how innate immune cells affect adaptive immunity after lung transplantation. The proposed research project, which focuses on how interleukin-1β (IL-1β) promotes the differentiation, release, and trafficking of immunosuppressive neutrophils that downregulate alloimmune responses after lung transplantation, will take advantage of clinical and scientific expertise and unique resources available at Washington University School of Medicine. The proposed career development and training plan will strengthen Dr. Bery’s fund of knowledge and develop important skills in bioinformatics and advanced imaging techniques that will promote his successful transition to independence. Dr. Bery has generated preliminary data showing that recipients deficient IL-1β uniformly reject pulmonary allografts. These preliminary data show that IL-1β signaling is necessary for G-CSF release which results in an intragraft neutrophilia that is abrogated in IL-1β-deficient recipients. Dr. Bery has also shown that these graft infiltrating neutrophils express markers associated with an immunosuppressive phenotype and that these neutrophils can suppress T cell responses in vitro. The short-term goals of this proposal include evaluating mechanisms how IL-1β signaling induces the differentiation and release of these neutrophils from the bone marrow (Aim 1), and to define trafficking requirements that promote the homing of these neutrophils to pulmonary allografts (Aim 2). He will utilize the techniques of flow cytometry, histopathology, in vitro colony forming cell assays, and advanced downstream analyses of single cell RNA sequencing data (Aim 1) along with in vitro transmigration assays, flow cytometry, histopathology, and real-time intravital 2-photon imaging (Aim 2) to achieve these short-term goals. The long-term goal of this work is to develop immunosuppressive strategies that are tailored to the lung. While outcomes after lung transplantation rank the worst among all transplanted solid organs, immunosuppressive strategies for recipients of pulmonary allografts have been derived from studies in other organs, mainly kidney transplantation. Thus, there is a major need to identify lung-specific immunosuppressive strategies to improve the longevity of pulmonary allografts and overall survival after lung transplantation. As many lung transplant candidates and nearly all lung transplant recipients are treated with immunosuppressives that may limit their ability to mount an emergency myelopoietic response to transplantation, the proposed work carries the potential to influence how immunosuppression is utilized after lung transplantation and may provide new insights on the effects of therapies for lung transplant candidates on the waitlist.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY Chronic neuropathic pain (CNP) is characterized by emotional-affective symptoms, which afflict between 27- 75% of patients and are associated greater pain intensity, reduce compliance with pain management interventions and drive reduced quality of life in CPN patients. Despite this, there are no effective therapies for depression symptoms of chronic pain, and these symptoms do not respond to classical antidepressant therapies. Neuroimaging studies have shown blunted accumbal dopamine release in response to reward in chronic pain patients, prompting the hypothesis that a hypodopaminergic state underlies affective symptoms in chronic pain. However, direct evidence for the involvement of altered dopaminergic function in CNP is contradictory; and the precise mechanisms underlying the emergence of a hypodopaminergic state remain unknown, preventing the development of targeted therapies for its reversal. Our long-term goal is to inform novel pharmacological or neuromodulation therapies to treat affective symptoms of CNP. To this end, we must first understand whether altered function of dopamine neurons drives changes in reward-related behavior in CNP, as well as the cellular and circuit mechanisms underlying this altered function. To this end, we will first quantify changes in dopamine release in the nucleus accumbens core (NAcC) across the development of CNP. We will take advantage of novel optical approaches to longitudinally quantify changes in tonic and reward-evoked NAcC dopamine release in the same animals over the acute to chronic pain transition (Aim 1). We will then use patch-clamp electrophysiology with intersectional genetic labeling to record from NAcC-projecting dopamine neurons at acute- and chronic- timepoints following nerve injury to characterize the cellular mechanisms by which dopamine dysfunction is instantiated in CNP, which will ultimately be necessary to design therapies to reverse this dysfunction (Aim 2). Finally, we will determine the role of NAcC-projecting dopamine neurons in altered reward-related behavior in CNP. We will track behavior for consecutive weeks with operant tasks designed to capture RDoC-defined behavioral constructs relevant to depression symptoms. This longitudinal design will establish a high-resolution profile of altered reward valuation and sensitivity across the transition to CNP and will establish causal links between NAcC-projecting dopamine neuron adaptations and the emergence of behavioral changes (Aim 3). How adaptations within the mesolimbic dopamine system evolve over the transition from acute to chronic pain, and whether these adaptations drive affective symptoms in CNP is unknown. Our proposal will fill this gap by applying novel approaches to probe dopamine neuron function in vivo and ex vivo over the transition over acute to chronic phases post- nerve injury. It will also lay the foundation to reverse these adaptations as a therapeutic strategy for affective symptoms of CNP.

NSF Awards · FY 2025 · 2025-08

Healthy bone growth is essential to attaining adult stature and maintaining good health, including appropriate levels of physical activity, through life. However, bone growth can be compromised by other essential life functions (e.g., immune function), especially among individuals with restricted access to resources (low-resource environments). Accordingly, the purpose of this doctoral dissertation project is to examine how variation in living conditions impact bone growth and immune function in children. The study uses minimally invasive methods, and the data generated aid the identification of factors that affect bone growth during childhood. Participants are informed of their bone growth and immunological status, and research findings are shared with public health agencies. In addition to training the graduate student researcher, the study provides additional student and public training and educational opportunities. This project studies energetic trade-offs in children with varying access to resources to better understand how factors related to living conditions and environmental circumstances relate to childhood bone growth. Quantitative ultrasonometry and accelerometry are used to measure bone growth and physical activity, respectively. Data on food is collected using surveys. Child growth is evaluated through height and skinfolds. Dried blood spots, from fingerpicks, are implemented to measure markers of immune function (e.g., immunoglobulin E and C-reactive protein). In measuring biomarkers of immune function, the project advances NSF investment in biotechnology. 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 · 2025-08

