Stanford University

NIH Research Projects · FY 2026 · 2024-04

Project Summary Dysregulation of RNA-binding protein (RBP)-RNA interactions impacts the coordination of RNA fate with severe implications for human health. RBPs interact directly with RNA processing machinery to modulate RNA localization, degradation, and translation independently of transcriptional regulation. Disruptions in RBPs or their RNA binding sites can lead to fluctuations in oncogene or tumor-suppressor gene expression and malignant changes in cellular homeostasis. Understanding the molecular functions of RBPs in regulating gene expression is then imperative for predicting metastatic progression in cancers with RBP mutant gene signatures. Recent work has led to the discovery of thousands of human RBPs, but much less has been done to systematically describe the function that each protein and its individual domains have on regulating their RNA partners. Additionally, the functional organization of RBPs beyond their distinct and generally well-characterized RNA-binding domains (RBDs) is not fully understood. Until recently, it was assumed that RBPs were effectively non-modular outside of RBDs; however, current studies suggest that RBPs may be composed of separable ‘effector’ domains that interact with processing machinery and promote regulatory activity. I hypothesize that most RBPs contain distinct effector domains that post-transcriptionally regulate protein abundance through interactions with cellular machinery that controls RNA decay or translation. The Bintu lab has developed a high-throughput recruitment assay to recruit 80 amino acid protein portions, or tiles, to a synthetic DNA reporter gene. I propose to extend this methodology to identify functional effector domains within human RBPs, where they also may be more sensitive to oncogenic mutation. First, I will use a high-throughput assay I recently developed to recruit tens of thousands of protein tiles to a synthetic reporter RNA, evaluate their effects on downstream protein expression, and determine the mechanism by which they regulate RNA levels. This will allow me to identify annotated oncogenic mutations that overlap effector domains and likely cause disease by disrupting RBP regulatory activity. Second, it is currently difficult to predict how loss of RBP activity affects a single bound transcript, largely because RBPs bind at different positions and in different copy numbers on each of their multiple partner RNAs. I will quantitatively compare how variation in RBP and effector domain positioning and occupancy along transcripts affects gene expression and eventual different cancer phenotypes. Finally, I will conduct a mechanistic investigation into METTL3, a known RNA-modifying enzyme whose overexpression is believed to drive the development of multiple cancers. I will determine its RNA regulatory potential independent of modification writing and query the direct correlation between RNA modifications and overall expression. This work will be the first to systematically identify functional domains in RBPs and will increase understanding of how dysregulation of RBP function leads to cancer through altered interactions with crucial cellular machinery.

