Washington University

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract: The neuronal ceroid lipofuscinosis (NCL or Batten disease), are a group of rare neurodegenerative lysosomal storage disorders (LSDs). Through mechanisms that remain poorly understood, inherited mutations that cause lysosomal dysfunction in the NCLs result in profound neurodegeneration within the central nervous system (CNS). Although progress has been made recently to produce disease-modifying treatments, none of these CNS-directed therapies are completely effective. We hypothesized that one reason CNS-directed therapies are incompletely effective is that lysosomal dysfunction also results in loss of neurons outside the CNS, specifically within the enteric nervous system (ENS). The ENS is the intrinsic nervous system of the bowel and controls most aspects of bowel function, with ENS defects causing life-threatening disease. These underappreciated effects of lysosomal dysfunction upon the bowel are unlikely to be treated effectively by CNS-directed therapies. Our preliminary data reveal profound ENS neurodegeneration in mouse models of CLN1, CLN2 and CLN3 disease, the major forms of NCL. These mice have bowel transit defects, that can be partly treated by neonatal delivery of intravenous gene therapy using adeno-associated viral vectors (AAV9). However, optimal therapy will require a better understanding of which bowel cell types are affected by lysosomal dysfunction and the cellular mechanisms underlying ENS neurodegeneration. Since our data show CNS-directed therapies do not treat the bowel, and bowel-directed therapies do not effectively treat the CNS, we will also test the hypothesis that optimal outcomes will require combined CNS- and ENS-directed therapy. For logistical reasons we shall explore these issues in a mouse model of CLN2 disease, which has rapidly progressing and severe enteric neurodegeneration and dies at an earlier age than mice with other major forms of NCL. In Aim 1 we shall define how different bowel cell types are affected by CLN2 deficiency, identifying cellular targets for gene therapy or that may be druggable. In Aim 2 we shall use novel transgenic mice to address which cell types critically need treating to preserve ENS function. In Aim 3 we will test combined approaches to simultaneously treat bowel and brain to define those most suited for subsequent clinical trials. Taken together these studies will provide important information for devising targeted therapeutic strategies to treat the effects of disease in the bowel and brain.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, particularly in Western countries, where it accounts for approximately 10% of all cancer fatalities. In the United States, CRC is the third most common cancer (excluding skin cancers), with around 160,000 new cases reported in 2023. Among these cases, metastatic CRC is present in 20% of patients at initial diagnosis and constitutes half of all CRC cases, with a 5- year survival rate of only 14%. Understanding the mechanisms driving CRC progression is crucial for improving diagnostic and therapeutic strategies. Recent research highlights the role of METTL3, an m6A (N6- methyladenosine) methyltransferase, in promoting CRC. These studies underscore the importance of epitranscriptomic regulatory pathways, particularly those related to m6A, in CRC tumorigenesis. However, the specific mechanisms underlying these pathways remain largely unexplored. The primary objective of this proposal is to elucidate how METTL3 and m6A modifications facilitate CRC progression, identify novel METTL3 coordinating factors and their targets in CRC, with the long-term goal to develop new diagnostic and therapeutic interventions. To achieve this, we used an innovative sequencing approach that combines long-read Nanopore Direct RNA sequencing (RNA-seq) with short-read Illumina RNA-seq in METTL3 knockdown CRC cells. Our long-read sequencing accurately mapped METTL3-dependent m6A sites across the CRC transcriptome. By integrating these results with short-read RNA-seq and patient data from TCGA, we identified several clinically relevant genes whose RNA stability and alternative splicing (AS) events are regulated by METTL3. Additionally, we pinpointed factors that mediate METTL3's functions in CRC, particularly the m6A readers involved in RNA stability and AS regulation. We also experimentally validated some METTL3 targets and confirmed YTHDF1 as an m6A reader that mediates RNA stability regulation. These preliminary findings provide a strong rationale for further exploring METTL3's roles in CRC, with the following specific aims: (1) To understand how METTL3 regulate RNA stability to promote CRC, and (2) To investigate the effect of METTL3 in regulating AS in CRC. The study will be conducted under the mentorship of Dr. Christopher Maher, co-mentorship of Dr. Jason Weber, and through the Cancer Biology program at Washington University, known for its leading genetics research. The Maher lab is a great fit for this project, with their expertise in molecular biology, bioinformatics, genomics, and CRC research. Dr. Weber’s expertise in RNA biology and cancer biology aligns perfectly with the objectives of this study. Training will focus on (1) advancing experimental biology and bioinformatics skills, (2) enhancing clinical engagement and translational medicine knowledge, and (3) developing scientific communication, leadership, and mentorship capabilities, all essential for a successful career in cancer research. This research is poised to make a significant impact by providing a comprehensive understanding of how METTL3 promotes CRC progression and holds substantial potential for clinical translation to improve patient care in CRC.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Millions of individuals in the United States suffer from post-acute symptoms of COVID-19 (PASC), many of which are neurological in origin. Although SARS-CoV-2 does not cause widespread infection of the central nervous system (CNS), significant neuroinflammation, decreased adult neurogenesis, alterations in synaptic proteins, decreased total brain volume, and poor performance on tests of learning and memory have all been observed in COVID-19 patients. However, the mechanisms by which a respiratory virus is causing persistent neurological dysfunction are not well understood. Studies of PASC patients identified ongoing dysregulation of T cell responses in the serum and imaging studies confirmed that PASC patients have activated T cells in the CNS. Homeostatic T cells in the meninges are known to influence learning, memory, and anxiety-like behavior through the secretion of cytokines, however the impact of T cells on neurological symptoms after COVID-19 is unknown. Thus, I hypothesize that systemic inflammation promotes recruitment of antigen-specific T cells to the CNS after SARS-CoV-2 infection, and these cells contribute to persistent deficits in learning and memory via secretion of cytokines and other pro-inflammatory mediators. To investigate this, I will utilize my recently developed mouse model of neurological dysfunction after COVID-19, in which intranasal infection of wild-type mice with SARS-CoV-2 causes robust infection of the respiratory tract, but not the CNS, post-acute memory deficits, loss of hippocampal neurogenesis, and increased pro-inflammatory cytokines in the CNS. Preliminary data found that CD4+ T cells are recruited to the meningeal dura after infection and promote post-acute memory deficits as determined via the Novel Object recognition test. In the K99 phase of this proposal, (Aim 1) I will investigate the phenotype of CD4+ T cells in the CNS after SARS-CoV-2 infection and determine how cytokines secreted by CD4+ T cells may contribute to memory deficits using a behavioral test battery and investigation of neurogenesis and tri-synaptic circuit function. In Aim 2, I will test if T cell priming and differentiation impact pathogenicity in the CNS after COVID-19 by first, determining if antigen specificity is required for recruitment to and effector functions in the CNS and second, investigating which CD4+ T cell effector subset is driving post-acute memory deficits. In the R00 phase, I will utilize a previously established murine model of breakthrough vaccination to investigate how priming of CD4+ T cells in the periphery influences differentiation into a pathogenic CD4+ effector subset. In Aim 3, I will explore how severe, systemic inflammation may be driving recruitment of these pathogenic T cells to the CNS and which chemokine: receptor pairs are critical for this process. Combined, this project will determine the mechanisms by which pathogenic CD4+ T cells are recruited to the CNS after COVID-19 and how they promote post-acute learning and memory deficits. These data could identify fundamental mechanisms by which immunity to viral infections controls neurological function and potential druggable targets for treatment of PASC.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY There is a sex disparity seen in cancers throughout the body where males have both a higher incidence and a higher mortality than females. This phenomenon is seen in glioblastoma (GBM) that comprises over 50% of brain tumors and are characterized by resistance to therapies and a dismal prognosis. This is not only true for adults, but children as well, suggesting that factors other than the effects of sex hormones may be playing a role in this phenomenon. Because enhanced nutrient consumption and metabolism are critical to enhanced tumorigenesis and its effect on poor patient outcomes, our group investigates the effects of biological sex on cancer metabolism as a driver for the sex disparity where male tumors engage in more carbohydrate and amino acid metabolism concomitant with higher PI3K-Akt-mTOR signaling that are associated with faster tumor growth and worse survival. Intriguingly, the metabolic sex differences that we observe in human and rodent GBM are also present in cell lines, suggesting a cell-intrinsic component. This could be due to direct sex chromosome effects and/or epigenetic tissue patterning effects from exposure to in utero gonadal sex hormones. Unfortunately, laboratory models for rigorous mechanistic sex differences research in GBM are lacking. This proposal will develop novel GBM models to address the hypothesis that sex chromosomes and sex hormones are both drivers of GBM phenotype, measured by proliferation and cellular metabolism. In the first Aim, we will use the Four Core Genotypes (FCG) mouse model to define the contributions of sex chromosomes and organizational effects of in utero exposure to sex hormones to astrocyte metabolism before and after mesenchymal GBM transformation. First, wildtype FCG astrocytes will be harvested from P1 pups and assayed for sex differences across key metabolic and signaling pathways important for cancer. CRISPR-Cas9 of FCG astrocytes will then be used to generate the mesenchymal subtype GBM models Nf1-/-Tp53-/- (proof of principle with strong sex-specific phenotype) and Pten-/-Tp53-/- (to test effects of Pten deletion on sex differences in PI3K- Akt-mTOR signaling). Cell lines and allografted tumors will be assayed for growth, metabolism and gene expression. Novel, advanced diffusion MR imaging from our lab will be used to noninvasively assay changes in the tumor microenvironment. In the second aim, we will develop human induced pluripotent stem cell (iPSC) models to evaluate the contributions of sex chromosomes to cellular differentiation and mesenchymal GBM transformation. NF1-/-TP53-/- transformed NPC will then be generated from iPSC using published methods developed by our team. NF1-/-TP53-/- NPC will be orthotopically xenografted into mice. Cell lines and tumors will be assayed for growth, metabolism and gene expression including imaging-based growth and tumor microenvironment assessment as above. This research proposal is innovative because it develops the necessary models to investigate the effects of biological sex on cancer biology and patient outcomes. This R21 presents a clear path to a future R01 that will involve the expansion of cell and animal models to study sex chromosome and gonadal effects on GBM, advancing the NIH’s initiative to incorporate sex as a critical biological variable.