PROJECT SUMMARY/ABSTRACT While older age is the greatest known risk factor for Alzheimer disease (AD), it is not causative. Increased risk of AD is likely driven in part by cumulative, progressive physiological processes across biological levels, termed biological aging. Biological age can be estimated through machine learning-based “clock” models using rich, multivariate physiological signatures, including DNA methylation (epigenetic age), protein concentration (proteomic age), and brain structure (brain-predicted age). By characterizing biological age at the epigenetic, proteomic, and endophenotypic layers, these clocks may detect complementary signatures of biological aging, which could identify unique processes of AD progression or resilience. The proposed K01 research study will rigorously compare state-of-the-art biological age clocks across epigenetic, proteomic, and neuroimaging modalities using available, deeply-phenotyped, longitudinal human AD datasets. The study will test whether epigenetic, proteomic, and brain-predicted age clocks represent complementary signatures of biological aging, disease progression, and resilience in sporadic late onset AD (sLOAD), which is the most common form of AD (Aim 1), and in autosomal dominant AD (ADAD), in which the accelerated aging hypothesis can be tested with minimal age-related confounding factors (Aim 2). The final aim will test whether biological age estimates from these clocks differ between sLOAD and ADAD (Aim 3). As protein expression inherently links genetic and phenotypic layers, we predict that proteomic age will mediate the association between epigenetic and brain-predicted age clocks in both sLOAD and ADAD. If these multimodal clocks capture complementary signals of AD risk and resilience, we predict that each clock will explain unique related variance in both sLOAD and ADAD. If pathological severity is greater in ADAD than sLOAD, we predict that age clocks will exhibit greater sensitivity to ADAD than sLOAD. This study will address an important knowledge gap on whether multimodal age clocks provide additive measures of AD staging or resilience. Future independent research projects may explore multimodal biological age longitudinally, in relation to additional pathological, risk, and resilience factors, or using more advanced modeling approaches. This K01 award will support the candidate’s transition towards an independent career with a unique, transdisciplinary research program in aging, AD, and AD-related dementias (ADRD). A carefully-tailored training plan will build upon the candidate’s established expertise in cognitive psychology, neuroimaging, and machine learning with additional expertise in the analysis and interpretation of epigenetic (Goal 1) and proteomic datasets (Goal 2), frameworks of resilience and protective factors in AD (Goal 3), and development of the professional skills required to lead and support an independent research lab (Goal 4). These training goals will be supported by individualized mentorship from a team of domain experts, didactic coursework, practical skill-building, and completion of the research aims.

NSF Awards · FY 2025 · 2025-08

The doctoral dissertation project explores how healthcare providers and patients are navigating biotechnological advances and economic changes within the fertility healthcare industry. In addition to training a graduate student in anthropological science, broader impacts could lead to a better understanding of how lower cost IVF may be expanded. Data and findings will be widely disseminated to improve the public's understanding of IVF biotechnological innovation. The researcher asks what variables drive the promotion and adoption of new fertility technologies. The researcher will conduct interviews with physicians, embryologists, nurses, genetic counselors, and patients across three separate clinical sites, extended behavioral/participant observation within these settings, and a content analysis of materials produced across the clinical settings. The project makes clear contributions to scientific work on dimensions of assisted reproductive technologies, and their access, use, and transformation. The data gathered from the project could also lead to a better understanding of how lower cost IVF may be expanded, in line with the February 18, 2025, Executive Order, "Expanding Access to In Vitro Fertilization." 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 · 2025-08