NIH Research Projects · FY 2025 · 2024-04

SUMMARY In the United States, Native American communities face the greatest burden of chronic diseases among all ethnic groups and high rates of cardiovascular disease (CVD) incidence and mortality. Elevated disease risk may be in part attributed to arsenic in drinking water, which is a key environmental risk factor among rural households that rely on private wells. Arsenic-related CVD risk may be modified by the biomethylation of arsenic, a pathway that decreases arsenic toxicity and increases urinary excretion. Arsenic methylation efficiency varies between individuals and populations and is influenced by genetic variation. However, the role of pre- and post- transcriptional gene regulatory factors, including DNA methylation (DNAm) and microRNAs, on arsenic methylation efficiency and arsenic-induced CVD is not fully understood. This study will leverage data and biospecimens representing multiple omics layers from the Strong Heart Study (SHS) and Strong Heart Family Study (SHFS), large, prospective, well characterized cohorts of Native American adults with longitudinal data on CVD outcomes and risk biomarkers. The aims of this project are to (K00, Aim 1) determine the relationship between DNAm, arsenic methylation efficiency, and CVD to identify epigenetic biomarkers of arsenic toxicity and arsenic-related disease risk; (K99, Aim 2) determine the effect of genetic variation on DNAm associated with arsenic methylation efficiency to distinguish molecular mechanisms underlying arsenic methylation phenotypes; and (R00, Aim 3) investigate the role of microRNAs in mediating the association between arsenic exposure and methylation efficiency and CVD risk biomarkers to elucidate molecular processes underlying arsenic-related CVD. To accomplish these aims, Dr. Bozack will be receive mentorship from experts in environmental, molecular, and genetic epidemiology. In the K99 phase, Dr. Bozack will also receive training in bioinformatics and machine learning, including approaches for developing DNAm biomarkers and investigating gene-epigene interactions. In the R00 phase, she will generate circulating microRNA expression data and will further apply her training in clustering and network analyses to identify microRNA signatures linking arsenic exposure and methylation efficiency to CVD risk. The proposed training and research will enable Dr. Bozack to establish an independent research path focusing on biomarker development and applying multiple omics approaches to environmental molecular epidemiology. Furthermore, mentorship and career development activities will facilitate her transition to an independent researcher. Overall, this study will advance the understanding of gene regulatory factors involved in arsenic-related CVD risk through a multiple omics perspective, which is necessary to unravel the relationship between environmental and biological factors involved in the etiology of complex diseases. Findings will contribute the development of noninvasive biomarkers of arsenic-related CVD risk and may aid in targeting arsenic mitigation and public health interventions.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT The microtubule cytoskeleton is a critical regulator of cell differentiation and must be spatially organized by subcellular sites called microtubule organizing centers (MTOCs) to fulfill cellular functions. Since the discovery of MTOCs over 50 years ago, the majority of MTOC research has focused on the centrosome, an organelle that organizes microtubules during animal cell mitosis. However, MTOC function is reassigned to non- centrosomal sites during cell differentiation: non-centrosomal MTOCs (ncMTOCs) form at the apical membrane of epithelial cells, the axons and dendrites of neurons, and the nuclear envelope in skeletal muscle cells, and non-centrosomal microtubules are critical for the development and function of the tissues that these cell types comprise. The generation of non-centrosomal microtubule networks is associated with the simultaneous inactivation of MTOC function at the centrosome and defects in centrosome inactivation are prevalent in epithelial cancer and linked to invasive cell behavior. Thus, a dramatic reorganization of the microtubule cytoskeleton is a fundamental aspect of cell differentiation, and my lab has used support from NIGMS over the last 5 years to uncover mechanisms of ncMTOC establishment and centrosome inactivation. We established C. elegans as an in vivo model of non-centrosomal microtubule organization, identifying and characterizing ncMTOCs at the apical surface of intestinal epithelial cells and at the tips of the dendrites in sensory neurons. By developing tools for tissue-specific degradation and proximity labeling, we found that essential MTOC proteins can vary across subcellular locations and cell types and identified novel ncMTOC components, including the spectraplakin protein VAB-10B and primary microcephaly protein WDR-62. We uncovered the modes and mechanisms by which MTOC function is removed from the centrosome and identified an essential SPD-5-based module of proteins required for centrosomal MTOC function across cell types. With these technical and conceptual advances in hand, our goal over the next 5 years is to answer fundamental questions related to microtubule organization: 1. How do differentiated cell types build specialized microtubule networks? We will use VAB-10B and WDR-62 as genetic handles to define comprehensive proximity maps of ncMTOCs across cell types with the goal of understanding how ncMTOCs first arise during differentiation; 2. How and why is MTOC function inactivated at the centrosome? We will uncover the molecular mechanism by which SPD-5 transforms the centrosome into an MTOC and test the physiological relevance of centrosome inactivation; 3. How do compound microtubules form? Using novel genetic tools, we will determine how microtubules build onto one another to form doublet microtubules, conserved features of organelles called cilia. These questions are fundamental to our understanding of cell differentiation and leverage unique properties of C. elegans to address this important, but understudied topic in cell and developmental biology.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Type 2 diabetes (T2D) is a leading cause of death nationwide, with 65% of mortality due to cardiovascular disease. The term “diabetic cardiomyopathy (T2DCM)” refers to a condition with adverse myocardial remodeling in the absence of hypertension and vascular pathology. Although T2D and CVD are tightly intertwined, we lack a deeper understanding of T2DCM at the molecular and cellular levels. Pathological mechanisms within the primary constituents of the heart – cardiomyocytes, fibroblasts, and endothelial cells – are incompletely understood. Furthermore, how these metabolic signals converge within the cardiac microenvironment remains elusive. First developed to treat T2D, sodium-glucose cotransporter-2 inhibitors (SGLT2i) prevent glucose reabsorption by the kidney. However, recent clinical trials of SGLT2i (canagliflozin, dapagliflozin, and empagliflozin) further demonstrated an unexpected and substantial reduction in heart failure hospitalizations in patients with and without T2D. Since SGLT2 is lowly expressed in the heart, its off-target mechanisms present a fascinating opportunity to elucidate cardiac protective targets beyond glycemic control. I hypothesize that metabolic interplay between cardiomyocytes, endothelial cells, and fibroblasts play a role in T2DCM pathogenesis, and SGLT2 inhibition is a tool to dissect cell-specific protective mechanisms. Since access to human cardiac samples is limited by primary culture or post-mortem autopsy, the pre-clinical testing of cardiovascular drugs difficult. Thus, induced pluripotent stem cells (iPSCs) have