NIH Research Projects · FY 2026 · 2025-07

ABSTRACT Substantial diarrhea is noted in about 50% of human immunodeficiency virus (HIV)-infected subjects with or without antiretroviral therapy (ART) in the U.S., of which the pathogenesis remains unclear. One of the challenges in studying HIV-associated diarrhea is that these patients often have comorbidities, such as malnutrition and co- infection with other gut pathogens, and hence it is difficult to dissect out the contributions of each cellular and microbial pathogenic components and their mechanisms. While a mouse model would provide a clean platform to study HIV-associated diarrhea, HIV does not infect murine immune cells, thus limiting the development of in vivo models. We recently established a novel humanized mouse model that supports robust reconstitution of human innate and adaptive immune systems in both lymphoid and non-lymphoid tissues, which provides an opportunity to investigate the pathogenesis of HIV enteropathy in vivo. The scientific premise of the findings is that the humanized mouse model faithfully recapitulates the molecular dynamics of HIV infection in immune cells. We also discovered that CARD8 inflammasome is activated in by the HIV protease. CARD8 inflammasome has been implicated in modulating the severity of inflammatory bowel disease (IBD) and inflammasome-mediated pyroptosis is known to trigger gut inflammation. In the gut, while HIV only infects immune cells (CD4+ T cells and macrophages), these cells also interact with intestinal epithelial cells (IEC), and could contribute to the homeostasis of gut barrier integrity. The knowledge gaps for advancing our understanding of HIV enteropathy are the clinical relevance of HIV infection and/or ART per se on gut barrier function, and if CARD8 inflammasome also adversely impacts IEC homeostasis. Our overarching hypothesis is that HIV-infected immune cells impede gut epithelial integrity through pyroptosis induction. We aim to understand the molecular mechanism governing gut inflammation and barrier leakiness during HIV infection and whether such damage persists despite suppressive ART, which impairs wound healing during subsequent injuries. We propose to define the interplay between IEC and HIV-infected immune cells in vitro, to define the how HIV infection dysregulates gut barrier function under injury using humanized mouse models, and to define the molecular crosstalk between IEC and immune cells in gut samples from HIV patients. Collectively, we will establish that HIV infection could negatively impact gut barrier integrity. We will additionally establish that HIV-activated inflammasome mediates the disruption of get epithelial barrier by recruitment of additional proinflammatory cytokines and chemokines that target the IEC. Other deliverables will also include the potential of targeting pyroptosis for HIV enteropathy.

NIH Research Projects · FY 2026 · 2025-07

Project Summary/Abstract Eye fixation enables us to see details of objects that require current attention using a specialized region in the retina called high-acuity area (or the fovea in humans). During eye fixation, visual inputs detected in the peripheral retina that may induce disruptive reflexive eye movements, such as optokinetic reflex (OKR), are suppressed. Eye movement and fixation are often disrupted in degenerative diseases in the early visual system (e.g. partial macular degeneration) as well as in high-order neural disorders. However, how the visual signals that evoke these two eye movements interact at the beginning of the vision, in the eye, is not well understood. In the eye, photoreceptors capture pixel representation of the visual scene. These signals are processed and transformed into visual feature representation, such as object motion, which triggers eye fixation, and background movement, which triggers OKR, in the retina before being sent to the brain. We hypothesize that these two visual feature signals interact with each other in the retina. To investigate this, we will use larval zebrafish as a model system. As larval zebrafish are transparent and easy to generate transgenic animals, this system uniquely allows us to optically record and optogenetically control neural activity throughout the visual system in behaving animals. This research will be the first to carry out such analysis in the retina. We will first behaviorally assess whether larval zebrafish can perform eye fixation in the presence of background movements. Next, using two-photon imaging of neural activity, we will identify the neural pathways that process eye fixation and OKR-evoking signals in the retina and retinal-recipient areas. Finally, by simultaneously recording behavior and neural activity in the eye and the brain, we will reveal how and where along these pathways, eye fixation and OKR-evoking signals interact with each other for selecting behaviors. Neural mechanisms underlying individual feature extraction in the retina have been extensively studied. Furthermore, these studies revealed that some of the feature signals are directly linked to behavior, such as OKR and eye fixation. However, in nature, we face visual scenes with multiple features that may compete with each other for behaviors, and the extent to which the neural basis of this computation is based in the early visual system, including the retina and retinal recipient areas of the brain, is less understood. By providing new fundamental knowledge about the circuitry and logic underlying the competition between central and peripheral vision, this project has broad implications for both normal and pathological vision.