PROJECT SUMMARY Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous connective tissue disorder resulting in muscle weakness, bone deformity and increased fragility, primarily due to type I collagen gene mutations. Severe OI is detected by standard American College of Obstetricians and Gynecologists (ACOG) recommended ultrasound screening (18-22 weeks). Additionally, paternal OI and de novo mutations can be detected by commercial cell-free DNA screening in maternal serum as early as 10 weeks gestation- demonstrating efficacy of early screening technologies. Although OI can be diagnosed before birth, physicians currently lack tools for in utero intervention. The goal of this proposal is to evaluate an innovative in utero pharmacological approach to building bone and muscle strength during development and throughout the lifespan. Previously, we demonstrated that pharmacological inhibition of myostatin (a negative regulator of muscle mass) beginning at 5 weeks of age improved bone parameters in two mouse models of OI. However, more significant improvements were achieved when OI mice were genetically deficient for myostatin, suggesting prenatal and/or early life myostatin inhibition is critical for maximum efficacy. Furthermore, we demonstrated that reduced maternal myostatin during pregnancy improved bone geometry and biomechanical integrity offspring with unaltered myostatin levels. Together these results indicate that inhibiting maternal myostatin during pregnancy is an innovative strategy to improve bone and muscle strength in offspring with OI. Here, we will test the efficacy of pharmacological inhibition of maternal and fetal myostatin via anti-myostatin monoclonal antibody therapy in two molecularly distinct OI mouse models initiated at two critical developmental time frames equivalent to 1) prenatal screening/diagnoses at 18-22 months (ACOG recommended) ultrasound (mouse E14.5); 2) pre-conception planning by a couple affected by OI. Maternal anti-myostatin monoclonal antibody treatment will be continued throughout lactation, followed by direct delivery to the offspring from the time of weaning into adulthood. Aim 2 will begin to elucidate the mechanism by which inhibiting myostatin in the maternal environment alters pregnancy health, maternal-fetal transport, and fetal musculoskeletal development using multiomic single nucleus sequencing. Finally, maternal health is a key outcome for in utero therapeutic development and is paramount for clinical consideration. Therefore, Aim 3 will evaluate maternal safety, metabolic and musculoskeletal health during pregnancy and lactation in wildtype and OI mice. The proposed project will provide preclinical evaluation of innovative in utero therapies for OI during critical developmental windows to maximize lifelong musculoskeletal health. Finaly, the project deliverables will provide key evidence for early therapeutic intervention as a strong rationale for implementing early first-trimester screening for OI.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC), including oral cavity squamous cell carcinoma (OCSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of OCSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed single cell RNA-sequencing (scRNA-seq) in OCSCC and identified a hybrid epithelial/mesenchymal state (HEM) with some features of classical EMT, yet persistent expression of epithelial markers (Puram et al., Cell). Further investigation into HEM demonstrated its localization at the leading edge of tumors where it appears to drive invasion and metastasis. Accordingly, HEM is associated with adverse pathology and poor prognosis, with a stronger effect on survival than even smoking. Strikingly, HEM appears to be a strong predictor of outcomes in both Black and White Americans (Mazul et al., Med), suggesting it may be useful in diverse sociodemographic groups. Based on these findings, we hypothesize that the HEM state is regulated by the complex interplay between malignant, immune, and stromal cells, and will reliably predict unfavorable outcomes in diverse OCSCC patients in treatment-naïve and recurrent/metastatic settings. First, we will validate a RNA-seq based HEM heterogeneity score to predict outcomes in a diverse cohort of 400 treatment naïve (including 150 Black American) OCSCC patients (Aim 1). However, we are also curious if HEM may predict outcomes in the recurrent/metastatic setting (Aim 2). Because these patients typically have limited tissue sampling due to tumor accessibility, bulk RNA-seq methods on fresh frozen tissue may be challenging. Thus, we will first define the effect of HEM on immune infiltration using multiplexed, multispectral imaging (MSI) approaches, followed by determining if MSI metrics of HEM predict outcomes in recurrent/metastatic patients. In addition, preliminary MSI analyses reveal that HEM leads to exclusion of tumor infiltrating lymphocytes including T-cells. Thus, we will also test whether HEM may predict immunotherapy response, which is front line therapy in recurrent/metastatic OCSCC patients: We will utilize MSI to analyze patients tumors treated with immune checkpoint inhibitors (ICI) to establish imaging-based metrics that predict response. If successful, our studies will authenticate HEM as a critical biomarker in OCSCC to risk stratify biologically favorable and unfavorable disease, improving patient expectations regarding disease outcomes, better informing patient-physician interactions regarding treatment decisions, and enabling future clinical trials for high risk OCSCC tumors using targeted therapy or potentially intensifying adjuvant therapy beyond standard-of-care treatment. Because no molecular markers of OCSCC are routinely used in clinical practice, these efforts represent an urgent and unmet need, which if addressed could significantly impact the management of these challenging patients and create opportunities for more personalized treatment through future studies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Cryptosporidiosis is a common cause of severe, chronic diarrheal disease in immunocompromised patients and infants from resource poor settings where infections are a major cause of morbidity and mortality. The only FDA approved drug for treatment of cryptosporidiosis, nitazoxanide, has limited effectiveness in those most at risk including immunocompromised patients and infants. As an enteric pathogen, Cryptosporidium grows at the apex of the gut epithelium proximal to the microbiota and metabolites produced at this interface influence infection. In previous studies, we defined three classes of microbial metabolites that inhibit C. parvum growth: secondary bile salts, indoles, and the vitamin B6 precursor pyridoxal. Indoles inhibit oxidative phosphorylation in the host mitochondria and depolarize the remnant C. parvum mitochondrion called the mitosome. The mitosome lacks most mitochondrial functions but retains an alternative electron transport chain as well as pathways for iron- sulfur cluster and ubiquinone biosynthesis. Very little is known about the physiology of this organelle, although the inhibitory actions of indole suggest it performs essential functions. In the proposed studies, we will elucidate the role of an ADP/ATP carrier in generating the membrane potential of the mitosome and characterize the action of indole in inhibiting this pathway. In addition, we will determine the function of a putative pyridoxal phosphate transporter that may bypass salvage pathways for vitamin B6 that are absent in C. parvum. Finally, we will explore the iron sulfur cluster biosynthesis pathway, which is normally dependent on vitamin B6, by testing the essentiality of key enzymes in this pathway. To facilitate future studies, we will define import and functional pathways in the mitosome using permissive biotin ligase coupled to different organellar components. Collectively, these studies will define essential functions in the mitosome and identify potential targets that may lead to alternative treatments for cryptosporidiosis.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Overview: DNA double-strand breaks (DSBs) that are present during mitosis can lead to severe genome instability. While cells have robust DNA repair mechanisms in interphase, these pathways, including non- homologous end joining (NHEJ) and homologous recombination (HR), are suppressed in mitosis. Recent discoveries, including my work, revealed that the alternative mutagenic Polymerase θ-mediated end joining (TMEJ) pathway is active during mitosis. However, the regulatory mechanisms determining when and how TMEJ or other repair processes are activated in mitosis remain unclear. My laboratory identified the E3 ubiquitin ligase RNF168 as a key regulator of mitotic DNA repair, which forms the foundation for further exploration of how mitotic DNA damage responses are controlled. RNF168 activity is suppressed in mitosis, but during G1, S, and G2 cell cycle phases RNF168 ubiquitinates histone H2A (ub-H2A) at DSBs to recruit NHEJ and HR machinery. RNF168-generated ub-H2A regulates DNA end resection at DSBs, a process critical to determining whether NHEJ or HR is activated in interphase cells or if DSBs are protected or subject to TMEJ repair in mitosis. I propose a model where ub-H2A acts as a molecular switch from interphase to mitotic DNA repair programs, and that unresolved S/G2 phase resection elicits mitotic TMEJ. Goals: The overall objective of this research is to unravel the molecular mechanisms that regulate the mitotic DNA damage response. To investigate these mechanisms and address key gaps in knowledge, my laboratory will focus on two major project areas: 1) regulation of the switch from interphase to mitotic DNA damage responses, and 2) regulation of DNA end resection during mitosis and the impact on DNA repair pathway activation. To understand how the mitotic DNA damage response program is activated, the goals of project 1 are to: a) reveal how ub-H2A levels and DNA repair activities impact DSB resolution for S/G2 phase breaks that enter mitosis, b) identify the deubiquitinating enzyme responsible for ub-H2A elimination from DSBs at the G2- mitosis transition, and c) determine how chromatin remodeling impacts ub-H2A levels upon mitotic entry. DNA end resection dictates repair pathway selection at interphase and mitotic DSBs. Because ub-H2A is absent during mitosis, the processes regulating resection and repair pathway selection are unclear. The goals of project 2 are to: a) quantify short- and long-range end resection at DSBs generated prior to and during mitosis, b) reveal the relationship between the DSB genomic location, resection distance, and mutagenesis, and c) identify the factors responsible for limiting resection at mitotic DSBs. Vision: The proposed work builds towards a detailed molecular understanding of how mitotic cells process DSBs. By dissecting the regulatory mechanisms governing the DNA damage response during mitosis, this research will advance our understanding of how genome stability is maintained. Insights gained from these studies could inform strategies to mitigate genome instability.