become a valuable platform for biomedical research by providing tissue-specific human cells that retain patients' genetic integrity and display disease phenotypes in a dish. In this F32 proposal, I will harness iPSC technology to generate T2DCM models of cardiovascular cell types for cellular and metabolic phenotyping with and without SGLT2 inhibition (Aim 1). The iPSCs of T2D patients (10 healthy, 20 T2D) are readily available from the Stanford Cardiovascular Institute Biobank. They will be differentiated into three cardiovascular cell types using robust protocols followed by contractility, mitochondrial oxygen consumption rate, cellular (viability, migration, proliferation), and metabolic function (13C-metabolomics) measurements. Next, I will construct iPSC-derived engineered heart tissues for functional phenotyping of cellular interplay (Aim 2). I will further determine the SGLT2i-protein interactome using limited proteolysis coupled to liquid chromatography-mass spectrometry (LiP-MS). Using a systems-level approach compatible with complex biological samples will enable elucidation of drug-protein interactions relevant to T2DCM with peptide-level resolution. In summary, this research plan presents a novel, comprehensive view of metabolic mechanisms conferred by T2DCM pathogenesis, and SGLT2 inhibition and can be used as a springboard for discovering new cardiac protective agents. Taken together, this project will bolster an innovative direction for the cardiovascular community while providing me with the necessary training to become an independent researcher of cardiac metabolic disease.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Insulin resistance (IR) is necessary for the development of type 2 diabetes (T2D) and is a major cardiovascular diseases risk factor. IR has few therapeutic options and identification of new drug targets and novel IR mechanisms would have a huge impact on public health. Although genome wide association studies (GWAS) have identified hundreds IR-associated genetic loci, there has been limited progress towards identifying causal genes and mechanisms. Our colocalization analysis combined data from GWAS of IR-associated traits (T2D, fasting insulin, fasting glucose, waist-hip ratio adjusted for body mass index, triglycerides and high-density lipoprotein cholesterol) and expression quantitative trait loci (eQTL) within five tissues related to IR or T2D (subcutaneous and visceral adipose tissue, skeletal muscle, liver and pancreas). The analysis identified specific causal IR genes in about 25% of IR loci and in half of these cases, the effects are attributable to effects in adipose tissue. We will extend our findings through larger colocalization studies and use single cell data to identify a credible list of adipose cell specific IR causal genes (Aim 1). In Aim 2, we will functionally characterize IR causal genes for cellular mechanisms of action as well as define gene regulatory networks by employing single cell analyses of transcriptomes and epigenomes following CRISPR gene perturbation. In Aim 3, we will define the physiological role of causal genes in vivo by creating loss of function mouse models by combining a CRISPRi mouse with the delivery of sgRNA and Cre recombinase via the adipose tissue specific AAV delivery system. By combining human genetics, computational analysis, and functional genomics tools, we will establish causal genes and their mechanisms of action in development of IR. This effort will lead to novel mechanisms and drugs targets to address the unmet need posed by IR, T2D and cardiovascular disease.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Coronary heart disease is the leading cause of death in the US. The atherosclerotic process underlying coronary heart disease is exacerbated by stressors in the vascular environment, including hyperlipidemia, tobacco smoke, and air pollution. These stressors have been known to cause increased inflammation and oxidative stress in the vessel wall, but how these stressors affect the different cell types in the vascular wall has not been well-described. In our preliminary studies, we found that atherogenic stress can induce significant transcriptional and epigenetic changes consistent with proteotoxicity in the atherosclerotic vascular wall. In particular, we found that e-cigarettes activate the unfolded protein response (UPR) in smooth muscle cells (SMCs), and that this response may be dependent on the environment sensing aryl-hydrocarbon receptor (AHR) pathway. The overall objective of this proposal is to characterize the smooth muscle specific misfolded protein aggregates and their functionality. The central hypothesis is that the atherogenic environment can induce tissue-specific protein misfolding and proteotoxic stress in vascular SMCs and that AHR can protect against atherosclerosis by limiting the proteotoxic stress. In Aim 1, we will isolate and identify the misfolded protein aggregates that are associated with atherosclerosis and different disease-causing stimuli. In Aim 2, we will assess the role of AHR in modulating the vascular proteostasis network utilizing primary human coronary artery SMCs in-vitro. Extending the results obtained from PI Dr. Kim’s NHLBI K08 career development award, the proposed work will address a critical knowledge gap in understanding the role of proteotoxic stress in SMCs during atherosclerosis. Ultimately, this work will shed light on novel pathways associated with risk for coronary heart disease. The successful completion of this project will provide the foundation to apply for a larger grant (R01) to address the effect of specific components of the vascular proteostasis network in controlling atherosclerosis.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Multiple Sclerosis (MS) is the most common cause of non-traumatic disability in young adults affecting approximately one million individuals only in the United States. The pathophysiology of MS remains unclear. Infection with the Epstein-Barr Virus (EBV) is epidemiologically a pre- requisite for developing MS, as essentially all MS patients become infected with EBV before the onset of the disease. The Robinson lab at Stanford recently identified molecular mimicry between the EBV transcription factor EBNA1 and the glial cellular adhesion molecule GlialCAM in 20-25% of MS patients, which is likely a critical mechanism underlying the development of MS in this subset of patients. The proposed project will investigate the differences (presence/absence and the levels) in antibodies reactive to different EBV proteins with the potential for immunogenicity, using large patient cohorts. This study also aims to define the role of EBV reactivation and anti- EBV antibodies in relapse and progression of MS as well as response to treatment. The detailed education and training plan for this K23 Mentored Patient-Oriented Research Career Development Award will give the candidate, Dr. Sattarnezhad, the necessary skills to reach this goal by confirming three specific Aims. Aim 1 will characterize MS cohorts using proteomic analysis of the anti-EBV antibodies and neurodegenerative markers to investigate the role of EBV molecular mimicry in MS etiopathogenesis; Aim 2 will define the role of EBV reactivation in the disease activity in MS patients; and Aim 3 will perform integrated informatics analysis of the finding from Aims 1 and 2 to investigate the association between anti-EBV antibody levels with genetic, clinical, and imaging data of MS patients. Success of the proposed studies would elucidate the role of EBV in MS, which will transform our understanding of MS and can lead to fundamental therapies for the treatment of MS. The proposed career development plan will also provide Dr. Sattarnezhad with the support and training to become an independent clinician-scientist in neuroimmunology.