NSF Awards · FY 2025 · 2025-07

The activity and interconnectivity of neurons, a key type of brain cell, are crucial to the brain's ability to compute and process information. However, recent studies suggest that astrocytes, a different type of brain cell, may also play an important role. This project combines experiments and computational modeling to study how astrocytes contribute to brain function. Astrocytes affect many aspects of neuronal activity and communication, providing a potential mechanism by which they can alter signaling in the brain. The computational modeling and mathematical analysis within the project will enable a deeper biological understanding of these astrocyte-neuron interactions, generate new ideas for why they may be important for information processing in the brain, and suggest ways to integrate these principles into artificial intelligence systems. In conjunction with the modeling will be experiments to observe and manipulate astrocytes in living brains. In so doing, the project will validate new ideas about astrocytes' roles in the brain, providing an enhanced understanding of neural circuits and brain function. The scientific premise of this project is the "contextual guidance" hypothesis, which postulates that astrocytes act as switchboards that transmit information about the environment and the physiological state of the organism to neurons and networks thereof. As such, astrocytes may act as a force multiplier that can expand the repertoire of dynamics that neurons can realize, thus enabling computation. The project will explore two ideas in this regard: (i) that astrocytes actively modulate neuronal dynamics in response to signals sensed from the environment, and (ii) this modulation enables neuronal networks to tailor their dynamics in response to context-specific circumstances. To substantiate these ideas, the project will investigate the role of astrocytes in neuromodulatory systems and subsequent effects on neuronal activity and synaptic plasticity. Furthermore, the project will examine network-level interaction between neurons and astrocytes, exploring features like "tiling," where astrocytes overlay neuron clusters to influence signal routing. In addition to scientific insights, the research will examine how brain-inspired computing may be enhanced by new artificial neural network designs that incorporate astrocytes, with a focus on context-dependent computational paradigms. Additionally, the project includes initiatives to engage trainees in interdisciplinary neuroscience research and exchange, including new mini-courses that bridge neuroscience, engineering and artificial intelligence. A companion project is being funded by the French National Research Agency (ANR). 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.

NSF Awards · FY 2025 · 2025-07

Plants are very important because they provide us with food, fuel, and medicine. However, climate change and other environmental challenges constantly threaten their growth and yield. Therefore, it's crucial to create "climate resilient crops" that can adapt to changing climate conditions to support sustainable agriculture. The goal of this project is to understand how plants use biomolecular condensates - microscale compartments found in all forms of life - to sense their environment and reprogram their epigenomes to achieve fitness, resilience, and productivity under stress. The results of this study could lead to new ways to engineer biomolecular condensates to improve nutrient allocation, growth, and stress resistance - major determinants of crop yield and renewable energy production vital to human health and global environment. In addition, this project will have a significant educational impact by advancing science discovery to the public and training students and scientists. Another impact of this project will be the creation of outreach events to share the research with the public, including science stations for K-12 students and summer science camp for high school students from Missouri and southern Illinois. Biomolecular condensates are emerging as key players in sensing and translating environmental signals to direct diverse cellular functions. While many studies have focused on the physical properties and molecular compositions of biomolecular condensates, much less is known about how they form and contribute to cellular function under stress – particularly in plants. This project is poised to significantly enhance our understanding of the molecular mechanisms underlying nutrient-induced biomolecular condensates and their biological roles in epigenetic reprogramming and plant developmental transitions. Specifically, this project will investigate diverse nutrient and environment signals that trigger EARLY BOLTING IN SHORTDAY (EBS) condensate assembly, dissect the mechanism by which TOR signaling regulates EBS condensates, and determine the biological roles of EBS condensates in epigenome dynamics and floral transition. Findings from this project will offer important insights into how different signaling pathways interact with biomolecular condensates and chromatin dynamics to regulate cellular functions. Given the importance of biomolecular condensates and epigenetic regulation in many biological processes, understanding how these condensates reprogram the epigenome to cope with the various stresses is a fundamental question relevant to both plants and animals. 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.

NSF Awards · FY 2025 · 2025-07

The rapid rise in antimicrobial/antibiotic resistance in pathogenic bacteria is a global public health threat. Antimicrobial resistance occurs when bacteria and fungi evolve to stop responding to antibiotics and to continue to grow. Each year in the US alone, antimicrobial-resistant bacteria or fungi cause infection in more than 2.8 million people and more than 35,000 deaths. Developing alternatives to traditional antibiotics is critical for addressing this global challenge. This collaborative project supports fundamental research to develop a new type of antimicrobial nanoparticle to combat antibiotic-resistant bacteria. These nanoparticles are unique in that they display a non-uniform coating of hydrophobic and positively charged molecules. Such nanoparticles are expected to act through novel antibiotic mechanisms that are less likely to cause acquired drug resistance in bacteria. The research team will combine experiments with computational modeling to elucidate how interactions of these nanoparticles with bacteria depend on the non-uniform surface chemistry of nanoparticles and the cell wall chemistry of bacteria. The mechanistic understanding from this study will guide the rational design of antimicrobial nanoparticles against a wide range of pathogenic bacteria. By integrating nanoscience research with educational and outreach activities this collaborative project outlines interdisciplinary approaches to promote critical thinking and increase diversity in STEM. These approaches include the development of introductory undergraduate courses that integrate science teaching with art, a collaborative outreach project to K-12 students in rural areas, and training of the next generation of researchers, especially underrepresented minority groups. Developing broad-spectrum antimicrobial nanoparticles is challenging because a single nanoparticle design cannot be a one-size-fits-all solution effective against all bacteria. Instead, nanoparticles whose antimicrobial activity can be tuned to match the bacterial diversity are needed. This collaborative project is focused on developing a new strategy to tune nanoparticle-bacteria interactions by using the anisotropic organization of ligands on nanoparticles. Specifically, experiments will be combined with molecular dynamics simulations to investigate interactions of amphiphilic nanoparticles with model bacterial membranes and a diverse selection of Gram-negative bacterial strains, many of which are resistant to most available antibiotics. The expected results will establish the structure-activity relationship governing the antimicrobial mechanisms of the amphiphilic nanoparticles. Such new understanding will enable the development and optimization of antimicrobial nanoparticles that are potentially more potent than existing ones and whose effects are tunable. The educational and outreach goal of this project is to develop interdisciplinary approaches to promote critical thinking and increase diversity in STEM. These approaches include innovation of introductory undergraduate courses by integrating art with science teaching, a collaborative outreach project to K-12 students in rural areas, and training of next generation of researchers with a particular emphasis on the involvement of underrepresented minority groups. 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 · 2025-07

