Stanford University

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT: The overwhelming majority of cancer-associated deaths are caused by cancer metastasis rather than primary tumors. Cancer cells can metastasize, while normal cells cannot. This is due to unique characteristics that cancer cells acquire as they progress and become more aggressive. Cancer cells undergo genetic mutations that allow them to detach from the primary tumor, migrate through surrounding tissues, and enter the bloodstream or lymphatic system. Detached cancer cells must resist anoikis - a form of programmed death that occurs when cells detach from the extracellular matrix. In the absence of cell adhesions that provide survival signals for anchored cells, metastatic cancer cells must acquire special traits that enable them to persist and survive during invasion and in circulation. In this proposal, we aim to develop an effective therapy for solid tumors by targeting a survival characteristic that is newly discovered and crucial for cancer cell survival during metastasis. Very recently, we identified nanoscale membrane curvature as such a trait that is crucial for cancer cell survival in suspension but not when cells are in contact with substrates. Membrane curvature is a physical property of the lipid bilayer, but it is now recognized as an important and active constituent of biological processes. Curved membranes create distinct microenvironments that facilitate or inhibit the recruitment of specific proteins involved in signaling pathways. The discovery that the survival of cancer cells in suspension hinges on their nanoscale membrane curvature, and that this curvature facilitates essential survival signals through curvature-induced kinase activation, opens up new therapeutic avenues. In this proposal, we seek to unravel the molecular mechanisms underlying curvature-induced kinase activation, to explore the function of curvature-induced kinase activation in cancer metastasis, and to develop new therapeutic strategies by blocking curvature-induced kinase activation. Through these studies, we hope to develop curvature-blocking approaches that selectively kill metastatic cancer cells when they are detached from their surroundings without affecting normal cell survival when they are anchored to the basement membrane and other cells. These studies could lead to new treatments for combating metastatic cancer, addressing one of the most critical and challenging aspects of cancer therapy.

NIH Research Projects · FY 2025 · 2025-09

Transcriptional regulation and resulting development and health/disease states are shaped by the underlying 3D organization of the genome. Recent advances in single-cell genome-organization techniques (including Optical Reconstruction of Chromatin Architecture (ORCA) developed in the Boettiger lab), in parallel to recent theoretical models, have suggested the 3D genome is both flexible and dynamic. Thus, to understand the 3D-genome and its contribution to transcriptional regulation, we need kinetic data from live microscopy. Despite considerable recent growth of chromatin live imaging, substantial limitations in spatial and temporal resolution and difficulties in inserting required chromatin labels constrain the available kinetic data. Here, we propose a new generation of live-imaging approach to study 3D-genome motion, we call Transposon Accelerated Chromatin Kinetic Imaging Technology (TRACK-IT), which combines innovations in 1) ultrabright, miniaturized DNA labels, 2) efficient transposon-based tiling of target loci, and 3) optimized high-speed live microscopy. Our preliminary TRACK-IT data demonstrates ~10x improvement in spatial resolution, ~100x improvement in temporal resolution in live microscopy and enables rapid construction of >10 cell lines with a pair of optimized fluorescent labels tiling a target locus. We will investigate two model loci, one ‘simple’ and the other ‘complex’, with TRACK-IT, to understand how genomic separation and context shape 3D-genome motion (the frequency, speed, and timescales of genomic interaction). We will further test proposed molecular determinants of 3D-genome motion (cohesin loop extrusion, transcription, topoisomerase, etc) through genetic and pharmacological perturbations of these processes. In Aim 2, we will perform advanced biophysical modeling to simulate previously unexplored high-temporal-resolution 3D-genome motion. In particular, these simulations will determine how different parameters/conditions of the candidate molecular mechanisms can accelerate enhancer-promoter communications, and provide a theoretical framework for interpreting live-microscopy data from Aim 1. In Aim 3, we will expand on our TRACK-IT approach in Aim 1 to investigate the 3D motion of enhancers and promoters with respect to transcription busting, using a variation of TRACK-IT we call TRACK-EP. Critically, this aim will use inducible enhancers from the estrogen response pathway, providing a synchronized “time zero” in which to study how the signal flows from enhancer to nascent RNA, as well as contrast the motion of activated vs. inactive enhancers and promoters. Together, our ultra-spatiotemporal-resolution live-microscopy and biophysical simulations will enhance our understanding of 3D-genome biology and resolve key remaining questions in how the 3D genome regulates transcription.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Conotruncal heart malformations are the most common congenital heart disease. Genes associated with planar cell polarity(PCP) signaling are implicated in the etiologies of conotruncal heart malformations, as PCP signaling is necessary for the coordinated cell movements needed for proper outflow tract development. The broad objective of this proposal is to determine the basic molecular mechanisms of this key developmental signaling pathway, which are currently poorly understood. Establishment of PCP involves sorting PCP-associated proteins into complexes with distinct compositions that are distributed to mark a vector of polarity used for downstream readouts, such as coordinated cell movement. These complexes physically interact between adjacent cells to facilitate alignment of intracellular polarity vectors across whole tissues, aligning with broader axes, exemplified by the proximal-distal axis in the Drosophila wing. Complexes are linked by trans intercellular homodimers of the PCP protein Flamingo (Fmi), with PCP proteins Frizzled(Fz) recruited to one side of the cell-cell junction and Van Gogh (Vang) recruited to the other. Previous work suggests that Fz interacts laterally with Fmi and changes Fmi homodimer affinity to preferentially bind Fmi:Vang complexes. Additional work suggests a novel, adhesion- independent role for Fmi in modulating Fz binding to its downstream signaling partner Dishevelled (Dsh). Fz- Dsh binding is known to be sensitive in a dose-dependent manner to PI(4,5)P2. Thus, this work investigates how interactions between Fmi and Fz drive two key aspects of early establishment of planar cell polarity. I hypothesize that lateral interactions between Fmi and Fz I. promote interactions favoring the formation of intercellular trans interactions with Fmi:Vang complexes, and II. amplify PCP signaling through an adhesion-independent, PI(4,5)P2-mediated feedback loop that recruits cytosolic signaling partners. Aim 1 will characterize the asymmetric interactions between Fz:Fmi and Fmi:Vang complexes by reconstituting the proteins into a lipid- nanodisc system, using total internal reflection fluorescence (TIRF) microscopy to measure asymmetric binding affinity, and using cryogenic electron microscopy to identify structural determinants of asymmetry. Aim 2 will characterize a potential PI(4,5)P2-mediated feedback loop by characterizing PI(4,5)P2 availability, determining the Fmi-dependence of PI(4,5)P2 levels near PCP complexes, examining whether recruitment of Skittles, a lipid kinase that produces PI(4,5)P2 can rescue Dsh binding in a Fmi null phenotype, and investigating whether Fmi can directly bind Skittles. This project thus sets the stage for the development of a detailed mechanistic understanding of PCP, which is essential for understanding normal heart development as well as the origins of conotruncal heart disorders. The applicant will receive additional training from expert mentors in membrane protein biochemistry and cryogenic-electron microscopy, in single-molecule biophysics, and training in Drosophila genetics, building an excellent foundation for an independent research career working on the molecular mechanisms that underpin human development and physiology.