NIH Research Projects · FY 2026 · 2024-04

Summary/Abstract Planar cell polarity (PCP) signaling controls the polarization of cells within the plane of an epithelium, orienting asymmetric cellular structures, cell divisions, and cell migration and is well conserved from Drosophila to vertebrates. In vertebrates, defects in the core PCP mechanism result in a range of developmental anomalies and diseases including conotruncal heart defects, open neural tube defects, deafness, and situs inversus and heterotaxy, among others. Congenital heart disease (CHD) comprises close to half of all birth defects, and conotruncal heart malformations are the most common CHDs in humans, and include anomalies such as double outlet right ventricle, transposition of the great arteries, and overriding aorta. Despite its critical role in the development of the heart and other organs, the molecular mechanisms that drive PCP signaling remain remarkably poorly understood. Although a mechanistic dissection of PCP signaling would be prohibitively difficult in the mammalian heart, mechanistic conservation in PCP signaling allows findings from highly tractable model systems to translate readily to PCP in mammalian systems. Drosophila is an ideal system for studying PCP due to its highly developed genetic tools, its history as a PCP model system, and its suitability for high-resolution microscopy. In this project, we will leverage the experimental tractability of the Drosophila model system, in combination with cutting-edge biochemical and biophysical approaches, to elucidate the mechanistic basis for PCP signaling. We propose two independent but interrelated Aims that will i) elucidate the feedback circuits and molecular interactions that lead to the induction of asymmetry, and ii) determine how clustering contributes to the amplification of asymmetry, to polarity readout, or both. To accomplish this, we will employ novel genetic approaches to unlock understanding of extensive feedback mechanisms in PCP signaling, single-molecule biophysical approaches that will reveal how clustering is coupled to the amplification and readout of asymmetry, and advanced biochemical approaches that will provide direct molecular insight into how intrinsic asymmetry in PCP complexes is achieved and into how oligomerization may result in clustering. The proposed work will both enhance our knowledge of fundamental mechanisms underlying PCP signaling, as well as lay the groundwork for potential therapeutic interventions for PCP-related heart pathologies.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Vision is an important driver of our evolution and adaptation to different environments. It is a complex process that begins with photoreceptor signal transduction in retinal circuitry before transmitting to central brain targets to drive a range of image-forming visual functions, from color discrimination to navigation. Canonically, studies on image forming vision in the retina and cortex have largely focused on rod and cone inputs that encode pattered visual images. However, additional inputs that contribute to complex retinal and cortical computations from melanopsin-expressing intrinsically photosensitive retinal ganglion cells (iPRGCs) or sleep are largely unexplored. Therefore, there is a need to understand how multiplexed photoreceptor inputs mediate retinal and cortical processes and how such responses are altered with sleep. I hypothesize that multiplexing of rod, cone, and melanopsin input will allow cortical neurons to respond to visual stimuli with a large range of irradiance under complex visual features like natural scenes, and that these processes will be modulated by sleep. My objectives are to measure melanopsin-specific retinal and cortical responses, use that information to build a predictive computational model of the early visual system that incorporates multiplexed photoreceptor inputs, and determine how sleep alters cortical computations for visual processing. I will begin by isolating and measuring melanopsin-specific responses in the retina and cortex under natural scenes in Aim 1. Then, I will record responses in the visual cortex under natural scenes at different points of circadian time-of-day and sleep deprivation in Aim 2. By understanding a detailed quantitative description of how visual experience is represented in the retina and visual cortex, we will better understand how and why vision loss occurs in diseases and disorders that affect the early visual system. Furthermore, my work will contribute to the development of accurate and sophisticated computational models that could improve the design of cortical prosthesis systems that aim to restore lost vision due to damages or disorders to the visual centers of the brain. My Sponsor, Dr. Stephen Baccus, and I have created a training plan to focus on developing my technical, writing and communication, and mentorship skills. My technical skills will focus heavily on in vivo and in vitro electrophysiology, microscopy, behavioral assays, computational analysis, and computational modeling. I plan to register for relevant courses, attend workshops and training events, and network with experts in the field. My writing and communication skills will be developed by applying for grants/fellowships, manuscript development, and presenting at conferences. I will develop my mentorship skills by training undergraduate and graduate students to help run experiments and analyze data. I am part of a highly collaborative research environment with many world renown experts in the visual neurosciences within my department and sleep neurosciences through collaborations with adjacent departments. I plan to fully utilize the resources and facilities available to accomplish the goals of this proposal, as well as achieve my goal of becoming an independent research scientist.