ABSTRACT Washington University in St. Louis has had a funded Medical Scientist Training Program (MSTP) for more than fifty years that has evolved into a national source of highly successful physician-scientists. Herein they present a new application to continue this evolution to train the next generation of physician-scientists by providing in-depth pre-doctoral training integrating clinical medicine and biomedical research for individuals desiring careers as physician-scientists. MD-PhD students are selected from a strong pool of applicants and comprise an ever-increasingly diverse student body. Medical training is provided by the School of Medicine and research training is primarily carried out in the interdisciplinary graduate programs of the Division of Biology and Biomedical Sciences, or in the Department of Biomedical Engineering. Together, these graduate programs encompass a wide breadth of biomedical research. The basic components of the training program are: 1) pre-clinical medical school curriculum with a “MSTP Thread” that explores the scientific evidence for topics covered in the pre-clinical curriculum, including discussion of primary basic and clinical research papers; 2) PhD coursework in a biomedically relevant basic science discipline; 3) training in laboratory safety, responsible conduct of research, and rigor and reproducibility in research; 4) three or more years of original research, overseen by the training faculty and thesis advisory committees, leading to completion of a PhD thesis; 5) clinical training; and 6) a wealth of academic and social programs designed to enhance physician-scientist development and identity, and train students to think critically, and speak and write effectively. Individual students are extensively monitored, mentored, and guided. Policies are in place to ensure training faculty are providing appropriate mentorship. The MD and PhD degrees are awarded jointly upon successful completion which now averages 8.28 years, a period shortened by integrating both medical and graduate curricula which are continually evaluated and modified to enhance student success. The majority of our graduates join the faculty of the nation’s medical schools while others will contribute to the nation’s biomedical research enterprise in government laboratories, and the pharmaceutical and biotechnology industries. Regardless of their career path, their MSTP training will allow them to bridge the gap between the basic laboratory and the clinic. The success of this program is the product of strong institutional and NIH support that has allowed us to develop a supportive and effective training environment for physician-scientists.

NSF Awards · FY 2025 · 2025-07

The purpose of this project is to bring techniques from Hodge theory to bear on the properties of solutions of differential equations found throughout mathematics and physics. To elaborate, algebraic geometry is the study of systems of polynomial equations. Hodge theory seeks to understand the shape of the algebraic spaces defined by their solution sets. In so doing, it produces powerful connections between algebraic geometry and other parts of mathematics and physics, such as number theory, differential equations, and string theory. Normal functions are solutions to differential equations arising from algebraic cycles (or subspaces) on an algebraic space. By recognizing a solution as a normal function, which is a nontrivial task, one gets access to its behavior at numerous "special" points. In the scenarios to be considered in this project, these "special values" have important applications to identities and conjectures in number theory, the classification of algebraic spaces such as Fano varieties and algebraic curves, and the spectra of operators in topological string theory. This project will also help to train the next generation of researchers in pure mathematics, by integrating the PI's graduate students in work on specific problems in Hodge theory and bringing outside consultants to Washington University. Results will be disseminated through conferences, summer schools, journal articles, and websites. Normal functions originated in the work of Poincare and Lefschetz in the early 20th century. They are to families of algebraic cycles what period maps are to families of algebraic varieties: the basic Hodge-theoretic invariant. Informally, they are given by integrals of differential forms on non-closed chains instead of topological cycles, and satisfy inhomogeneous differential equations instead of homogeneous ones. The PI will study the properties, invariants, classification, and geometric realization of normal functions. The settings and applications range from arithmetic geometry and number theory to mirror symmetry, moduli, and mathematical physics. The specific goals of this project are to: (I) produce new evidence for the Beilinson and Bloch-Kato conjectures on special values of L-functions, and provide motivic realizations of biextensions; (II) describe an A-model variation of mixed Hodge structure on the cohomology of a Fano variety, and a B-model Lefschetz principle governing normal functions on LG-models; and (III) classify and realize normal functions with values in Hermitian and hypergeometric variations of Hodge structure, with applications to moduli of curves, hypergeometric identities, and quantization. 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 · 2025-07