NIH Research Projects · FY 2025 · 2025-09

Progress in translational microbiome science has been slow, despite the clear importance of the gut microbiome on human health and tremendous investment. Lack of understanding of the small intestinal microbiota is a primary obstacle—the small intestine (SI) is where nutrients are absorbed, the interface for interaction between microbes and the mucosal immune system, and the site for rising disorders such as Crohn’s and small intestine bacterial overgrowth (SIBO). The SI is a highly distinct environment from the colon, yet the human SI microbiota is poorly characterized due to our reliance on analyzing feces, which is easily accessible but inadequately represents the SI. The natural hypothesis that SI regional specificity arises from distinct microbial physiologies remains untested due to lack of SI-focused resources. Here, I lay out an ambitious roadmap to close this knowledge gap and enable engineering of therapies to improve human health through the SI microbiota. These efforts include a comprehensive species atlas that deconstructs the regional specificity and function of SI-resident microbes, a synthetic community that resists pathogen colonization in the SI, and capabilities for engineering the SI microbiota using phage editing and location-specific delivery. Our recent work has successfully reduced the major obstacles to studying the SI microbiota: regional sampling, microbial and phage isolation, and synthetic community design strategies. We have developed a revolutionary capsule device for safely and non-invasively collecting luminal samples from specific gut regions of humans during normal digestion. Our findings highlight the frontier nature of the SI: the microbiome, virome, metabolome, and host proteome differed dramatically from that of feces, and bile acids displayed region- specific signatures correlated with the abundance of specific species. To reduce the labor of strain isolation, we developed a cell sorting protocol to assemble personalized isolate libraries that is fast, inexpensive, and effective for even strict anaerobes. Finally, we used an iterative, ecology-based “backfill” process to develop a near-complete defined community as a model of the fecal microbiota. Using these tools, we aim to generate comprehensive strain libraries of bacteria from specific regions of the intestinal tract across donors with a wide range of diets and disease states. These personalized strain libraries will enable systematic functional characterization, genetic engineering, and utilization in therapies such as fecal microbiota transplants. We will construct diverse synthetic communities that capture microbiota function in both healthy and diseased humans throughout the intestines, not just the colon. Finally, we will generate phage libraries to enable precise editing of SI communities, and devices that enable precision delivery of specific microbes targeted locations. These efforts will refocus my lab’s expertise in bacterial physiology, imaging, systems biology, and big data toward a major scientific and societal challenge and lead to a new therapeutic paradigm: knowledge-based and probiotic solutions centered on SI-specific microbes.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Maintenance of gut epithelial homeostasis requires signals from a specialized set of RORγt+ innate lymphoid cells (ILCs) that reside in mucosa. RORγt+ ILCs directly regulate the gut barrier by secreting cytokines that activate epithelial host defense mechanisms and reinforce gut barrier integrity. Among the RORγt+ ILCs, there exists a set of phenotypically diverse cells characterized by distinct patterns of cytokine secretion, transcription factor expression, and physical distribution. Although a comprehensive understanding of the diversity of RORγt+ ILCs is required to mechanistically determine how to manipulate these cells, the full spectrum of ILCs remains to be examined. Our objective is to characterize the diversity of ILC subtypes, and define their development, function, and spatial regulation. Our long-term goal is to exploit mechanisms that control distinct innate lymphocyte subtypes to selectively manipulate these cells in vivo and restore mucosal homeostasis after intestinal dysregulation. We identified a specialized population of RORγt+T-bet+ ILCs that exhibited distinct patterns of expression of the chemokine receptor CCR6 compared to related ILCs. We also found that this chemokine receptor expression is associated with enhanced secretion of epithelial cell-active cytokines and was strongly regulated by genetic variation. We hypothesize that genetic variation in mouse strains results in distinct patterns of spatially distributed ILCs in tissue, which alter the capacity of RORγt+ ILCs to secrete mucosal cytokines. In Aim 1, we will investigate the developmental pathway of these newly described ILCs and identify genetic mechanisms regulating their expression of CCR6. In Aim 2, we will determine the impact of CCR6 on tissue localization of natural ILCs and their contribution to barrier immunity. These studies will provide insight into how diverse configurations of innate immune cells caused by genetic variation can shape underlying mucosal fitness.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Abdominal Aortic Aneurysm (AAA) repair is typically recommended when the aneurysm diameter reaches 5.5 cm, based on studies that primarily included men. Women, however, face a fourfold higher risk of AAA rupture at smaller diameters and nearly double the mortality following surgery. This discrepancy raises critical questions about whether women should undergo elective AAA repair at smaller diameters than men to improve survival and quality of life. The WARRIORS trial (Women’s Aneurysm Research: Repair Immediately Or Routine Surveillance) is an international, randomized controlled trial designed to address this issue by comparing early elective endovascular aortic repair (EVAR) with routine surveillance in women aged 50+ with small (4.0-5.4 cm) asymptomatic AAAs suitable for EVAR. The trial will enroll 1,112 women globally, including 350 in the United States, with 1:1 randomization stratified by age, country, and aneurysm size. The primary outcome is AAA-related mortality and rupture over five years, while secondary outcomes include quality-adjusted life years (QALYs), operative mortality, loss of EVAR eligibility, and cost-effectiveness. The trial’s innovative design incorporates registry-supported data collection, novel electronic health record screening for patient identification, and extensive patient and community engagement to ensure broad representation and minimal loss to follow-up. The trial meets criteria for NHLBI’s strategic goals and objectives i.e. Objective 3: Investigate factors that account for differences in health among populations. By determining whether women should have different criteria for AAA repair, the WARRIORS trial aims to establish sex-specific clinical guidelines, ultimately advancing health equity and improving outcomes for women with AAAs.