NIH Research Projects · FY 2026 · 2024-03

Peri- and post-menopausal women experience dysbiosis of the vaginal microbiota, characterized by a non-Lactobacillus dominant microbiome and higher microbial richness. However, there is a critical need to better characterize how the initiation and cessation of interventions used to manage menopause symptoms impact the vaginal microbiome of peri- and post-menopausal women. The overall objective of this project is to investigate the epidemiological and biological factors that influence vaginal dysbiosis and its clinical management among peri- and post-menopausal women. Our central hypothesis is that treatments for managing menopause symptoms produce significant changes to the vaginal microbiome, and that these changes are mediated by the stage of the menopause transition. In Aim 1, we will recruit a longitudinal clinical cohort of peri-menopausal, early post-menopausal, and late post-menopausal women to characterize changes in the vaginal microbiota composition and taxonomic richness following initiation of menopause treatments. In Aim 2, we will characterize longitudinal changes in the vaginal microbiome following cessation of menopause treatments. We will use 16S rRNA gene amplicon sequencing (V3-V4 hypervariable region) from self-collected vaginal swabs. These aims constitute the mentored research component of the candidate’s career development plan for this K01. In parallel with this research, the candidate will pursue training in translational microbiome science, supported by an exemplary team of renowned investigators with expertise in gynecological health (Primary Mentor, Dr. Juno Obedin-Maliver), the vaginal microbiome (Co-Mentors Drs. David Relman, Christina Muzny, and Christopher Taylor), and the impact of menopause on genital tissues (Co- Mentor Dr. Bertha Chen). Stanford University offers a world-class research infrastructure that fosters outstanding collaborative and innovative translational research. This research also leverages the expertise of Stanford’s OBGYN Department, including its Menopause and Healthy Aging Programs. In summary, the strong mentoring environment and training plan are anticipated to comprehensively prepare Dr. Tordoff to launch an independent research career. The proposed studies promise to address critical gaps in our understanding of how pharmacological treatments for menopause impact vaginal health, and the results of this research will improve decision making for clinicians and patients regarding menopause treatments.