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) causes 1.6 million tuberculosis (TB) related deaths per year, and current TB therapeutic strategies are becoming inadequate due to the emergence of drug resistant strains. One of the primary antibiotics used to treat TB infection acts at the level of transcription, inhibiting the RNA polymerase (RNAP). Sigma factors (σ) are accessory subunits that associate with the RNAP, directing a transcription- competent RNAP holoenzyme to unique genes. The σA RNAP holoenzyme is responsible for transcribing the majority of the Mtb genome, and as such, represents the major RNAP-dependent drug target. Of all the σs found in Mtb, only σA contains a N-terminal intrinsically disordered region (IDR) with strong charge segregation (i.e. clustering of positively/negatively charged residues). The propensity for disorder and charge segregation is conserved not only across mycobacteria, but more broadly across the whole Actinobacteria phylum, suggesting a functional, yet uncharacterized role of these σA IDRs in bacteria. This proposal seeks to understand the role of the Mtb σA IDR in transcriptional regulation, assessing what sequence and conformational features of the IDR encode functionality. Leveraging both experimental and computational approaches that overcome the technical challenges associated with the structural characterization of IDRs, I will assess how the conformational dynamics and interactions of the σA IDR impart transcriptional activity. I will employ a combination of in vitro kinetic measurements for transcriptional activity, single-molecule fluorescence techniques for measurements of IDR conformations and dynamics, and molecular dynamics simulations and cross-linking mass spectrometry for identifying IDR- mediated interactions within the RNAP holoenzyme. Measurements will specifically investigate the role of charge-segregated disordered regions and post-translational modifications and be performed under conditions that mimic intracellular ionic concentrations during Mtb infection and in the presence/absence of antibiotics. Given the broad conservation of sequence features, conclusions made will likely be applicable beyond Mtb, leading to a new paradigm for how bacteria use IDRs in transcription. The research and training objectives in this proposal will further the understanding of mechanisms of gene regulation unique to disease-causing bacteria, while enabling me to obtain additional technical training to foster the future generation of academic biomedical investigators.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ ABSTRACT The biological circadian clock generates a series of time markers across the 24 h day, by which different aspects of physiology and behavior (e.g., sleep, hormone release, temperature dynamics) may be aligned to local time for optimal efficiency. The molecular mechanisms of the PER-dependent circadian clock (which is a cell autonomous timekeeping system), and how the clock is set to local time, are both well-studied. Less is known about how the molecular clock couples within pacemaker cells to cell physiology, and how the clock uses intercellular communication to generate the multiple phase (markers) that are used by other clock cells, and by non-clock bearing downstream cells and circuits. This laboratory studies circadian neurophysiology in the model system Drosophila and the overall goal of this project is to understand how intercellular communication among pacemakers is used to generate different phases of circadian output. The fly’s daily rhythmic locomotor behavior is controlled by ~150 dedicated circadian pacemaker neurons whose internal molecular clocks are highly synchronized. Neuropeptide signaling in circadian neural circuits of both insects and mammals supports molecular synchrony: in Drosophila, the neuropeptide PDF (via the PDF receptor, a G Protein-Coupled Receptor (GPCR)) serves this function. Importantly, PDF modulation supports normal pacemaker circuitry in a second critical way: it inhibits the neuronal activity of identified pacemaker groups to specific non-morning phases. Prior work generated a model whereby the cell-intrinsic molecular clock in all pacemakers drives a common morning phase of enhanced neuronal activity. Yet in vivo, real-time measurements of calcium dynamics reveal that different pacemaker groups in the Drosophila brain are not synchronously active in the morning. Instead the different groups exhibit a stereotyped sequence of activity periods, at different times of the day and night. Hence this diversity of active periods (circadian phases) is generated primarily by cell interactions (especially neuropeptide modulation). Together these activity periods represent the poly-phasic outputs of the pacemaker system. Now, to test and extend the model, this research program will evaluate the hypothesis that two distinct forms of the PDF receptor may differentially contribute to molecular clock synchrony versus to inhibition of pacemaker neuronal activity. Experiments are also designed to identify the signaling pathways used by the receptor isoforms. Finally, work will extend studies based on the recent identification of a membrane protein that interacts with PDF receptor: an evolutionarily conserved magnesium ion transporter which co-regulates locomotor behavior along with PDF receptor. Together, this work performed in Drosophila will also inform an understanding of circadian output mechanisms in the mammalian brain, because the fundamental mechanisms of GPCR modulation are highly conserved. More generally, it will be relevant to fundamental mechanisms of neural circuit modulation by neuropeptides.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Individuals with clonal hematopoiesis (CH) are at a significantly higher risk to develop solid tumors compared to age-matched non-CH carriers irrespective of prior therapy. While shared risk factors may contribute, the unique functions that CH- mutant hematopoietic cells play in solid tumor development remain unknown. Notably, CH carriers with mutations in the epigenetic regulator TET2 have a strong predilection for development of solid tumors such as lung cancer, underscoring a critical underexplored area of research with the potential to improve disease surveillance and treatment strategies. I hypothesize that Tet2-mutant hematopoietic cells play a critical role in tumor development by conditioning a favorable solid tumor environment into a conducive landscape for CH-mutant cell growth and proliferation. Aim 1 of this proposal will quantify the impact of Tet2- mutant hematopoietic cells on lung-specific tumor development and response to radiation therapy (RT) as Tet2 mutations are frequently found in solid tumors and lung cancer is among the most frequent solid cancer type to arise in a CH background. Building on preliminary data which displayed enhanced tumor growth in a Tet2-mutant background, I will create chimeric mice with a Tet2-mutant hematopoietic system followed with establishment of solid lung-specific tumors. After determining normal tumor growth and metastatic patterns, targeted RT will be utilized to capture tumor volumetric response to therapy. Aim 2 will identify the spectrum of CH-mutant immune cells that infiltrate the tumor and define the role(s) they play in the tumor microenvironment (TME). Pilot experiments revealed overrepresented tumor infiltration of Tet2-mutant monocytes. I will investigate these target cell populations by conditional activation and deletion in genetic mouse models to address if specific, mutant cell populations enhance tumor development. In parallel, tumor infiltrating monocytes will be evaluated for aberrant transcriptional statuses and could provide attractive targets to mitigate solid tumor growth/resistance to therapy. Aim 3 will evaluate the cytokine profiles within solid tumors and how this environment uniquely shifts in the presence of Tet2-mutant hematopoietic cells. Pilot studies reproduced known aberrant cytokine levels induced by TET2 CH in humans, with TNFα emerging as a highly upregulated cytokine which further proved amenable to pharmacological reduction in preliminary experiments. The primary focus will be on the impact of TNFα neutralization on tumor burden, while a comprehensive cytokine profiling will ensure no potential therapeutic targets are overlooked. This project will address a knowledge gap that affects a large patient population who develop solid tumors with a background of CH mutations. The results of this research proposal will identify new insights into the function of CH-mutant cells and how they drive solid tumor development which could unveil pivotal mechanisms offering new avenues for targeted therapies and intervention strategies.