NIH Research Projects · FY 2025 · 2025-09

Aging is associated with structural and functional changes in the heart, a condition commonly referred to as cardiac aging, which is a critical risk factor for cardiovascular diseases. These changes, including fibrosis, hypertrophy, impaired autophagy, and mitochondrial dysfunction, significantly increase the risk of cardiovascular disease. One key process affected by aging is mitophagy, a specialized form of cellular recycling that clears damaged mitochondria, the energy-producing components of cells. A decline in mitophagy leads to mitochondrial dysfunction, making the heart less adaptable to stress and more prone to damage. Emerging evidence suggests that RNA alternative splicing—a cellular process that generates different isoforms of proteins from a single gene—might play a crucial role in regulating mitophagy. Despite its potential significance, little is known about how changes in RNA splicing influence mitophagy, particularly in the aging heart. Preliminary analysis of long-read RNA sequencing has identified significant changes in mitophagy- related gene isoforms in the aging heart. Additionally, using enhanced RNA interactome capture, I identified RNA-binding proteins (RBPs) with significant changes in expression in the aging heart, including components of the spliceosome machinery. This project aims to uncover how alternative splicing regulates mitophagy and identify potential therapeutic targets to combat cardiac aging. Aim 1: Using human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), I will study the role of spliceosome machinery components in mitophagy. Mitophagy indicators, such as mito-Keima and GFP-LC3, CRISPR/Cas9 genome editing technology, along with electron microscopy and long-read RNA sequencing, will be used to investigate how RBPs regulate mitophagy. This aim could position RBPs as promising therapeutic targets to restore mitophagy in aging hearts. Aim 2: I will explore novel splicing isoforms of mitophagy-related genes using iPSC-CMs. This aim will investigate how these novel isoforms impact mitochondrial fission and mitophagy and how their manipulation could restore mitophagy in aging hearts. By uncovering the molecular mechanisms linking RNA splicing to mitophagy, this study could pave the way for innovative therapies to rejuvenate the aging heart and reduce the burden of cardiovascular diseases.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Lower urinary tract symptoms (LUTS), including daytime incontinence, frequency, urgency, and dysuria, affects up to 1 in 5 children, leading to decreased quality of life, increased parental stress, child embarrassment, urinary tract infections, and persistence of symptoms into adulthood. Regular bladder emptying (every 3 hours) effectively improves symptoms. However, students, parents and teachers report that the school setting is an obstacle to this behavior due to individual factors (e.g., knowledge of bladder health, perceptions of bathrooms) and bathroom environments (e.g., dirty bathrooms). Even though children spend a substantial time of their day in school, school-level factors that influence bathroom use and LUTS have not been studied in a U.S. school setting. Addressing factors that influence bathroom use in schools presents a valuable opportunity for early intervention in the disease lifecycle. My long-term goal is to gain skills and experience to transition to an independently funded investigator and leader who will pioneer impactful behavioral interventions to treat and prevent LUTS, reducing the burden of disease at a population level. The overall objective for this K23 proposal is to investigate environmental and individual factors associated with school bathroom use and use a community- engaged research approach to develop and pilot test the acceptability and feasibility of a behavioral intervention to promote school-day bathroom use to improve LUTS. The central hypothesis is that by identifying and addressing both individual and environmental factors that influence bathroom use in elementary schools, we can improve and prevent LUTS. This central hypothesis will be tested through the following specific aims: 1) the use of an observational tool to determine environmental factors that impact school-day bathroom use in elementary schools, 2) the use of Ecological Momentary Assessment to identify real-time factors that impact elementary student bathroom use during the school-day, and 3) the development and pilot test of the acceptability and feasibility of an elementary school-based behavioral intervention to promote student bathroom use. Completion of these aims will generate preliminary data for a fully-powered clustered randomized control trial that will test the impact of an elementary school-based intervention to promote student bathroom use and reduce LUTS symptoms through a competitive R01 application. This proposal supports my career development and training in mixed methods techniques, developing and sustaining partnerships with schools, clustered randomized control trial design, and advanced statistical analysis, in order to support my growth into an independent investigator.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract The objective of this proposal is to determine the molecular mechanisms by which Lac-Phe, a lactate-derived signaling metabolite, regulate energy balance. Lac-Phe is synthesized from lactate and phenylalanine via CNDP2, and was initially discovered as an exercise-inducible metabolite that suppresses food intake. I recently identified metformin, a widely-used anti-diabetic medicine, as a strong pharmacological inducer of Lac-Phe biosynthesis in both humans and mice (Xiao et al., Nature Metabolism, 2024). Cndp2-KO mice, which are genetically deficient in Lac-Phe biosynthesis, show resistance to the anti-obesity effects of metformin. Additionally, statistical mediation analysis provides evidence for Lac-Phe as a mediator of the metformin- associated weight loss in humans. These preliminary results establish the important role of Lac-Phe in regulation of energy balance. However, critical knowledge gaps remain regarding the locations of Lac-Phe production in vivo and the mechanisms through which Lac-Phe reduces food intake. In preliminary results, I found that gut- specific Cndp2-KO mice phenocopy the global Cndp2-KO mice in terms of both basal and metformin-inducible Lac-Phe production. These results suggest the gut as the primary source of Lac-Phe biosynthesis in vivo. Enteroendocrine cells scatter along the gut and secretes hormones to regulate metabolism. Vagal sensory neurons, which