NIH Research Projects · FY 2026 · 2024-03

Project Summary Food allergy (FA) impacts approximately 33 million people in the US, contributing to substantial economic and emotional burdens. Oral immunotherapy (OIT) is an effective treatment strategy to induce desensitization to specific food allergens, in which participants consume increasing amounts of allergenic foods daily. However, there are several remaining concerns that hinder its widespread implementation including the burdens of daily OIT maintenance dosing and gastrointestinal complications such as the development of eosinophilic esophagitis (EoE). OIT participants often find that continued daily OIT maintenance dosing is challenging, causing some participants to discontinue dosing and potentially revert to their pre-OIT allergic state with rapid loss of desensitization and increased risk of anaphylaxis. Therefore, there is an urgent need to identify whether less frequent OIT maintenance dosing regimens can allow participants to maintain desensitization. Long-term follow up studies for participants who have completed OIT clinical trials at our center show that 53% of participants had difficulty with continued OIT dosing, largely due to lifestyle restrictions of when they can safely consume their daily OIT maintenance dose. This led to 30% of participants discontinuing dosing for all their allergenic foods, highlighting the need for less burdensome maintenance dosing options. Non-daily dosing regimen with 300 mg peanut allergen has not been sufficient, however higher concentrations of non-daily dosing have not been investigated. In our CoFAR network-wide clinical trial we propose to evaluate the safety and efficacy of non-daily OIT maintenance doses in maintaining peanut desensitization. Gastrointestinal symptoms are common during oral immunotherapy (OIT) for food allergy (FA), with the majority of patients experiencing mild to moderate symptoms of abdominal pain, nausea, or vomiting at some point during the desensitization process. This raises concern for eosinophilic esophagitis which is a potential and serious side effect of OIT, estimated to occur in 2-5% of patients on OIT. The diagnosis of EoE during OIT requires an endoscopy with biopsy to confirm diagnosis, however, many patients forgo this procedure leading to inaccurate estimates of the true incidence. Additionally, in a pilot study conducted by our site (POISED study), baseline eosinophilia exists in FA patients avoiding the culprit antigen. Furthermore, OIT-induced gastrointestinal eosinophilia was mostly transient and asymptomatic. In contrast, although most cases of OIT-associated EoE resolve with dose modification or cessation of OIT, a small subset of FA participants have EoE symptoms that persist after stopping OIT. However, the mechanisms that drive these differences in the clinical heterogeneity of EoE during FA treatment are not understood. FA and EoE are associated with changes in the abundance and expression of key immune cells: eosinophils, mast cells, and gene expression profile of type 2 CD4+ T cells in prospectively collected biopsies. Exploring these characteristics can provide valuable insights into the underlying mechanisms and potentially help tailor treatment approaches and drive novel diagnostic techniques.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT With repeated Plasmodium infections, children eventually gain the ability to tolerate Plasmodium parasitemia without developing symptoms. However, preventing malaria with effective chemoprevention may delay development of this clinical immunity, such that children are at increased risk of more severe disease following cessation. Although mechanisms controlling development of clinical immunity remain unclear, effector and regulatory CD4+ T cells play a critical role in orchestrating a coordinated immune response to pathogens including malaria. Plasmodium-specific Type 1 regulatory CD4+ T cells (Tr1) are a specialized, tolerogenic CD4 subset that expand following Plasmodium infection in humans and mice, although genetic mechanisms that support Tr1 differentiation and maintenance remain unresolved. It also remains unclear whether these represent clonal expansions, contribute to a stable memory pool, and/or play a role in mediating clinical immunity to subsequent Plasmodium infections. We hypothesize that clonal populations of Plasmodium- specific Tr1 cells expand following Plasmodium infection, are critical mediators of clinical immunity to repeated infection, and are limited by effective chemoprevention. To test our hypotheses, we will leverage two extraordinary and complementary cohorts to longitudinally and comprehensively analyze the CD4+ T cell response in children. In Aim 1, we will analyze samples already obtained from children enrolled in the Ugandan International Centers of Excellence in Malaria cohorts before, during, and at multiple time points following a single and repeated malaria infection. We will study CD4+ T cell population dynamics within the same child over time using innovative single cell transcriptomic and epigenomic approaches that we have optimized. We will predict genes and other genetic loci that support or suppress Tr1 responses in children, and determine associations with antibody responses and clinical phenotypes of infection. In Aim 2, we will analyze samples collected as part of the Modifying Immunity in Children with Dihydroartemisin-Piperaquine (MIC-DroP) clinical trial, in which 924 infants are being randomized to receive artemisinin-based chemoprevention vs. placebo from birth to 2 years of age, then followed to 4 years of age. We will determine whether preventing repeated infection interferes with Tr1 development, and whether specific malaria-specific CD4+ T cell populations correlate with clinical outcomes following the cessation of chemoprevention. Finally, to complement the pediatric studies, in Aim 3 we will perform studies of experimental re-infection and chemoprevention in mice, since these systems permit detailed analysis of CD4+ T cell dynamics in the first few hours and days after a reinfection event, and can assess organ-specific CD4+ responses in both the spleen and liver. This study of Plasmodium-specific CD4+ T cell dynamics in humans and mice will provide critical insight into how the adaptive immune system tolerates foreign antigens, and lay the foundation for the development of therapeutic strategies aimed at reducing disease severity and/or enhancing parasite clearance.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY / ABSTRACT Neutrophils are the first line of host defense against invading pathogens that trap and kill pathogens through a process called NETosis. Upon stimulation by the pathogen, neutrophils extrude decondensed chromatin in the form of DNA fibers that trap invading cells and forms a scaffold to harbor associated proteins and antimicrobial peptides. Bactericidal activity has been attributed to the action of these associated proteins and peptides through oxidative stress resulting from the free radicals they generate. DNA however, the largest component of NETs, is only believed to offer the trapping scaffold despite a suicidal path a neutrophil undergoes to extrude the DNA. DNA is known to form non-canonical secondary structures like the i-motif, triple helices, and G-quadruplexes (G4). G4 is formed by the stacking of planar square structures of four guanine bases associated by Hoogsteen hydrogen binds. These secondary structures are concentrated around transcription start sites in promoter regions and genome-wide high-resolution sequencing has detected >700,000 G4s in the human genome. G4s are known to sequester free hemin to protect the cell from hemin toxicity and this complex (G4/H) subsequently regulates gene expression. In vitro, G4/H has been characterized as a DNAzyme that mimics peroxidases to decompose to hydrogen peroxide to produce hydroxyl radicals. Hydroxyl radicals are known to be most reactive and toxic to cells in vivo. While G4/H activity is well understood in vitro and the G4 is known to sequester free hemin within cells, the existence of G4 structures in the decondensed chromatin of NETs or the peroxidase like activity of G4/H in vivo is unknown. We propose to discern the existence of G4/H complexes in NETs using antibodies specific to hemin and G4 using multiple approaches like colocalization of immunofluorescence and ChIP-seq on NETs specific DNA pulled with myeloperoxidase specific antibodies from whole blood of healthy individuals after IL-8 or bacterial stimulation and patients with COVID-19. We propose to characterize the enzymatic activity of the G4/H DNAzyme in NETs and demonstrate the local concentrated effect of free radicals generated on trapped bacteria and prove that this phenomenon occurs naturally ex vivo. Finally, we propose to exhibit the biological outcomes from the G4/H DNAzyme generated free radicals by testing bactericidal activity, host cell injury, and posttranslational modifications of NETs associated proteins giving rise to autoimmune disorders like systemic lupus erythematosus.