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT We have shown that elective nodal irradiation (ENI) blunts systemic immunity in preclinical models of head and neck cancer (HNC), but without it, the risk of regional recurrence in the lymph nodes (LN) is high. Data from clinical trials showed that omission of ENI is feasible, but nodal progression or recurrence does vary by the choice of immunotherapy (IO) that is combined with the radiation (RT). Our previous trial showed that combination aPDL1 (Durvalumab) with tumor-only RT resulted in exceptional high pathological and clinical response rates and no nodal recurrences. However, in two other separate trials, when either another a- PDL1(Atezolizumab) or a-CTLA4 was added to Durvalumab-RT, it resulted in high rates of nodal recurrence leading to trial closures. This suggests that target cell activation or inhibition varies by the type of IO or combination IO used in the context of RT and dictates nodal outcome. Our preliminary data show that trial patient progressive nodes contained a high percentage of Tregs and, preclinically, genetic knockout of PD1 on Tregs increases their activation, which we have previously shown to be driven by STAT3. We demonstrate that nodal metastasis can be prevented by early depletion of Tregs prior to tumor implantation. This is accompanied by enhanced morphological maturation of high endothelial venules (HEVs), specialized postcapillary venules whose function within DLNs has been correlated with immune trafficking and activation. Localized within perivascular niches, and surrounded by Tregs and cancer cells, these HEVs, which express LTBR, are decreased in numbers and maturation on nodal recurrence, a phenomenon that is reversed by LTBR agonists. We also show that ST2 Tregs, a specific Tregs subset, are upregulated with RT and that genetic knockout of ST2 on Tregs results in marked tumor regression. Recombinant administration of IL33 (ST2's ligand), on the other hand, combined with RT and Treg depletion, leads to high rates of eradication of nodal recurrence. We hypothesize that activation of PD1 expressing Tregs in the RT-IO setting contributes to cancer immune evasion directly via STAT3 pathway activation, indirectly through LTA1B2-LTBR Treg-HEV interaction, thereby limiting the Teff infiltration into the nodal environment, and through the ST2-IL33 Treg-stromal interaction, abrogating Teff cells' expansion potential. In Aim1, we will examine the mechanistic impact of immune check point inhibition on Tregs; in Aim2, we will test how Treg-HEV interaction facilitates a tumor- promoting environment within the LN, leading to immunosuppression and cancer cell immune evasion; and in Aim 3, we will determine how ST2 expressing Tregs modulate tumor immune microenvironment in nodal progression. These will be tested using genetically engineered mouse models, multicompartmental mass cytometry, scRNA seq, metabolomic and proteomic analysis, and multispectral immunofluorescence preclinically and in clinical trial specimens from those with and without nodal failure. These studies will collectively elucidate mechanisms of nodal progression with profound impact for therapeutic discovery.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Although tyrosine kinase inhibitors (TKIs) are highly potent in treating malignancies, >60% of them have been reported to lead to adverse cardiovascular outcomes. Moreover, anti-angiogenic TKIs, such as sunitinib, preferentially induce microvascular toxicity followed by cardiac dysfunction. However, our knowledge of the underlying pathogenic mechanisms of TKI-induced vascular toxicity (TKI-VT) has been hampered partially by the limited access to human diseased vascular tissues for molecular and cellular analysis. As such, no effective strategies have been developed to prevent or treat these otherwise life-threatening cardiovascular complications. This proposed project will leverage patient-specific induced pluripotent stem cell (iPSC)-derived cardiac pericytes (PCs) and endothelial cells (ECs) to understand the molecular and cellular basis of TKI-VT and discover personalized therapies for cancer patients who are suffering TKI-induced cardiovascular disease. In Aim 1, both monoculture and vessel-on-chip (VoC) coculture systems will be employed to characterize sunitinib-induced cell type-specific cytotoxicity profiles and aberrant cellular crosstalk between iPSC-PCs and iPSC-ECs that can contribute to TKI-VT. Overlapping upregulated and downregulated differentially expressed genes (DEGs) triggered by sunitinib in both cell types will serve as the candidate genes for large-scale druggable target screens. In Aim 2, CRISPR interference/activation (CRISPRi/a) survival screens will be performed in sunitinib-treated iPSC-PCs using a customized lentiviral sgRNA library targeting the overlapping DEGs identified in Aim 1. Top 10 hit genes in CRISPRi and CRSIPRa machineries will be subjected to structure-based virtual screens (SBVS) to discover candidate compounds that can mitigate TKI-VT phenotypes in iPSC-VoCs. In Aim 3, 3D iPSC- engineered vascular tissues (EVTs) and a mouse model will be used to validate the mitigation efficacy of candidate compounds on TKI-VT in a more physiological setting. The ex vivo plasma proteome generated from human whole blood-perfused 3D iPSC-EVTs will be correlated with those identified in patients to discover reliable disease-relevant biomarkers in predicting individual cancer patients’ susceptibility to TKI-VT. The research and career development training plans during the K99 phase, under the mentorship of Drs. Wu and Ky, as well as an expert interdisciplinary advisory committee, will provide Dr. Shen with advanced knowledge in stem cell biology, vascular biology, cardio-oncology, CRISPR technology, and bioengineering. The development of CRISPRi/a and SBVS screen platforms (K99) and 3D iPSC-EVTs (R00) will enable him to conduct disease modeling and drug discovery research in cardio-oncology specifically and vascular disease in general. The new skills and experience gained during this K99/R00 career development award, combined with Dr. Shen’s prior expertise in vascular biology, will facilitate his transition to an independent career conducting basic and translational research in cardio-oncology with a particular focus on the vascular aspect.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT Since 2020, over 600 million COVID-19 vaccinations and 500 million influenza vaccinations have been administered in the United States. These vaccines have saved millions of lives by inducing the differentiation and maturation of antigen-specific B cells into antibody-secreting cells, resulting in the generation of high-affinity, protective antibodies. However, little is known about how these antibodies could regulate the ongoing B cell response or subsequent responses to vaccination. Notably, a durable B cell response that produces long-lived antibody-secreting plasma cells and poised memory B cells relies on the processes of affinity maturation and clonal expansion, which occur in anatomical structures called germinal centers (GCs). This proposal aims to provide insight into antibodies' role in regulating germinal center responses. This is a particularly important concept for vaccinations that occur in the context of pre-existing humoral immunity, as in seasonal influenza or COVID-19 booster vaccinations. In Aim 1, the hypothesis of antibody feedback on vaccine-elicited GC B responses will be tested in a newly generated mouse model where IgG1 antibody secretion can be inducibly blocked. The GC response's quality, magnitude, and duration will be characterized in primary and heterologous boost systems. In Aim 2, the same overarching hypothesis will be examined in a human rabies vaccination model. Here, the impact of polyclonal rabies immunoglobulin administration on vaccine-induced GC B cells will be examined for epitope specificity, magnitude, and kinetics changes. Additionally, GC products, long-lived memory B cells, and bone marrow plasma cells will be characterized in the presence and absence of exogenous antibodies. The overall goal of this proposal is to test hypotheses of antibody feedback on GC B cells in complementary systems that combine unique mechanistic probing in mice with authentic human vaccine responses. These findings will have broad public health implications for the millions of vaccinations administered yearly in the United States. During the fellowship, the applicant will acquire fundamental skills that will serve as a steppingstone toward becoming an independent investigator in vaccine research. This will include developing expertise in both rodent and human vaccination models. As described in the proposed training plan, the applicant will be supported by Drs. Ellebedy and Diamond who have demonstrated experience in B cell vaccine responses and mentorship of scientific trainees. With Washington University's track record of excellence in training immunologists and physician-scientists, the applicant will be well-equipped for success in their training towards becoming a physician-scientist.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Bacteriophages (phages) are bacterial viruses that can influence human health by modulating bacterial populations. As such, phages have been associated with a number of diseases linked to bacterial dysbioses like inflammatory bowel disease. The number of known DNA and RNA phages has exponentially increased by more than 10,000-fold over the past 10 years due to rapid advances in metagenomics. However, since this culture- independent method does not preserve phage-host relationships, the hosts of many phages are unknown. This prevents experimental investigation of the mechanistic connections between phages, bacteria, and human disease. A number of phage-host pairing tools are available but lack the throughput and efficiency to rapidly screen thousands of bacterial cells in complex communities like the human enteric microbiome. The overall goal of this proposal is to define phage-host pairs in the context of complex communities. I will achieve this goal by establishing an innovative, high throughput phage-host pairing tool for complex, naturally occurring communities. Specifically, I will use an innovative strategy based on prosingle-cell (sc) RNA-sequencing (scRNA-seq) to rapidly screen complex communities for individual cells carrying phage-derived RNA transcripts, which are generated by DNA and RNA phages. However, current prokaryotic scRNA-seq protocols are not designed for profiling phage infections or multi-species scenarios. My preliminary data demonstrate that I can successfully recover the transcriptomes of noninfected and phage-infected bacterial species in a 10-species mixture using a scRNA-seq protocol originally intended for noninfected monocultures, which has not been previously shown in the literature. Therefore, to establish and implement a scRNA-seq-based, phage-host pairing tools for complex communities, I will first (Aim 1) adapt a published prokaryotic scRNA-seq protocol to identify RNA and DNA phage-infected cells in a synthetic community of bacteria representative of the human enteric microbiome to demonstrate proof of concept. (Aim 2) I will then apply my optimized scRNA-seq protocol to human stool samples to identify phage- host pairs. I will use multiple approaches to validate a subset of phage-host pairs (Aim 3) identified in Aim 2, including fluorescent in situ hybridization combined with whole genome sequencing and establishment of culture models. Collectively, this work will be instrumental in advancing our ability to functionally study phage-host interactions in the context of human health and disease.