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Every year there are two million new cases of cancer diagnosed in the United States and 600,000 cancer-related deaths. Preventing the development of cancer, when therapies are more effective, is one of the most potent methods to decrease cancer-related mortality. While the acquired genomic drivers of cancer are known, it is less well understood how those genomic changes arise and drive tumorigenesis. Understanding this process may yield new interventions to prevent malignant transformation from these pre-malignant states. Interestingly, in the hematologic system, somatic mutations that drive leukemia can be detected at low levels in nearly all disease free adults—a phenomenon termed clonal hematopoiesis. Clonal hematopoiesis increases in prevalence with age and is associated with increased cancer risk. However, clonal hematopoiesis is so common that it rarely progresses to blood cancer and the factors driving leukemic transformation are largely unknown. This also occurs in other solid organs such as skin and esophagus where clonal mutations are spatially constrained in the tissue. Prior work has focused on understanding how clonal hematopoiesis arises and evolves over time in the peripheral blood of healthy individuals. Less is known about how these clones exist and interact within the bone marrow—the source of hematopoiesis in humans. It is possible that these clones remodel their local niche to support clonal evolution and leukemic transformation. Intriguingly, given the prevalence and age-dependence of clonal hematopoiesis, these changes may also negatively affect physiological function to drive aging-associated changes in the hematologic system (e.g. anemia and declining immunity). This proposal seeks to understand how these clones exist and interact within the aging human BM niche in cancer-free adults. This builds upon prior techniques developed for rare mutation detection, single-cell mutation characterization and immunophenotyping, and spatial mutation detection. In a pilot study of a patient with hematologic malignancy, applying these techniques demonstrated marked spatial and clonal heterogeneity within their bone marrow. Now these tools will be applied to examine clonal organization and heterogeneity within the bone marrow to study “healthy” aging hematopoiesis before blood cancer develops. This study is possible because of a unique collaboration with Mid-America Transplant, the regional organ procurement organization, to provide multiple bone marrow containing bones and peripheral blood from brain-dead organ/tissue donors for this study. This study has the potential to uncover how somatic mutation and aging conspire to drive hematologic dysfunction, support clone persistence for decades, and drive leukemogenesis directly in human specimens. The tools developed for this project are broadly applicable to future studies discovering how aging affects all of the organs within the human body. Long-term, this will identify potentially testable mechanisms to reduce the risk of malignant transformation and maintain homeostatic organ function late in life.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal condition that affects an estimated 2.4-3.1 million people in the US. IBD is increasing in prevalence and cost, and linked to the development of colorectal cancer. While recent progress has improved our understanding of IBD pathophysiology, many unknowns remain about the relationship between host and microbe interactions in the intestinal microenvironment. A stronger understanding of the microscopic biochemical basis of IBD could lead more effective prevention and treatment. Established technologies lack the ability to observe bacteria and host cells with sufficient temporal and spatial resolution, with most methods performing a bulk analysis ex vivo. Many established imaging methods do not have sufficient spatial resolution to observe bacteria cells, and established super-resolution imaging methods are not well-suited for dynamic imaging in vivo. IBD is one of many driving forces that has led to a critical need for new methods to observe and quantify the intestinal microenvironment with high spatial and temporal resolution. This proposal seeks to address this need by developing and testing a novel optical imaging technology to observe the dynamics and heterogeneity of host-microbe interactions in the live mouse intestinal microenvironment. A new method for multiphoton super-resolution microscopy will be developed, compatible with label-free autofluorescence imaging of NAD(P)H, FAD, and tryptophan, all of which are essential to host and microbe metabolism in the intestine. Furthermore, this method will be used for super-resolution imaging of label-free biochemical information with hyperspectral coherent Raman scattering microscopy and structural information with sum-frequency generation of collagen. In order to accelerate the acquisition of 5D data with temporal and spectral information (x-y-z-t-λ), computational methods will be employed to acquire and save only the essential information, enabling imaging fast dynamics and large volumes of data. These newly established methods will be used to image and characterize the microenvironment of the large intestines of mice in vivo, examining differences between IBD and control mice, and metabolic changes associated with different grades of IBD. This project fulfills a critical need for new approaches to study the metabolism of the intestinal microenvironment in vivo with unprecedented spatial and temporal resolution. While focused on IBD, this technology has broad potential for studying other complex tissue microenvironments, and will be especially useful for bringing the next generation of microscopy methods to microbiology.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract The administration of exogenous oxytocin (OT) to prevent postpartum hemorrhage (PPH) is a cornerstone of obstetric care. However, the impact of this practice on oxytocinergic signaling in the offspring remains poorly understood. Our preliminary studies using a translational rat model that mimics clinical OT infusion for PPH prevention (PPH-OT) indicate that maternally administered OT may be transferred to the offspring via breast milk, leading to altered oxytocin receptor (OTR) expression and reduced sociability in male offspring. This project aims to address the potential consequences of this transfer for offspring neurodevelopment, with a specific focus on sex-specific outcomes in hypothalamic OTR-OT signaling. In Aim 1, we will quantify OT transfer from mother to offspring through breast milk. Using stable isotope-labeled OT (SIL-OT) administered to postpartum rats, we will use mass spectrometry to track SIL-OT in breast milk, neonatal plasma, and brain tissue (Aim 1a), and we will map the distribution of SIL-OT within the neonatal brain using MALDI-MSI (Aim 1b). These experiments will reveal the extent and specific brain regions affected by lactational OT exposure. In Aim 2, we will assess the impact of lactational OT exposure on OTR-OT signaling in offspring, with a focus on sex-specific responses in the hypothalamus. Guided by preliminary data showing decreased hypothalamic OTR expression and reduced sociability in male offspring, we will use RNAscope in situ hybridization to analyze hypothalamic region-specific OTR expression in male and female offspring at postnatal days 7, 14, and 21 (Aim 2a). Additionally, we will examine c-Fos expression in OT neurons within the paraventricular hypothalamus (PVH) following social interaction to assess potential sex-specific disruption in hypothalamic neuronal activation (Aim 2b). Collectively, this research will fill a significant knowledge gap in our understanding of the broader consequences of PPH-OT administration. By leveraging a novel animal model and cutting-edge analytical techniques, we aim to provide insights that could inform the optimization of OT dosing after childbirth, potentially reducing unintended neonatal exposure and informing the selection of safer uterotonic alternatives. Ultimately, our goal is to enhance maternal and neonatal wellbeing by ensuring that obstetric practices are informed by a comprehensive understanding of the physiological and behavioral effects of OT during a critical period of neurodevelopment.