NIH Research Projects · FY 2026 · 2024-03

Abstract To survive in the nutrient- and oxygen-deprived niche, cancer cells undergo a drastic metabolic reprogramming where they upregulate their endogenous lipid synthesis and exogenous lipid uptake. Population-wide mass spectrometry lipid analyses reveal that amounts of lipid stores, degree of lipid saturation, and acyl-chain length provide survival advantages by making cells metabolically independent of external sources of energy, protecting against oxidative stress, and stimulating immune response suppression. Hence, cancer cell lipid profiling emerges as a powerful diagnostic tool and fundamental research goal. However, current technologies lack single-cell resolution, do not provide spatial information, and are incapable of linking the cancer cell phenotype to the lipid composition. Here, we describe a unique platform for simultaneous lipid profiling of multiple cell types in intact tissue samples at sub-cellular level by probing inherent molecular vibrations using spectral coherent anti-Stokes Raman scattering (CARS) microscopy. It promises transformative information on how lipid profiles are shaped in different tumor microenvironments (TMEs) in both cancer cells and cancer- associated cells, and relates this quantitative, spatial information to patterns of protein and gene expression. We combine a custom, automatically tunable laser system with a commercial confocal microscope to enable convenient mapping of a range of molecular vibrations to assess (i) amounts of lipids with sub-femtoliter precision, (ii) degree of lipid unsaturation at single carbon double-bond precision, and (iii) acyl chain length at single methylene group precision, which can be scaled up by programmable multipoint measurements of multiple samples. Further, by implementing simultaneous single-cell RNA imaging, metabolic profiles can be linked to upstream RNA transcripts controlling cell metabolism, including known oncogenes. For this purpose, we will design a series of novel RNA probes, each with an isotope-specific nitrile group, which can be targeted with high specificity through its distinct molecular vibration by stimulated Raman excited fluorescence (SREF). Thanks to a multitude of possible combinations for isotope labeling, this innovative approach allows simultaneous detection of a higher number of RNA transcripts than can be done with current fluorescence-based RNA labeling techniques without disturbing the lipid landscape. Simultaneously, standard fluorophores can be used to probe cell-type markers at the protein level with conventional immunocytochemistry. As a result, multiple RNA transcripts can be mapped simultaneously with lipid profiles and protein profiles for multiple different cell types. The development will be conducted in collaboration with cancer biologists, cancer metabolism experts and clinicians, ensuring the biological and clinical relevance of our approach and data analysis.

NIH Research Projects · FY 2026 · 2024-03

Abstract: The rapid diffusion of genetic testing across adult cancer diagnoses is a unique opportunity to personalize cancer treatment and prevention at the population level. Germline genetic testing guidelines have broadened to encompass nearly all patients with breast, ovarian and pancreatic cancer; all patients with advanced prostate cancer; and many patients with colorectal, endometrial, and other cancer types. The growing use of genetic testing is driving the development of precision oncology: a new paradigm for personalizing prevention and treatment based on genetic testing results in addition to or instead of traditionally- measured tumor features. Increasingly, an inherited pathogenic variant in a specific gene serves as an essential common thread that connects diverse cancer diagnoses and enables genetically-targeted cancer therapy. However, we know virtually nothing about how genetic test results are managed across cancer types. We pioneered the Georgia-California (GACA) Genetic Testing Linkage Initiative, linking industry-provided genetic testing data to SEER registry records for all adults diagnosed with cancer in Georgia and California from 2013-19. We found that genetic testing across cancer conditions has increased but rates are too low relative to clinical recommendations. Our foundational work underscores the urgent need for research about how well genetic testing results are broadly integrated into the management of cancer. We are now completing the next phase of the GACA Genetic Testing Linkage Initiative: merging SEER data for all adults diagnosed with cancer in Georgia or California from 2013-21 (N=1,826,000), with an update planned for cancers diagnosed in 2022-23 (N=456,000), to genetic results through 2025. We will use this unique, population-based data infrastructure to determine whether genetic testing results management is effectively personalized across common cancers with high testing rates. We will also examine cancer mortality by genetic testing results to inform communication about prognosis and personalized treatment. Our hypotheses are as follows: Extensiveness of surgery (e.g., organ preservation vs. removal) is well-personalized: it is strongly associated with clinical indications derived from tumor features, host factors and actionable genetic results, but not with non-actionable genetic results, socioeconomic or healthcare system factors. Receipt of chemotherapy and genetically targeted systemic therapies (immune checkpoint inhibitors and poly(ADP-ribose) polymerase inhibitors) is well-personalized: these treatments are strongly associated with clinical indications and actionable genetic results, but not with non-actionable genetic results, socioeconomic or healthcare system factors. Among cancer patients treated with systemic therapies, patients with PVs have lower cancer-specific mortality, controlling for tumor features, treatments, comorbidities, socioeconomic and healthcare system factors.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT The overarching goal of the proposed study is to better understand COVID-19 vaccine immunogenicity in immunocompromised individuals, particularly patients with connective tissue diseases (CTDs) and other musculoskeletal disorders. A paucity of data exists regarding vaccine safety, immunogenicity, efficacy, durability and especially mechanisms of vaccine responses in AID patients, leading to an unmet need to elucidate these critical mechanisms. We performed a “systems vaccinology” study of the BNT162b2 (Pfizer-BioNTech) mRNA vaccine in 18 patients with systemic lupus erythematosus (SLE), using multiple methods including single cell profiling technologies. We showed vaccination was generally safe but led to minor disease flares in a subset of patients. Vaccine efficacy, based on anti-spike IgG responses and pseudovirus neutralization assays for multiple SARS-CoV-2 variants of concern (VoC), were highly variable and not fully explained by immunosuppressive medications. Vaccine nonresponders (NR) had severely blunted induction of IFN-γ, CXCL9, CXCL10, and CXCL11 proteins following vaccine boost. SLE patients had universally lower frequency of CD8+ and CD4+ spike-specific T cells throughout the vaccination series. This R01 application proposes 3 aims. Aim 1 will enroll an expanded SLE longitudinal COVID-19 vaccine cohort and a new SSc vaccine cohort. We will continue to follow the SLE inception cohort to determine how their immune responses evolve with future COVID vaccines. In parallel, we will enroll two new cohorts, one in SLE and the second in SSc, a disease which shares several features including AAb, anti-cytokine AAbs (ACA), and lung and skin pathology. We will test the hypothesis that immune mechanisms of vaccine responses will be shared between diseases, but that differences in autoreactive B and T cell receptors, antibody repertoires, and innate immunity will be discovered. We will compare CTDs with each other and with healthy people, across vaccine platforms (e.g., mRNA vaccines, protein vaccines, and DNA vaccines) currently in development for COVID-19 prevention. Aim 2 will characterize adaptive immune responses. We will characterize antibodies, T cells, and B cells. Antibodies will be measured using custom, bead-based arrays for measurement of AAbs and ACA to ensure vaccines do not enhance secretion of existing or new AAb after vaccination; and anti-viral IgG responses, including in vitro and pseudoviral functional blocking assays against emerging VoC over time. B cell studies will include B cell receptor (BCR) repertoires and transcription modules, and creation and analysis of recombinant monoclonal Ab (rMAb); T cell studies will include phenotyping of virus-specific T cells using MHC Class I and II spheromers. T cell populations known to be associated with AID, will be studied. Aim 3 will characterize COVID-19 vaccine responses in the innate immune compartment. The role of innate immune cells will be explored using bulk-RNA-seq, measurement of plasma analytes using “omics” technologies, and Epigenetic profiling by Time of Flight (EpiToF) to measure “innate memory”. CITE-seq will then be used to deeply phenotype innate immune cells from responders and NR.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Acute respiratory distress syndrome (ARDS) is a major source of morbidity and mortality in patients ventilated for acute respiratory failure. Despite extensive study, little is known about the pathogenesis of ARDS in humans. One salient finding is that barotrauma or ventilator-associated lung injury (VALI) from high-pressure ventilation is a consistent signal for ARDS risk regardless of the underlying disease. Beyond lung protective (low pressure) ventilation, ARDS lacks specific treatments in large part due to the lack of knowledge about the cellular and molecular mechanisms of pathology. Here, we will take advantage of an established ex vivo ventilation and lung perfusion (EVLP) system to directly assess the initial cellular and molecular responses to VALI in human lung. Importantly, each individual will serve as their own control, achieved by ventilating the right and left lung under high and low pressure, respectively. Some studies will incorporate hyperoxia, elevated PEEP, or live bacteria with VALI to provide even more clinical relevance. Single cell RNA sequencing on the experimental and control lungs following ventilation will provide deep molecular profiles of alveolar cells subjected to barotrauma. This tissue-dissociative analysis will be complemented with multimodal (protein and RNA) and multiplex staining of intact lung tissue using two complementary technologies, to correlate the molecular with spatial and morphological features of pathology. We will also test the capacity of human lung to recover from acute VALI by culturing lung slice cultures at air-liquid interface following ventilation, and how this is impacted by pan and endothelial-specific Wnt inhibition, lipopolysaccharide, and targeted pathway manipulations. In summary, our studies will open a window into the dynamic cellular and molecular responses of human lung in acute lung injury.