NIH Research Projects · FY 2025 · 2025-06

Project Summary Rotaviruses (RVs) are the leading infectious agent of severe gastroenteritis in infants and young children. RVs encode a small non-structural protein 6 (NSP6) with no known function. Studying NSP6 function is difficult as it is expressed as a frameshifted product from a double-stranded RNA gene segment that also encodes another RV protein NSP5 and the two open reading frames completely overlap. To overcome these hurdles, we developed a robust RV reverse genetics system and generated a recombinant NSP6-deletion RV with all possible NSP6 start codons removed without affecting NSP5 expression. For the first time, we have the opportunity to examine the physiological role of NSP6 in RV replication, pathogenesis, and immunity in organoid cultures and in vivo. In preliminary studies, we found that in neonatal mice, oral inoculation of an NSP6-deletion RV resulted in significantly lower intestinal replication and fewer diarrhea incidences than the parental virus starting at 6 days post infection. Single-cell RNA-sequencing, further validated by multi-color flow cytometry, revealed that NSP6 expression correlated with reduced T cell numbers at the site of RV infection. NSP6 was secreted from RV- infected intestinal epithelial cells and blocked chemotactic migration of T cells in a transwell assay. Our long- term goal is to delineate a novel immune evasion role of RV NSP6 and use the knowledge towards preventing and treating RV infection. In aim 1, we hypothesize that a predicted four-α-helix structure of NSP6 is critical for its immunoinhibitory activities. We aim to: (1) define the key amino acids within NSP6 responsible for secretion and chemoattractant binding; and (2) identify how NSP6 sequence variation between human and animal RV strains regulates virulence and zoonotic infection. We expect that critical mutations in α-helices will disrupt secretion and that truncated NSP6 proteins from porcine and attenuated human RV strains cannot functionally replace the full- length murine RV NSP6 protein in vivo. In aim 2, we hypothesize that RV NSP6 is a novel virulence factor that acts as a molecular mimicry of chemokine receptors and inhibits T cell recruitment. We aim to: (1) characterize the effect of NSP6 on systemic and local intestinal CD4+ and CD8+ T cell responses; (2) identify the migratory patterns of T cells towards RV-infected epithelial cells by two-photon microscopy; and (3) determine the immunogenicity and protective efficacy of an NSP6-deletion RV as a vaccine candidate. We expect that NSP6 is secreted basolaterally from infected intestinal epithelial cells and suppresses T cell chemotaxis. We also expect that immunization with an NSP6-deficient RV will induce cross-reactive T cell responses and confer heterotypic immunity against subsequent challenges. By interrogating RV NSP6-T cell interactions, we will gain new information that aids in development of improved live-attenuated RV vaccines and design of novel therapeutics that dampen undesired T cell-mediated inflammation in the intestine.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract Adipose tissue (AT) is a complex metabolic organ that functions to protect other tissues from lipotoxicity during overnutrition. It does this by storing lipids and modulating systemic metabolism though the release of adipokines. In obesity, AT becomes highly dysfunctional, leading to ectopic deposition of lipids in other tissues, systemic inflammation, insulin resistance, and oxidative stress. For this reason, dysfunctional AT is thought to contribute to most comorbidities of obesity including the development of type 2 diabetes and cardiovascular disease. From the discovery of leptin almost 30 years ago we have come to appreciate the robust role that adipokines have in inter-organ crosstalk to regulate metabolism in physiology and pathophysiology. Recently, us, and others, have demonstrated a new language that adipocytes use to communicate with other organs, extracellular vesicles (EVs). The language of EVs is powerful as these membrane-enclosed vesicles can carry any macromolecule including miRNAs, RNA, or enzymes, resulting in robust functional modulation of receiving cells at all levels of regulation, transcription, translation, and signaling. In the obese state, AT produces more EVs that, in vitro, propagate disease signals like inflammation and insulin resistance to other cells. The field of EVs in metabolic regulation is still in its infancy. In our published work we demonstrated that energetically stressed adipocytes release pieces of damaged mitochondria in EVs (mitoEVs) that enter circulation and have the capacity to respire and produce ATP. Adipocyte mitoEVs target the heart inducing mild oxidative stress, which is not pathogenic during acute exposure. Instead, a single injection of mitoEVs preconditions the heart to protect it from lethal levels of free radicals generated during ischemia/reperfusion. However, the preliminary data in this proposal suggests that long term treatment of mice with EVs from energetically stressed adipocytes has a negative impact on systemic metabolism, which may promote cardiac pathology during chronic obesity. Therefore, this proposal aims to understand the fundamental regulation of adipocyte-derived mitoEVs and if circulating mitoEVs become maladaptive over time, eventually contributing to cardiovascular disease. Ultimately, our goal is to determine if adipocyte EVs and mitoEVs are a viable target for therapeutic intervention. To do this we will focus on 2 aims: 1) Determine how the production and release of adipocyte mitoEVs is regulated under energetic stress and 2) Establish the consequences of chronically circulating adipocyte mitoEVs on obesity-related cardiometabolic disease in vivo.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT / PROJECT SUMMARY Xylazine is an alpha 2 adrenergic receptor agonist with sedative and analgesic properties which is increasingly being found in the US illegal drug supply and linked to numerous adverse health outcomes including overdose deaths, skin ulcerations and infections. Public health efforts are limited by a lack of data on the type of illicit controlled substances that have been adulterated by xylazine, while at the same time clinicians face an increasing number of patients presenting with large painful skin ulcerations, many of which are complicated by necrotizing infections or underlying osteomyelitis. However, it is unclear to what degree these xylazine related complications are the result of infections due to pathogens directly injected from a contaminated drug supply, compared with secondary superinfections from skin colonizing microbes of vasoconstriction mediated chemical injuries. Our proposed grant application seeks to fill this critical gap in the literature. In aim 1 we will conduct active surveillance of the local illicit drug supply in St. Louis Missouri using mass spectrometry and microbiologic cultures of syringe residue. This will allow us to identify the full spectrum of illicit drugs associated with xylazine while also performing ongoing surveillance for the potential emergence of any other novel adulterants. Microbiologic analyses of syringe residue will allow us to determine if xylazine contaminated illicit drugs support increased levels of bacterial contamination compared to other illicit drugs which might explain the high degree of invasive infections associated with this novel adulterant. Aim 2 will consent hospitalized patients with xylazine related invasive infections and compare bacterial pathogens isolated from clinical specimens with skin colonizing microflora and bacterial and fungal species isolated from xylazine containing syringe residue collected in Aim 1. By comparing core genome single nucleotide polymorphism (cgSNP) distances we will determine if clinical sites of infection are the result of bacterial superinfection from skin colonizing organisms or arose directly from bacterial species innoculated along with xylazine containing illicit drugs. The work proposed in this application has the potential to directly impact public health and patient care. A clear understanding of what pathogens people who use xylazine are routinely exposed to, and an increased understanding of the etiology of xylazine related infectious complications is urgently needed to guide public health policy and target harm reduction interventions.