NIH Research Projects · FY 2026 · 2025-08

Cancer predominantly affects the elderly, with a significant incidence of breast cancer in women aged 50-70. While cell mutations contribute to tumorigenesis, age-related changes in the tumor microenvironment, particularly in cancer-associated fibroblasts (CAFs), which themselves are diverse subsets with distinct roles, including inflammatory, vascular, and myofibroblast CAFs (iCAF, vCAF, and myCAF, respectively)1, may play a crucial role. Importantly, CAF subsets display substantial variability between different tissues and even within the same tissue2. Our study identifies senescent CAFs (senCAFs) as key players in breast cancer progression. In exciting new data we find that 1) senCAFs are frequently found in DCIS where they predict recurrence and in triple negative breast cancer (TNBC), estrogen receptor positive (ER+) BC, and Her2/Neu+ BC. 2) senCAFs are restricted to the myCAF population, raising the possibility that they are a developmental endpoint and do not simply appear stochastically in response to tissue level stress. 3) Depletion of senCAFs significantly reduces primary tumor progression and metastasis. 4) senCAFs modify the biophysical features of the extracellular matrix (ECM), which can impact host immune responses and tumor progression2. 5) senCAFs modify NK cell killing activity, which supports tumor growth. 6) senCAFs increase BC lung metastasis. Together, these data lead to our hypothesis that senCAFs alter the biophysical properties of the ECM and immune responses to increase tumor growth and metastasis. To address this exciting hypothesis, we will use state of the art immunological and biophysical techniques in novel genetically engineered mouse models (GEMM) that we have built and human BC specimens in the following Aims. 1) Determine the organ specific effects of senCAFs on metastatic BC progression; 2) Determine how senCAFs impact ECM dynamics and tumor cell migration; and 3) Determine the impact of senCAFs on NK cell function in BC tumor progression. Our study on senCAFs, a novel component of the breast cancer microenvironment, may uncover critical mechanisms in breast cancer progression, guiding the development of immunotherapeutic and senolytic strategies.

NIH Research Projects · FY 2025 · 2025-08

Project Summary This application addresses critically unmet needs in the field of targeted radiopharmaceutical therapy, an emerging treatment paradigm for metastatic cancers. Therapeutic radiopharmaceuticals localize to and deposit cytotoxic beta and alpha particles at sites of disease. Despite the great potential of these cytotoxic emitters, a one-size-fits-all approach is used to determine administered activities that ignores features of an individual’s disease phenotype. Precision and personalized dosimetry is a long-sought goal that is complicated by the inherently low imaging signal produced by these therapeutic radionuclides and low administered activities used for these drugs. For example, in contrast to diagnostic procedures, the administered activities of alpha particle therapies are in the 100 µCi range, orders of magnitude lower than a typical SPECT or PET imaging agent; and isotopes of interest (Actinium-225, Radium-223 and Lead-212) release only 5% of their decay energy by photons. At such low photon fluxes, it is crucial to collect as many photons as possible in order to reconstruct accurate, precise, and high-resolution imaging data. Conventional SPECT systems employ a lead collimator (to provide positional information) in front of a NaI(Tl) crystal based camera (with optimal sensitivity for the detection of ~140keV photons). Such a device is sufficient for diagnostic imaging with Tc-99m despite rejection of 99% of incident photons. Here, we develop a novel imaging detector module in which the collimator section is composed of active sensing detectors to provide spatial and directional information without rejecting photons. Predicated on extensive preliminary data and proof of concept prototype detectors we put forwards a mathematic framework using accurate physics and state-of-the-art anthropomorphic models to optimize this Sensing-Collimator Imaging (SCI) SPECT technology. We rigorously validate and evaluate this platform in order to develop the next generation SPECT imager that can reliably measure the distribution of therapeutic radiopharmaceuticals with orders of magnitude greater sensitivity and innovate the field towards the goal of direct, sensitive, and accurate imaging of alpha-emitting targeted radiopharmaceutical therapies.

NIH Research Projects · FY 2026 · 2025-08

Project Summary Bioactive molecules from nature are valuable templates for essential medicines. Filamentous actinobacteria are gifted producers of such molecules and rank among the richest sources yet discovered. They produce >50% of clinical antibiotics, plus numerous other medicines including immunotherapeutics and anticancer agents. These organisms dedicate a substantial fraction of their genomes to gene clusters predicted to encode drug-like compounds. Our research centers on a critical question: Why do the vast majority of actinobacterial biosynthetic gene clusters fail to yield anticipated products? There is an urgent need for new drug scaffolds and this problem transects multiple therapeutic areas. Gaining access to the plethora of yet-undiscovered drug-like molecules encoded within actinobacterial genomes could broadly revolutionize medicinal discovery and development. Coaxing actinobacteria to “turn-on” quiescent pathways is a challenge whose solution is fundamentally rooted in microbial physiology. Building on methodology and research insights established in our prior works, we here outline a multidisciplinary program that investigates two families of antibiotic molecules as models to reveal new mechanisms controlling production phenotypes. These molecules, polycyclic tetramate macrolactams and piperazyl-peptides, are broadly distributed among filamentous actinomycetes. They are largely chemically and biosynthetically distinct but share a common link via ornithine metabolism. Goal 1) Our prior works establish small nucleotide changes in transcriptional promoters can “tune” antibiotic production. How do these promoter polymorphisms exert their control? How can this knowledge be applied to override poor production traits? Goal 2) Certain piperazyl pathways respond positively to ornithine and related pathway intermediates. This suggests a mechanism of metabolic control. We ask: Because both types of antibiotics we study require ornithine precursors, might cellular ornithine status constitute an important regulatory component that could empower discovery strategies for both? Goal 3) We recently established a positive relationship between cellular growth on solid surfaces and polycyclic tetramate macrolactam production in certain strains. We hypothesize this intriguing relationship may extend to other antibiotic production pathways as well. We ask: How do actinobacterial surface interactions modulate bioactive molecule production, and might answering this deliver novel insights for broad-scale biosynthetic activation?