innervate the gut, send signals to the nucleus of the solitary tract (NTS). Considering the gut’s crucial role in feeding control, my central hypothesis is that gut-derived Lac-Phe targets NTS neurons to regulate energy balance. I will test the hypothesis with three specific aims: 1) Determine the contribution of gut-derived Lac-Phe to the anti-obesity effects of metformin and exercise; 2) Determine the crosstalk between Lac-Phe and GLP-1 through a fatty acid receptor, FFAR2; 3) Determine the role of NTS neurons as downstream effectors of Lac-Phe’s effect on food intake. I will pursue these aims using a combination of innovative and interdisciplinary approaches spanning physiology, endocrinology, and neurobiology to reveal the neuronal basis of the anorexigenic effects of Lac-Phe. This proposal is significant and impactful because it will determine the mechanisms by which a lactate-derived metabolite affects energy balance. Successful completion of the proposal will functionally expand our understanding of lactate metabolism in energy homeostasis, and reveal a Lac-Phe-mediated gut-to-brain communication pathway. More importantly, this research provide new opportunities for developing treatments, a similar comparison of Semaglutide and native GLP-1 to combat obesity, diabetes, and other metabolic disorders. My career goal is to become an independent investigator to study the tissue crosstalk in regulation of metabolic homeostasis and energy balance. This K99/R00 award presents an outstanding opportunity: the scientific and career goals will be fulfilled by having a multidisciplinary mentor and advisory team, which possesses extensive experience in areas related to the proposed research and in mentoring trainees towards independence.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Ischemic heart disease remains the leading cause of death in the United States. Treating its associated pathologies is complicated by ischemia-reperfusion (I/R) injury, which further damages affected tissue due, in part, to mishandling of mitochondrial Ca2+. While physiological mitochondrial Ca2+ is an important component of cellular metabolism and signaling, aberrant Ca2+ accumulation during I/R injury is a key contributor to apoptotic and necrotic cell death. Targeting the molecular agents mediating mitochondrial Ca2+ extrusion thus poses a promising therapeutic avenue against I/R injury. However, the underlying agents and mechanisms facilitating mitochondrial Ca2+ extrusion remain poorly characterized. Here, TMEM65 is an emerging target implicated in the process of mitochondrial sodium-dependent calcium extrusion (mNCX) as its overexpression was recently reported to enhance cellular mNCX activity. However, the molecular basis of this important function remains to be elucidated. The proposed project will investigate the molecular mechanisms bridging TMEM65 to mNCX. The Research Training Plan proposes an innovative, multidisciplinary approach incorporating reductive biochemistry (Aim 1), structural studies (Aim 2.1), and cell biology (Aim 2.2) to investigate the role of TMEM65 in Na+-coupled Ca2+ transport and its relevance to cellular Ca2+ signaling. In Aim 1, the Applicant will functionally interrogate and quantitatively analyze the activity of purified TMEM65 molecules in a chemically defined, liposomal environment to unambiguously characterize the function of TMEM65. In Aim 2, the Applicant will determine the molecular- basis for TMEM65 function and modulation at the atomic level via cryogenic electron microscopic studies and perform structure-function analyses. Completion of the proposed aims will yield critical insights on mitochondrial Ca2+ signaling and cellular energetics. The Applicant has suitable prior training in protein biochemistry and strong preliminary data to support the feasibility of his proposed work. Additionally, the Applicant will receive further training in membrane protein biochemistry, functional assays, and structural analysis as well as career development skills in scientific communication, project management, and mentorship. The Project Sponsor and surrounding Training Environment are well-suited to provide the guidance and resources necessary for completing the proposed scientific and training aims, which will additionally prepare the Applicant for a career as an independent research scientist. Ultimately, the research proposed here will improve our understanding of cellular physiology and, by furthering our understanding of mitochondrial Ca2+ extrusion, effectively provide a new avenue for the development of therapeutics against I/R injury.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY / ABSTRACT Human neuroscience does not currently have a noninvasive tool to focally modulate activity anywhere in the brain. This is a critical unmet need: Without a focal brain-wide neuromodulation technique, hy- potheses on neural circuit function generated from neuroimaging and behavioral studies remain corre- lational, and circuit-based treatments for central nervous system disease are limited by lack of depth and anatomical specificity. The overall objective of the current proposal is to validate the ability of transcranial ultrasound stimulation (TUS), an emerging noninvasive neuromodulation technique, to target deep areas at high focality in humans, using the visual system as an ideal test-bed. The ex- pected outcome of completing these aims is the delineation of the optimal parameters and spatial specificity of targeting that is possible with this technique. Aim 1 tests TUS parameters, efficacy and focality in the early subcortical visual system (thalamic lateral geniculate nucleus), with concurrent EEG and behavioral measures of image contrast responsiveness. Aim 2 tests TUS efficacy and focali- ty in adjacent (1-2 cm apart) visual category recognition areas in ventral temporal cortex, with EEG and behavioral measures of visual category processing. Additional innovations of this proposal are that it includes measuring and modeling how a sensory input-output function – the contrast response function – is modulated by TUS protocols, and it implements computational linking models to relate TUS-induced changes in visual pathway activity to changes in associated perceptual behavior. The significance of this work is that, if successful, it provides vision researchers with a precise brain-wide tool to causally test hypotheses of visual system function. Moreover, It will lead eventually to safer and more effective circuit-based therapeutics for CNS visual disorders including amblyopia, visual halluci- nations, dyslexia, and visual agnosias.