NIH Research Projects · FY 2026 · 2025-06

Solving the puzzle of beta-1,6-glucan in the cryptococcal cell wall ABSTRACT Cryptococcus neoformans is a global pathogen responsible for roughly 150,000 deaths each year, mainly in HIV+ individuals but with increasing morbidity in non-AIDS patient populations. Like other fungi, C. neoformans is bounded by a complex and resilient cell wall, which maintains cell integrity while allowing morphologic flexibility. Wall synthesis is a compelling research area, because it is a validated target of antifungal drugs. Unfortunately, these do not work against C. neoformans infection, revealing a pressing need to elucidate the biosynthesis - and potential vulnerabilities - of the cryptococcal wall. Our long-term goal is to define cell wall biosynthesis in C. neoformans, to deepen our understanding of this critical process and potentially identify targets for antifungal therapy. Much of the wall is composed of various polymers of glucose, or glucans, which differ in their linkage and branching patterns. Our focus here is on β-1,6- glucan, which is a key component of cell walls because it helps interconnect other wall components but whose synthesis is poorly understood. This material is particularly abundant in C. neoformans, even though the crypto- coccal genome does not encode homologs of several proteins that have been implicated in its synthesis in other yeast. We have discovered that cryptococcal cells lacking a putative glycosyltransferase called Ggt2 are dra- matically impaired in wall β-1,6-glucan, cell integrity, and virulence. Based on our preliminary results and struc- tural features of this protein, we hypothesize that Ggt2 acts directly in the formation of β-1,6-glucan. To pursue our promising initial findings, we propose to determine how Ggt2 affects cryptococcal wall structure, its cellular role, and its biochemical activity. We further propose to identify the C. neoformans machinery required for β-1,6-glucan synthesis. We plan to achieve these goals by pursuing three aims in parallel: Aim 1 is to combine biochemical and biophysical methods to define the cell wall of wild-type and ggt2∆ cells. Aim 2 is to establish the biochemical function of Ggt2 and its role in cell wall synthesis. Aim 3 is to apply genetic strategies to discover proteins that either participate in β-1,6-glucan synthesis or compensate for its loss, and to initiate mechanistic studies of these candidates. We propose highly feasible studies in pursuit of aims that are designed to be independent yet complementary. Our results will increase fundamental understanding of the cryptococcal wall and processes that are crucial for its integrity, and possibly open the way to new therapeutic strategies. Achieving our aims will also leave us poised for exciting future studies, including further structural studies of fungal walls, potential screens for inhibitors of Ggt2, and mechanistic dissection of β-1,6-glucan synthesis with potential application to other fungi.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract Cardiac contraction is a tightly regulated process, where the heart needs to generate sufficient power to perfuse the body during systole and then relax to allow for filling of the ventricles during diastole. Disruption of this regulation can lead to diseases including cardiomyopathy and heart failure. At the molecular scale, cardiac muscle contraction is powered by myosin molecular motors pulling on thin filaments consisting of actin, troponin, and tropomyosin. The troponin complex, comprised of troponins I, T, and C, plays a central role in regulating contraction by positioning tropomyosin to enable or disable the calcium-dependent interactions between myosin and thin filaments. Altering the sequence composition of troponin subunits has direct effects on its function, and point mutations in troponin are prominent causes of cardiomyopathies; however, it is still a major challenge for the field to connect how changes in sequence affect troponin's functio n. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of the troponin complex; however, there are large, unresolved segments of troponin, which include critical phosphorylation sites and the locations of many pathogenic mutations. These unresolved yet functionally-significant regions likely contain intrinsically disordered regions (IDRs) with behaviors that are not well described by traditional structural approaches. Advances in the study of protein disorder have revealed new paradigms connecting sequence, disorder, and function, and it clear that sequence changes in IDRs could impact function by disrupting binding motifs, altering IDR dynamics, tuning conformational biases, introducing new aberrant interaction sites, and/or affecting interactions with binding partners. Our central hypothesis is that mutations and key post-translational modifications within troponin's IDRs alter: (1) intramolecular interactions, (2) intermolecular interactions, or (3) both. Changes in intramolecular interactions will alter intrinsic IDR conformational biases and/or dynamics, while changes in intermolecular interactions will alter interactions between troponin subunits and/or the well defined troponin binding partners: calcium, tropomyosin, actin, myosin. Here, we have assembled a team with expertise in the sequence and simulation of IDRs (Holehouse), single-molecule structural dynamics of IDRs (Soranno), and cardiac troponin (Greenberg) that will apply cutting-edge experimental and computational tools to study the connections between sequence, disorder, and function in key IDRs within troponin. Together, we will test the following specific hypotheses: (1) Regions predicted to be disordered within troponins T and I are indeed disordered in isolation and in the context of the thin filament. (2) Both phosphorylation and pathogenic mutations within troponin IDRs lead to interpretable/predictable changes in the underlying structural ensemble, conformational dynamics, and/or intermolecular interactions with well-established binding partners, which in turn alter thin filament activation. (3) Applying integrative tools across scales will enable us to better predict whether key VUS within IDRs are likely pathogenic or benign. These studies will provide new insights into the biophysical properties of IDRs and the structure-function relationship in troponin. Moreover, these studies have the potential to aid in the classification of newly identified variants of unknown significance found in patients with cardiomyopathy. .

NIH Research Projects · FY 2025 · 2025-06

Our research focuses on understanding how long noncoding RNAs (lncRNAs) promote non-small cell lung cancer (NSCLC) to address the critical need for improved biomarkers and targeted therapies. Lung cancer is currently the leading cause of death worldwide, accounting for more than 150,000 deaths a year in just the United States. Approximately 85% of lung cancer patients have NSCLC and over 50% of these patients present with metastasis at their initial diagnosis. Historically, NSCLC research has primarily focused on the deregulation of protein-coding genes to identify oncogenes and tumor suppressors as potential diagnostic and therapeutic targets, thereby under-representing the emerging roles of long non-coding RNAs (lncRNAs). To address this critical knowledge gap, we focused on the discovery and functional characterization of novel lncRNAs that promote tumorigenesis and aggressive phenotypes in NSCLC patients. Through a pan-cancer computational analysis we discovered that the expression of our recently discovered oncogenic lncRNA, RNA associated with metastasis 11 (RAMS11), was elevated in NSCLC patients harboring mutations in the NRF2 signaling pathway (NRF2 activating mutations and KEAP1 inactivating mutations). Our preliminary data shows: (1) RAMS11 promotes aggressive phenotypes, (2) NRF2 transcriptionally activates RAMS11 by binding to its promoter region, (3) RNA-Seq analysis of models manipulating NRF2 and/or RAMS11 revealed overlapping downstream regulatory programs, (4) NRF2 and RAMS11 interact, and (5) NRF2 transcriptional regulation of canonical targets was dependent on RAMS11 expression. Notably, while NRF2 is dependent on RAMS11 to transcriptionally regulate canonical target genes activated in lung epithelial cells and NSCLC cells, we also demonstrated that RAMS11 may mediate a cancer specific NRF2 regulatory program. Collectively, this serves as a strong rationale for our hypothesis that RAMS11 is necessary to confer NRF2-dependent oncogenic phenotypes in NRF2/KEAP1 mutant NSCLC lung cancer patients. Here we will determine the role of RAMS11 dependent NRF2 transcriptional regulation of cancer-specific, non-canonical target genes and evaluate how RAMS11 interacts with NRF2. Our proposal will significantly advance the lncRNA tumor biology field by providing the first evidence of RAMS11-dependent NRF2 regulation to confer oncogenic phenotypes in vitro and in vivo.

NSF Awards · FY 2025 · 2025-06

Rainfall patterns in Central America and northern South America are changing as global climate warms, and are associated with disease outbreaks such as Dengue fever and agricultural losses from drought and flooding. The shifts in rainfall have previously been attributed to migration of the Intertropical Convergence Zone (ITCZ), a band of clouds and rain around the equator that tracks the Sun north and south through the seasons. However, it is becoming clear that the observed rainfall patterns can not be fully explained by ITCZ migration. Drivers of the change in rainfall are key uncertainties in future climate projections in this region and, consequently, the projected impacts on human livelihood and safety. This project will use sediments collected from lakes in Guatemala and Venezuela to reconstruct rainfall through the last 10,000 years by measuring the hydrogen isotope ratios of leaf wax molecules, which originate from the surrounding vegetation and reflect the local rainfall. These data will be synthesized with other climate datasets, and the spatial pattern in rainfall and isotopes will be used along with climate models to investigate the contributions of a variety of atmospheric phenomena. The foundation of this research project is investigation of the rainfall patterns in Earth's climate zones. These foundations map to many of the Missouri grade 6-8 learning standards, for which science curriculum resources are scarce. This project will partner with teachers to identify needs and co-develop, field test and improve curriculum, and disseminate materials across the Midwest. The goals of this project are to reconstruct Holocene hydroclimate and vegetation change in the Central America, northern South America (CANSA) region with leaf waxes from six previous collected and dated sediment cores; synthesize the multiple proxy records from these cores to identify spatiotemporal patterns in centennial, millennial and Holocene variability; analyze new and existing model simulations for the dynamics that drive rainfall changes; and compare the data and model simulations to identify mechanisms of rainfall change in the Holocene. The Broader Impacts are to develop grade 6-8 climate science lesson plans in partnership with teachers and disseminate the resources through the Midwest Climate Collaborative's educator network and design a lab for an undergraduate class. 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.