NIH Research Projects · FY 2025 · 2025-08

Treatment-resistant depression (TRD) is a debilitating mental health condition in which patients experience persistent symptoms despite medical intervention, leading to adverse outcomes and an overall diminished quality of life. This condition imposes a significant financial burden on both individuals and healthcare systems due to frequent medical consultations, ongoing therapies, and lost productivity. TRD is typically defined by the failure to respond to at least two different classes of oral antidepressant medications, underscoring the critical need for new therapeutic approaches. One promising avenue for treating TRD and other neuropsychiatric disorders is the use of pharmacological agents that are active in the brain, such as general anesthetics like propofol. Emerging theories suggest that these treatments may work by inducing or modulating electrophysiological biomarkers (e.g., neural synchronization and oscillations) that have been linked to disease phenotypes. However, the development of these alternative treatments faces several technical bottlenecks. First is the substantial variation in drug dosing requirements between and within individuals. More fundamentally, manipulating an electrophysiological biomarker is mechanistically ambiguous, since it is unclear what circuit dynamics are being engaged by the drug. To obviate these issues, this proposal aims to develop personalized medicine strategies that will enable tailored drug dose titrations based not only on patient demographics but also on data-driven inferences of their individual brain dynamics and neural circuitry. Our specific motivation arises from the study of slow oscillatory activity, particularly slow waves (SWs) in the EEG that can be elicited by anesthetic drugs. Empirical evidence indicates that there may be an ‘optimal’ dosing range that promotes these SWs, and that doing so may lead to therapeutic effects in patients with TRD. To realize this clinical potential, we seek several computational advances: (i) A data-driven dynamical systems model to identify the neural dynamics that underlie EEG SWs, thereby resolving mechanistic ambiguity in detecting these oscillations; (ii) Modeling general anesthetics as perturbations to these latent neural dynamics, allowing for dose titration to be formulated as a manipulation of neural mechanisms, thus enabling (iii) Dosing strategies that induce the desired neural dynamics by means of a bifurcation. Our approach uses brain electrophysiology to create personalized dosing models, introducing a new paradigm for pharmacodynamics based on dynamical systems theory. RELEVANCE (See instructions): Treatment-resistant depression (TRD) is a severe form of major depressive disorder that is associated with persistent symptoms and reduced quality of life. A promising new treatment strategy for TRD involves using anesthetic drugs like propofol to modify pathological brain activity. This study will enable and optimize these treatments for TRD by developing new computational techniques to systematically predict how anesthetic drugs modify brain activity at the level of individual patients.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Bacteria often grow non-planktonically in aggregates termed biofilms, a growth state in which they are highly tolerant of antibiotics and host immune defenses, and are even protected from the viruses that target bacteria, phage. These aggregates are encased within a complex extracellular matrix that typically includes exopolysaccharides (EPS), extracellular DNA (eDNA), and secreted proteins. Biofilm-involved infections are a particularly grave danger from the opportunistic pathogen, Pseudomonas aeruginosa, that chronically infects the lungs of people with cystic fibrosis, chronic wounds, and medical devices. Thus, alternative therapies are urgently needed, and phage therapy, the treatment of bacterial infections with phage, is one promising option. Biofilms are tolerant to many phages, which reduces the effectiveness of phage therapy. However, there are numerous phages that have evolved mechanisms to infect biofilm-associated bacteria. Mining these anti- biofilm strategies used by phages represents an exciting opportunity to improve phage therapy and to identify proteins that could be developed as stand-alone therapeutics. Previous studies have revealed that some phages encode depolymerase proteins associated with their tail spikes; however, the assays used for identifying these proteins are limited in scope, and as a result the diversity of antibiofilm strategies remains largely unexplored. In general, known depolymerases target the o-antigen LPS and capsule rather than bona fide matrix components such as EPS. Furthermore, there are few studies that investigate phage and phage- derived anti-biofilm proteins using in vitro biofilm models. As a result, we have very little knowledge of how phages are normally tolerated by biofilms and what mechanisms they use to overcome this barrier. We propose two unique strategies to isolate biofilm-interacting phages coupled with innovative methods to identify the phage proteins responsible for the interactions. We next propose dissecting the mechanism by which the phages and phage-derived anti-biofilm proteins interact with the biofilm matrix using in vitro biofilm models coupled with imaging and biophysical approaches such as solid-state NMR. These studies will begin to assign functions to previously unknown, hypothetical phage genes that make up the enormous amount of uncharacterized viral genetic material. Additionally, the proposed research will open new exciting avenues for future studies with additional biofilm-forming pathogens. We anticipate the outcome of this work will move the field forward by shedding unprecedented light on basic phage and biofilm biology and provide essential information to better treat biofilm-associated infections.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Seasonal respiratory RNA viruses, including human parainfluenza viruses (HPIVs) infect every child and can reinfect individuals later in life. Globally, HPIVs are responsible for 725,000 hospitalizations and >34,000 deaths per year with HPIV1 being the major cause of croup. In addition to acute complications, severe disease from HPIV infection early in life associates with the development of chronic lung pathologies, including asthma, impacting a large fraction of the adult population. Based on observations in immunocompromised patients, persistence of respiratory RNA viruses is suspected to drive the development and maintenance of these chronic lung diseases. However, respiratory RNA virus persistence in immunocompetent hosts remains largely understudied. Studies on the mechanisms and consequences of persistent RNA virus infections are critical to expand our understanding of the biology, ecology, and evolution of common viruses of clinical importance and may reveal solutions to debilitating chronic illnesses. Infection of mice with the murine parainfluenza virus type 1 (best known as Sendai virus; SeV) results in acute disease and virus clearance that is followed by the development of expanding pathology with characteristics of type 2 inflammation, similar to what is observed during asthma in humans. Using immunocompetent mice, we recently demonstrated the persistence of viral proteins and viral RNA in a subset of hematopoietic cells infiltrating the lung. Persistently infected cells were found long after the acute infection was cleared. We also showed that cells expressing persistent viral proteins had transcriptomic signatures consistent with pathogenic chronic inflammation. Importantly, ablation of cells exposed to the virus significantly decreased the severity of chronic lung inflammation, suggesting that viral persistence is a key factor in the establishment and maintenance of chronic post-viral lower respiratory tract disease. Here, we will use this murine model to determine where and how the virus persists in hematopoietic cells (Aim 1). We will also study the long-term impact of SeV infection in the structural cells of the upper and lower respiratory tracts (Aim 2), and we will further explore the long-term impact of parainfluenza virus infection on chronic post-viral disease and susceptibility to future viral infections (Aim 3). Overall, our studies will advance our understanding of the mechanism mediating the establishment and maintenance of persistent viral products, as well as the long-term impact of parainfluenza virus infection on the host’s health. We expect that knowledge gained from these studies will be applicable to other RNA respiratory viral infections that affect every child, including respiratory syncytial virus, human metapneumovirus, and rhinoviruses.