NSF Awards · FY 2025 · 2025-09

Processes that involve the transport of electrically charged species in fluids consisting of multiple phases, such as liquids and gases, are fundamental in energy production, energy storage, healthcare, and manufacturing. Important examples include energy storage systems such as batteries, hydrogen production, medical diagnostic devices, and manufacturing processes. Despite their importance, accurately modeling these processes remains challenging due to their complex interactions and varied scales ranging from microscopic interfaces to large-scale systems. Addressing these challenges can significantly enhance the performance, efficiency, and affordability of critical technologies, particularly in energy production and storage. This project develops an accessible, advanced simulation framework that enables scientists and engineers to effectively model and optimize these vital electrochemical processes. By simplifying complex computational challenges, the project accelerates innovations across several crucial sectors, benefiting society through improved energy technologies, healthcare applications, and industrial processes. Educational activities and community training are integral components, aimed at increasing STEM participation and training a workforce skilled in cutting-edge technologies. This project develops an architecture-agnostic computational framework called FASTEST (Framework for Advanced Simulation of multiphaSe ElecTrochemical Systems). FASTEST provides scalable, robust, and accurate simulation capabilities for the complex, multiscale dynamics of multiphase electrochemical systems. FASTEST employs a domain-specific language (DSL) to enable domain experts to focus on scientific modeling while computational experts optimize performance and scalability. The framework combines scalable adaptive meshing, implicit numerical methods, and architecture-aware portability, leveraging modern computing resources such as multicore CPUs and GPUs. It addresses longstanding computational challenges in the modeling of electrochemical systems, such as stiff equations, multiscale adaptivity, and implicit solvers, with optimized numerical algorithms and iterative solvers. FASTEST improves computational speed and accuracy compared to current commercial and open-source solutions. The project emphasizes the development of robust numerical methods, scalable parallel algorithms, and a user-friendly interface to facilitate widespread adoption and application. Comprehensive validation, verification, and benchmarking efforts ensure accuracy and reliability, supporting broad applications in energy production, energy storage, manufacturing, and bioengineering. The outcomes advance simulation-based understanding and optimization of multiphase electrochemical systems, fostering innovation across multiple critical technological domains. This project is co-funded by the Office of Advanced Cyberinfrastructure (OAC) and the Division of Civil, Mechanical, and Manufacturing Innovation (CMMI). 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 · 2025-09

Abstract Overdose deaths are on the rise, with 107,941 reported overdose deaths in the US in 2022 alone. Strikingly, 73,838 of those deaths were associated with synthetic opioids, most of which involved fentanyl. Drugs of abuse are thought to “hijack” synaptic plasticity mechanisms and drive molecular, synaptic, and circuit remodeling within the ventral tegmental area (VTA)-nucleus accumbens (NAc) mesolimbic dopamine system. These neuroadaptive responses are hypothesized to underlie the development of addictive behaviors and to promote negative emotional and motivational states during withdrawal through strengthening anti-reward systems. Evidence suggests that there are dissociable circuits within the striatum and NAc that mediate distinct aspects of opioid reward, seeking, somatic withdrawal, and dependence and that each of these may be associated with their own forms of drug-induced plasticity mechanisms. NAc medium spiny neurons (MSNs) are typically characterized by their expression of dopamine receptors, however, the standard dopamine receptor (D)1/D2 classifications do not fully capture the role of MSNs in addiction. Manipulations to these populations based on dopamine receptor expression has led to conflicting and ambiguous results. Single cell transcriptomic data has identified several novel classes of MSNs, however, limited studies on these cell types exist, especially in the context of opioid use. Additionally, single cell sequencing is unable to capture key features of these newly identified subtypes, including topography and input-output organization. This proposal seeks to utilize “Barcoded Anatomy Resolved by Sequencing” (BARseq) to address these technical challenges while elucidating the active cells and circuits during remifentanil taking and seeking. BARseq provides a means to trace thousands of single neurons, providing gene expression and topographical information of the cells in the injection site(s), and producing brain-wide projection patterns. In a diverse, heterogeneous, and anatomically indistinct region with multiple output projection pathways such as the NAc, this technique is essential to characterize the multiple distinct subclasses of MSNs and begin to understand their function in addiction. The studies proposed will provide novel mechanistic insights into the cells, circuits, and neuroadaptive mechanisms that mediate the progression of addiction, with greater precision than previously available, resulting in novel opportunities for developing treatments for addiction.

NSF Awards · FY 2025 · 2025-09

Synthetic biology can be used to create and improve treatments for many diseases. Genetic techniques are used to modify T cells to create CAR-T cells that can kill some types of cancer cells. Gene therapy offers the promise of curing genetic diseases for which there are no treatments. A significant problem with these technologies is the variability of gene delivery to the targeted cells. Viral particles are often used to deliver the desired genes. A cell could be infected by a single viral particle, or many, meaning the cell could receive one or many copies of the gene to be expressed. The variability in the resulting response of the infected cells means the treatment results could vary widely. The objective of this project is to develop protein circuits that can self-regulate. This would remove the effect of infection variability on cellular performance, and thereby even out the therapeutic effectiveness. The reproducibility this would introduce would accelerate the development process for new biotherapeutic strategies. This project will develop dosage-controlled synthetic circuits by implementing proteolysis-based incoherent feedforward loops (IFFLs). The primary goal is to create self-regulating circuits that maintain consistent performance regardless of delivery variations. The approach combines proteolytic regulation with secreted protein engineering. The research will proceed in two phases: first, establishing the technical foundation using synthetic reporters to develop and characterize the basic circuit components, followed by demonstrating functional feasibility using cytokine outputs. An iterative strategy of computational modeling and experimental validation to optimize circuit design and performance will be employed. The methodology includes developing proteolytic control mechanisms, engineering secreted protein systems, and implementing circuit-on-circuit dosage control through careful characterization and tuning of individual components. The experimental approach will systematically progress through several key stages. Initially, individual proteolytic regulatory elements will be designed and optimized, establishing their kinetic parameters and dose-response characteristics. These components will then be integrated into IFFL circuits, carefully validating their function using fluorescent reporters. Computational modeling will guide the design process and help predict circuit behavior under various conditions. The project will advance to testing with therapeutic proteins, specifically cytokines, as output molecules. This phase will include extensive characterization of circuit performance in therapeutically relevant cell types and delivery vectors. 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 · 2025-09