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Our goal in this new WRHR is to leverage the significant resources, mentorship, and track record of successful physician-scientists within WashU and the Department of OB/GYN to train and promote the development of future leaders in interdisciplinary research in women's reproductive health. The WashU WRHR program will have three core content themes – 1) Pregnancy and pregnancy complications, 2) Gynecologic cancer, and 3) Benign gynecology and reproductive health – and four methodologic tracks: 1) Clinical trials and epidemiology, 2) Implementation science, 3) Basic and translational science, and 4) Engineering and data analytics. This program, designed with input from successful OB/GYN physician-scientists who launched their careers at WashU, will allow the WRHR Scholars to immerse themselves in reproductive sciences and use the skills and knowledge of diverse scientific disciplines to conduct rigorous, high-impact research aimed at improving women's reproductive health. We will accomplish our goal by pursuing the following four objectives: 1) Provide robust interdisciplinary research experiences in women’s reproductive health. Each Scholar, with the guidance of Mentors, the Program Director (PD), and the Research Director (RD), will design a research project centered in one of our three core content themes. The Scholars' research projects will be the foundation for subsequent grant proposals as independent investigators directing research teams. 2) Provide effective mentoring to promote Scholars’ retention and productivity. Each Scholar will have an interdisciplinary mentor team that includes a primary mentor and at least two secondary extra-disciplinary and external mentors. The mentors, PD, and RD will guide the Scholars to understand research and the research landscape, develop critical assessment skills, ask creative questions, and identify and overcome challenges. The PD and RD will regularly monitor each Scholar’s progress, provide feedback to overcome obstacles to success, and work to retain them in the pursuit of women’s reproductive health research. 3) Provide formal didactic training and professional development opportunities. We will help each Scholar choose the most relevant discipline-specific courses, seminars, and symposia within one of our four methodologic tracks. Additionally, all Scholars will participate in core professional development programs and peer networking to master essential research execution, management, and leadership skills. And 4) Rigorously and comprehensively evaluate and improve our program. The Advisory Committee will annually use milestone tracking to evaluate each Scholar and the program as a whole. Additionally, WashU-WRHR leaders will participate in the WashU Council of NIH-funded Training programs to learn and share best practices, exchange ideas, and work to continually enhance our program.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY/ABSTRACT Despite advances in sickle cell disease (SCD) treatment, patients continue to experience stroke, endure lifelong cognitive disability, and have a reduced life expectancy of 43 years. While previous imaging studies have focused on anemia and hypoxia as risk factors for stroke, they have not adequately explored other potential disease mechanisms in SCD, such as systemic thromboinflammation and blood-brain barrier (BBB) disruption, which may contribute to increased stroke risk. Although thromboinflammation-targeted approaches have shown promise in mitigating BBB disruption and reducing brain injury in animal models of SCD, their effectiveness in humans remains unexplored. To bridge this gap, this K23 proposes a prospective cohort study to investigate the central hypothesis that BBB dysfunction, associated with specific thromboinflammatory pathways, predicts silent infarct progression and cognitive decline in patients with SCD. The study has three main aims. In Aim 1, using both a well-validated MR sequence and a novel MR sequence, it will assess BBB permeability in patients with SCD compared to healthy controls, stratified in relation to existing infarct burden. In Aim 2, the study will investigate specific thromboinflammatory pathways associated with BBB dysfunction by examining unique gene expressions associated with elevated BBB permeability. Finally, in Aim 3, during a 30-month follow-up period, the study will determine whether baseline BBB permeability can predict the progression of ischemic brain injury and cognitive decline (executive function). This K23 proposal will be implemented with the support of an interdisciplinary team comprising the PI, as well as, mentors and consultants who are experts in relevant fields including: SCD, cerebral small vessel disease, advanced MRI methods, platelets and thromboinflammatory disorders, cognitive function in SCD, and biostatistics. The candidate, an adult cerebrovascular neurologist, has a long term goal to become an independent physician-scientist and to integrate advanced neuroimaging with blood-based markers of thromboinflammation to best achieve a comprehensive understanding of cerebral microvasculopathy and its cognitive consequences. The career development plan outlines three key areas: (1) expertise in advanced neuroimaging acquisition, data processing, and application; (2) foundation in systemic thromboinflammation; and (3) foundation in cognitive testing to translate MRI findings into clinically relevant outcomes. Successful completion of the proposed research and career development activities will fill knowledge gaps for the developing PI and provide compelling evidence on the role of BBB integrity and thromboinflammation in ischemic injury and cognitive decline in SCD. Furthermore, it will guide the development of an independent R01 proposal aimed at investigating BBB integrity as a novel neuroprotective strategy for treatment of cerebral small vessel diseases.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Chronic microglial activation is an early and critical driver of neurodegenerative diseases. Hence, there is an urgent need to measure microglial responses in vivo. Positron emission tomography (PET) provides a non- invasive means to track and quantify microglial responses. However, current approaches are limited by the lack of microglial-specific imaging biomarkers. To address this gap, we propose the development of the first Tandem pore domain halothane-inhibited K+ channel 1 (THIK-1)-targeted PET radioligands. THIK-1 is a K+ channel which exhibits high microglial specificity. Moreover, THIK-1 was recently associated with microglial functions such as proinflammatory IL-1b release and phagocytosis and identified as a therapeutic target for neurodegenerative diseases. We have identified two lead small molecule candidates as the first THIK-1-targeted PET probes, exhibiting THIK-1 specificity and high potential to cross the blood brain barrier. First, we will 3H-radiolabel these lead compounds and evaluate their specificity and concordance with neuroinflammatory markers in vitro. We will subsequently develop 18F-radiolabeled ligands and assess their in vivo biodistribution and brain penetrance in healthy C57/bl6 mice. The ability of these THIK-1 radiotracers to detect changes in microglial responses within disease will be assessed in vitro and in vivo using animal models of Alzheimer’s disease and multiple sclerosis. The specificity and sensitivity of these THIK-1 PET ligands for imaging microglia will be further assessed through blocking and microglial depletion studies. The innovation of this multi-disciplinary proposal lies in the development of the novel targeting of THIK-1 for highly specific and functionally relevant imaging of microglia. This will be the first report of THIK-1-PET approaches. Additionally, PET imaging of THIK-1 will provide critical information on the metabolism, efficacy, and safety of THIK-1 therapeutics. The novel approach proposed here has high potential for significant impact in both fields of neuroinflammation and neurodegeneration research. Enhancing understanding of the in vivo dynamics of microglia in neurological disease will improve disease monitoring, screening of potential disease-modifying therapeutics, and development of effective strategies to reduce risk of disease development.