Tuberculosis (TB) is a chronic pulmonary disease caused by Mycobacterium tuberculosis (Mtb), which infects approximately one quarter of the world’s population. A variety of drugs have been identified that rapidly kill Mtb and its relatives in vitro, yet clinical treatment requires at least 6 months of combination therapy and resistance is rampant. Furthermore, the current state-of-the-art for detection of Mtb infection employs cumbersome methods that were developed more than 80 years ago. Herein we propose to develop new methods for detection of Mtb that can be employed in low resource settings, and to develop new screens for potential TB drugs, as well as to perform fundamental studies on the role of mycobacterial lipids in virulence. In this renewal application of R37 AI051622 entitled “Chemical Mycobacteriology”, we propose the following four Aims: (1) to develop probes based on the fluorogenic Nile Red and 3-hydroxychromone dyes, which can be used to detect Mtb with low-power, low-cost microscopes; (2) to establish a magnetic bead- based enrichment platform that can be deployed at the point-of-care to enhance detection of fluorescently labeled Mtb cells; (3) to deploy metabolic labeling as a readout for high-throughput drug screens to decrease time and expense in discovery of new TB drugs; and (4) to employ bioorthogonal labeling and chemical biology approaches to elucidate the role of phthiocerol dimycocerosates (PDIM) lipids in mycobacterial virulence.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY Millions of children in the United States contract respiratory infections such as influenza, RSV, and COVID-19 every year, with those under the age of five affected the most severely1,2. Respiratory infections can have long- term effects on the central nervous system in both adults and children3–11. In fact, currently, 6 million children are suffering from long COVID, and up to 44% of these children experience cognitive impairment3. Childhood is a critical period for synaptic formation and myelination, making the brain particularly vulnerable to insults that can lead to permanent and long-lasting neurocognitive changes12. In rodents, postnatal day (P)14 is a crucial period of peak synaptogenesis and myelination12–14. As a result, during this time, a respiratory immune challenge such as influenza can result in persistent neurodevelopmental dysfunction. While many studies have investigated the neurological effects of systemic immune challenges during the prenatal and adult periods15–18, the impact of influenza infection during early postnatal life remains understudied. The proposed study aims to investigate the effect of influenza infection at P14 on glial dysregulation in mice. The overarching hypothesis is that influenza infection results in multicellular dysfunction driven by aberrant microglia. My preliminary data show that influenza infection at P14 leads to an increase in reactive microglia one-week post-infection in the cingulum and dentate gyrus, along with elevated expression of inflammatory genes in microglia as revealed by single-nuclei RNA sequencing. I will employ advanced transcriptomic analysis to examine changes in glial sub-states, and to identify alterations in synapse/pruning-associated genes (Aim 1A). Additionally, changes in microglia-mediated synaptic pruning will be assessed using Imaris 3D reconstructions to quantify microglial engulfment of synapses following infection. My preliminary findings indicate that influenza infection results in a decrease in OPCs and oligodendrocytes in the cingulum and dentate gyrus one-week post-infection. I will use an established optogenetics paradigm19,20 to stimulate excitatory dentate gyrus neurons and test for changes in activity- dependent myelination following infection (Aim 2). Finally, based on the hypothesis that reactive microglia dysregulate oligodendroglial dynamics, microglia will be depleted between P7-P21 to determine if this rescues the observed loss of oligodendroglial cells (Aim 3). Given my experiences investigating the impact of prenatal environmental immune challenges on microglia and behavior, I am well-prepared to execute these experiments. This work will be conducted under the sponsorship of Michelle Monje, MD/PhD, a leading expert in glial-neuron interactions and myelin plasticity. The co-sponsorship of Catherine Blish, MD/PhD, will ensure comprehensive guidance on virologic aspects, and collaborations with Karl Deisseroth, MD/PhD, Akiko Iwasaki, PhD, and Beth Stevens, PhD, will provide the necessary support to successfully achieve the aims of this project. Stanford's rigorous training environment and the collective support from these experts will ensure the project's success and my growth into an innovative physician-scientist capable of developing therapies for neuroimmune diseases.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Cardiac fibrosis, a hallmark of aging, leads to tissue stiffness, impaired cardiac function, and heart failure, significantly reducing quality of life in elderly populations. Despite its clinical burden, no FDA-approved therapies exist for treating cardiac fibrosis, underscoring an urgent need for innovative solutions. This UG3/UH3 study aims to discover and validate novel antifibrotic compounds by leveraging cutting-edge technologies, including human induced pluripotent stem cells (iPSCs), artificial intelligence (AI), and advanced preclinical models. The UG3 phase will focus on high-throughput screening (HTS) of over 225,000 compounds using iPSC-derived cardiac fibroblast (iPSC-CF) reporter lines to identify lead candidates. The ADMET-AI platform will filter these candidates for favorable drug-like properties and low toxicity. In the UH3 phase, selected compounds will undergo robust in vitro and in vivo validation. This includes testing in “cell villages” for population-scale evaluations and 3D cardiac organoids to assess efficacy and safety in tissue-like environments. The top two hits, including a repurposed drug and a novel compound, will be tested in aging mice to examine their therapeutic potential in restoring cardiac function and reducing fibrosis. This multidisciplinary approach integrates fibrosis research, AI/ML tools, and iPSC technology to address a critical unmet need in aging cardiovascular health. By targeting senescent fibroblasts and employing innovative drug screening platforms, this UG3/UH3 study has the potential to advance the discovery of effective antifibrotic therapies, paving the way for clinical translation and improving outcomes in aging populations.

NSF Awards · FY 2025 · 2025-09

This project aims to fill the gap in foundational knowledge between well-established sampling and estimation methods and Artificial Intelligence (AI)-inspired ones, referred to as ‘generative sampling.’ Generative AI algorithms are capable of producing plausible instances of objects from complex distributions, such as ‘naturally occurring’ sentences or ‘naturally occurring’ images. Rather than learning a probability distribution, these methods typically learn an ‘algorithm’ to generate samples with the desired distribution. This project has two main goals: (1) Determine the fundamental computational and statistical limitations of generative Artificial Intelligence (AI) methods, addressing what the classes of outputs (probability distributions) can and cannot be generated by these methods; (2) Design algorithms to accelerate the generation process. The project also involves training activities in this area through the involvement of undergraduate and graduate students in this research and the development of topics courses. More specifically, the project’s focus is on denoising diffusions, their generalization via stochastic localization, and related approaches. It appears that the scope and limitations of these methods are dictated by subtle properties of the target probability distribution. For instance, it can happen that a distribution can be sampled in polynomial time, and yet reasonable polynomial time generative processes, e.g., all denoising diffusions in a broad class, fail to sample correctly. The project aims to determine fundamental limitations both in terms of computational resources at generation time (computational bottlenecks) and the required sample size to estimate the denoiser (statistical bottlenecks), as well as optimal parallel generation algorithms when efficient generation is possible. 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 · 2025-09

The goal of this project is to use a newly developed, transparent ultrasound transducer together with in vivo optical imaging to study the direct effects of ultrasound on retinal ganglion cell (RGC) activity. Ultrasound stimulation of the eye has been demonstrated to preserve RGCs after optic nerve injury, making it a promising strategy for neuroprotection and vision preservation. Although the physical mechanisms of ultrasound stimulation have been explored in the ex vivo retina, its range of effective parameters in vivo and underlying biophysical mechanisms are not well understood. Due to the optical accessibility of the retina, RGC activity can be noninvasively monitored in vivo using Ca2+ imaging with genetically encoded calcium indicators. However, conventional ultrasound transducers are opaque and will block the light path for Ca2+ imaging by microscopy. To address this limitation, we have developed a novel class of transparent ultrasound transducers that facilitates in vivo Ca2+ imaging while providing ultrasound stimulation. With this new tool, we will study the mechanisms and therapeutic efficacy of ultrasound retinal stimulation. We propose to determine effective parameters for ultrasound retinal stimulation, and test its physical mechanisms and clinical applications for neuroprotection using in vivo 2-photon imaging and animal models. A multi-disciplinary team with complementary expertise is assembled to perform the proposed aims. The team consists of experts in transducer fabrication, ultrasound neuromodulation, retinal physiology and optic nerve disease. Ultrasound is an emerging, noninvasive technology explored for vision-preserving therapies. To apply ultrasound retinal stimulation in a safe and predictable manner, a detailed understanding of the effects of ultrasound on neural activity is required. By using new technology, this project addresses a critical need to understand the effects of ultrasound neuromodulation in the retina and other nervous systems in vivo, and also carries significant translational potential. Information about how ultrasound modulates RGC activity should allow us to develop safer and more effective ultrasound-based therapies for optic nerve disorders and retinal degenerations.

NIH Research Projects · FY 2025 · 2025-09

ComponentAutoimmune diseases comprise a large set of disorders that together afflict almost 50 million Americans; four out of five patients with autoimmunity are women. The mission of the Specialized Center of Research Excellence on Sex Differences in Autoimmunity (SCORE-X) is to serve as a national resource in understanding sex as a biological variable in autoimmunity. SCORE-X has three objectives. Objective 1 is to drive innovation and intellectual leadership of sex biased autoimmunity. Human organoids and spatial proteomics will be employed to dissect the newly recognized role of female-specific XIST RNA protein complex in driving autoimmunity. Objective 2 is to enable translational interdisciplinary advance of sex-biased autoimmunity, focusing on the diagnostic and prognostic opportunities from the recent discovery of anti-XIST RNP antibodies in patients with autoimmune diseases. Objective 3 is to enhance collaborative, and research educational opportunities. SCORE-X will create and coordinate research projects, pilot programs, and educational programs to support the SCORE consortium, and broadly disseminate new insights, technologies, and resources developed at SCORE-X. SCORE-X will promote training and education in sex differences in autoimmunity through the establishment of a Pilot grant program for early stage investigators and integrated educational program along the entire training pipeline. The end result will be a national resource for transformative advance in the mechanistic understanding, clinical translation, and educational opportunities for sex differences in autoimmunity.

NSF Awards · FY 2025 · 2025-09

The goal of this project is construct a rigorous framework for timelike Liouville theory. Timelike Liouville theory is a theory of quantum gravity with a "wrong sign" in the exponent, which makes it impossible to put it in the setting of ordinary probability theory. This project will aim to develop an extension of probability theory that accommodates Gaussian distributions with negative variance, and apply this extension to construct rigorous timelike Liouville theory. This project involves graduate students. While spacelike Liouville theory has received intense attention in the probability community over the last two decades, the timelike theory - which is closer to a true theory of quantum gravity - has remained beyond the reach of rigorous mathematics. The main reason, as in quantization efforts for other theories of gravity, is the appearance of Gaussian integrals with the "wrong sign" in front of the quadratic term. If the quadratic term comes with a positive sign, ordinary measure theory does not give the tools to make sense of it in a way that is consistent with physical calculations. This project will provide a rigorous framework for doing that, which has been outlined in near-complete detail in the project description. The long-term goal of this effort would be to derive the timelike DOZZ formula in the new rigorous setting, which has eluded mathematical understanding until now. 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 · 2025-09

PROJECT SUMMARY Otitis media (OM), or infection of the middle ear, is the one of most common illnesses diagnosed in children in the United States and is the leading reason for pediatric antibiotic prescriptions. Acute OM is most commonly caused by the bacteria Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus, while Pseudomonas aeruginosa is the most common cause of chronic OM. Diagnosis of OM currently relies on otoscopy, which has low sensitivity and specificity and is unable to differentiate bacterial infection from other causes of inflammation. These limitations result in over-diagnosis and over- prescription of antibiotics, contributing to increased antimicrobial resistance (AMR) and subsequent treatment failures. Therefore, there is a significant unmet need for improved diagnostic tools for OM that can enable faster and more effective treatment decisions and reduce the potential for AMR development. Photodynamic therapy (PDT), is a promising antimicrobial treatment option in which light-activated photosensitizers induce cell killing. However, prior applications of PDT to bacteria have been untargeted or used non-covalent methods of targeting, raising the risks of off-target toxicity and resistance development. Covalent targeting of enzyme active sites has the potential to address these current shortcomings and result in probes capable of both imaging and treatment applications. The overall objective of this proposal is to engineer a covalent theranostic probe targeting bacterial D,D-carboxypeptidases (DD-CPases) as a novel tool for imaging and treating bacterial OM using PDT. All five common OM-causing bacteria have a DD-CPase and humans lack homologs. Therefore, the primary aims of this project are to: (1) develop a covalent activity-based probe (ABP) suitable for imaging and treatment of the five primary OM-causing bacteria; (2) demonstrate that a single covalent probe can be used to image and kill the five most common OM-causing bacteria in vitro, and (3) use the top probe to confirm imaging and killing of the two most common OM-causing bacteria in vivo. Success with these aims will result in a strategy for diagnosis and treatment of OM that will reduce AMR development, reduce treatment failures, and prevent progression to chronic OM and its associated complications. In addition, the training plan outlines a comprehensive strategy for career advancement for the applicant, Dr. Emily Woods, under the mentorship of the sponsor, Dr. Matthew Bogyo. Dr. Woods will engage in a variety of seminars, courses, and experiences to develop her scientific and academic skills and enable her transition to independence. Overall, the proposed studies are expected to generate a novel tool for improved diagnosis and treatment of bacterial OM.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract: Catheter embolization is a critical treatment for multiple diseases, including hepatocellular carcinoma, uterine fibroids, renal cell carcinoma, and massive hemoptysis in cystic fibrosis. However, current embolization endpoint assessment is subjective, relying on intermittent X-ray imaging to evaluate blood flow qualitatively in the treated artery. Current X-ray embolization endpoint assessment methods are operator-dependent and not quantitative, leading to potential undertreatment or overtreatment. Our research aims to develop a novel method for quantitative flow assessment within the treated artery, providing a more accurate evaluation of embolization treatment. I propose integrating electrochemical impedance spectroscopy (EIS) sensors into catheters for real-time monitoring of embolization. Catheter sensing of embolization will reduce reliance on high-radiation techniques like quantitative digital subtraction angiography and avoid impracticalities associated with Doppler guidewires and intraoperative MRI flow assessment. In this project, I will miniaturize sensor catheter technology to microcatheters, that are commonly used for embolization and are able to track through small blood vessels. In addition, I will evaluate feasibility of new methods for continuous blood flow determination by the sensor catheters. Upon completion, this project will enhance the clinical translatability and utility of catheter embolization by realizing sensor microcatheters potentially providing continuous blood flow sensing. This innovation will have broad applications in intravascular flow sensing, benefiting fields such as interventional radiology and cardiology, ultimately improving the safety and efficacy of catheter procedures.