NIH Research Projects · FY 2025 · 2025-08

Abstract Bats harbor the unique ability to host a wide array of emerging viruses, such as Ebola virus, Nipah virus, Hendra virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). These RNA viruses are highly pathogenic and often lethal to humans and animals. Intriguingly, bats develop no/minimal signs of diseases in both natural and experimental infections. Significant progress has been made to suggest the altered immunological networks and dampened inflammatory signaling in bats. However, the direct viral sensing mechanisms in bats and the unique immunological features that distinguish bats from other mammals remain poorly studied. Inflammasomes are multi-protein signaling platforms that form in epithelial cells and myeloid cells upon stimulation by pathogen and damage signals. Their primary function is to active the inflammatory caspases such as caspase-1. Canonical inflammasome sensors consist mainly of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing (NLR) family proteins. Among these NLR proteins, NLRP6 is a unique pattern recognition receptor that is predominantly expressed in intestinal and liver system. The inflammasome function of NLRP6 has been reported to directly detect the RNA viruses (rotavirus and mouse hepatitis virus) that infect the gastrointestinal (GI) tract. On the other hand, the excessive activation of NLRP6 inflammasome may exacerbate the tissue damage and cause the autoinflammatory diseases. In bats, the GI tract represents one major organ for viral infection, while infections rarely cause symptoms. The long-term goal of our project is to understand the specific inflammasome sensing mechanisms in detecting RNA viruses in the intestinal epithelium of bats and gain the insights of how bats protect themselves from the pathogenesis of inflammation-induced intestinal barrier dysfunction. In this application, we propose to pursue the following specific aims: 1) Determine the cryo- EM structures of bat NLRP6 monomer, elucidate the biochemical foundation of bat NLRP6- dsRNA interaction, determine the cryo-EM structures of bat NLRP6 with viral dsRNA and compare the structural mechanisms of dsRNA sensing and inflammasome signaling among bat, mouse and human NLRP6; 2) Elucidate the RNA virus-induced bat NLRP6 inflammasome signaling in reconstituted intestinal epithelial cells (IECs), analyze the bat inflammasome signaling in Eonycteris spelaea (Es) in response to bat-borne RNA viruses, study the genetic role of bat NLRP6 in regulating inflammasome signaling in bat primary IECs/bat intestinal organoids. The proposed studies will guide the development of therapeutics to target GI inflammatory disorders in human based on the molecular details of bat NLRP6 inflammasome.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Title: Molecular basis of receptor interactions for Venezuelan equine encephalitis Venezuelan Equine Encephalitis Virus (VEEV) is a mosquito-transmitted alphavirus that causes severe neurological symptoms in South and Central America and threatens parts of the southern region of the United States. No therapies or licensed vaccines are currently available. Recently, our laboratory identified its major entry receptor, low-density lipoprotein receptor class A domain containing 3 (LDLRAD3). Ldlrad3-/- mice do not develop severe infection when the virus is administered by subcutaneous, intranasal, or even intracranial routes. Nonetheless, the infection still progresses in Ldlrad3-/- mice in peripheral organs and the central nervous system (CNS), albeit it at a lower level. This result highlights the existence of additional, uncharacterized subordinate entry pathways of VEEV. To identify alternative receptors, in this R03 application, we propose to perform a novel CRISPR activation screen targeting the plasma membrane proteins. The top ‘hits’ will be validated in the presence or absence of LDLRAD3 expression. A second component of this application will be to improve the possible therapeutic activity of an LDLRAD3 soluble decoy molecule through a forward genetic screen. Our prior structural and mutagenesis studies show that domain 1 (D1) of LDLRAD3 engages the VEEV in the cleft of the heterodimer of viral envelope proteins E1 and E2. Preliminary structure-guided experiments suggest that amino acid substitutions in D1 can result in variants with enhanced binding affinity for VEEV. We propose to conduct directed protein evolution by mammalian cell display and screening D1 mutants to identify variants with high binding and neutralization capacity to VEEV. Variant LDLRAD3-D1-Fc decoy molecules will be tested for inhibitory activity in cells. Relevance Venezuelan Equine Encephalitis Virus (VEEV) is a mosquito-transmitted alphavirus that causes devastating encephalitis with a high death rate in horses and humans. The presence of its mosquito vectors throughout the Americas makes it a potential threat to public health. No drugs or licensed vaccines are available for humans. Although LDLRAD3 was recently identified as a principal entry receptor of VEEV, additional unknown receptors still sustain infection. Decoy molecules designed based on the receptor LDLRAD3 can neutralize VEEV infection. Here, we will conduct a CRISPR-based screen to find alternative receptors for VEEV and a mutagenesis screen to identify LDLRAD3 D1 variants with greater binding and neutralizing activity. This study will enhance our knowledge of VEEV entry and provide potential avenues for VEEV infection and disease control.