NIH Research Projects · FY 2025 · 2025-09

Project summary Converging evidence suggests that dysfunction of the lysosomal system, responsible for degrading cellular waste, is a critical factor underlying neurodegenerative disease and neuronal aging. However, how this age- related impairment manifests in neurons is not well-understood. This proposal will leverage the power of in vivo subcellular imaging of neurons in the nematode Caenorhabditis elegans to investigate the dynamic behavior of neuronal endolysosomal organelles and their aging-related decline. In Aim 1, the applicant Zoe Cook will train with mentor Dr. Kang Shen to characterize in vivo neuronal endolysosomal dynamics across C. elegans aging, using the highly branched PVD sensory neuron as a model. Ms. Cook will first methodically characterize the flow of endolysosomal and autophagic degradative trafficking in a healthy neuron using multiple compartment marker proteins, a pH sensor, and an in vivo protein degradation assay. She will then utilize these tools to define aging-related dysfunction in aged worms and mutants with altered lifespans. In Aim 2, Ms. Cook will investigate the mechanism and function of endolysosomal dynamics in neurons, specifically tubulation, or the extension of elongated membrane tubules. In her preliminary work, the applicant has identified the transmembrane lysosomal protein PIPP-4P, homologous to human TMEM55B, as a negative regulator of age-dependent endolysosomal tubulation in neurons. While TMEM55B is a known mediator of lysosomal transport and stress response, this function has not been previously reported; furthermore, TMEM55B has not been investigated in neurons. Ms. Cook will leverage approaches developed in Aim 1 to determine the mechanism by which PIPP-4P regulates endolysosomal tubulation and then manipulate tubule dynamics in vivo to establish their functional relevance in neurons. The training plan is tailored to enable candidate Zoe Cook to develop subject area expertise in endolysosomal biology in the context of neurodegenerative disease, as well as practical skills for data analysis, scientific presentation, and team leadership. The collaborative and innovative Neurosciences Interdepartmental Program at Stanford University will be an outstanding environment for Ms. Cook to build a strong background in neuroscience research and learn from world-renowned scientists to reach her research and training goals. Mentor Dr. Kang Shen is a leading expert in neuronal cell biology. Dr. Shen's expertise in C. elegans neuroscience will be complemented by consultants Dr. Monther Abu-Remaileh, Dr. Marius Wernig, and Dr. Tom Clandinin, all experts in diverse approaches to investigating lysosomal biology in the context of neurodegenerative disease. The proposed research will generate insights into how the endolysosomal system is dynamically regulated in neurons and where aging-related dysfunction develops, potentially informing future therapeutic approaches. Furthermore, the strong mentoring team, institutional environment, and training plan will fully prepare Zoe Cook for a postdoctoral position, the next step in her journey towards becoming an independent investigator.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Malaria in pregnancy is one of the leading causes of infant death globally. Placental malaria (PM) is the main mechanism by which malaria in pregnancy causes birth complications, such as preterm birth, stillbirth, and low birth weight. During PM, Plasmodium falciparum (Pf)-infected red blood cells sequester to syncytiotrophoblasts (STB) within the placental intervillous space, stimulating maternal immune cell recruitment and leading to other placental pathologic changes. With increasing gravidity, the severity of PM decreases and birth outcomes improve as pregnant women acquire Pf-specific antibodies (Abs) against a variant surface protein – VAR2CSA – after repeated Pf exposures. Previous work shows that neutralizing Abs against VAR2CSA are important for protection but do not entirely prevent neonatal complications, which suggests a key role for Ab-mediated effector functions. Work by our group and others demonstrates that Ab-mediated effector functions are crucial for naturally acquired immunity to malaria in children. We hypothesize that myeloid cell state, phagocyte localization at the maternal-fetal interface, and Ab repertoire adapt with repeated Pf exposure in a gravidity-dependent manner, and that Ab-dependent phagocytosis (ADP) is necessary for limiting PM pathogenesis and improving neonatal outcomes. To test this hypothesis, we will leverage an extraordinary biobank of plasma and placental tissue collected from pregnant women enrolled in the DPSP clinical trial (U01 AI1431308). In Aim 1, using novel spatial proteomic (MIBI-TOF) and transcriptomic (NanoString DSP) imaging approaches, we will determine how Pf parasitemia and gravidity shape myeloid cell - STB spatial relationships that drive Pf clearance in placental tissue. We will test whether, with increasing gravidity, inflammatory maternal myeloid cell infiltration will decrease, and maternal phagocytes will preferentially localize to the Pf-infected STB. In Aim 2, we will utilize in vitro models to determine how gravidity-induced Ab modifications influence Fc-mediated protection from malaria in pregnancy. Together, successful completion of these aims will inform vaccine development and therapeutic strategies to reduce the global burden of malaria in pregnancy.