NIH Research Projects · FY 2026 · 2024-03

Abstract: Most cancer-related deaths are a consequence of metastatic disease. Yet, our understanding of the underlying mechanisms responsible for metastasis—especially the immune system's role in modulating metastatic progression—remains rudimentary. My sponsor’s lab recently discovered that tumor invasion into lymph nodes (LNs) evokes the induction of regulatory T cells (Tregs) that confers systemic, tumor-specific immune tolerance, thereby permitting distant (organ) metastases. While both human and murine tumor-involved lymph node tissues have demonstrated a conserved and dominant mechanism requiring Tregs for metastatic progression, the underlying mediator that governs their development remains enigmatic, and thus, warrants further investigation. In line with the notion that professional antigen-presenting cells (APCs) are critical for antigen-specific T cell development, my preliminary work revealed that professional APCs are differentially enriched in tumor-involved lymph nodes of mice. However, which APC population is responsible for promoting tumor-specific Treg development, and whether the tumor immune tolerance machinery can be therapeutically overcome remains to be investigated. To address these gaps in knowledge, I propose the following aims: 1) identify the APCs that potentiate LN metastasis-induced tumor-specific immune tolerance; and 2) assess therapeutic strategies that reprogram APCs to curtail metastatic progression. Using high-dimensional multiplexed imaging (CODEX), single-cell RNA-sequencing, various in vivo cell-depletion mouse models and adoptive cell transfer assays, as well as in vitro co-culture experiments, I will systematically dissect the key APC population that I hypothesize is responsible for mediating tumor-specific Treg development. To therapeutically reprogram the APCs away from tolerance induction, I will develop immunostimulatory antibody conjugates that drain into LNs and test them in mouse models of melanoma and pancreatic ductal adenocarcinoma. The proposed work aims to delineate the APC population that is responsible for mediating tumor-specific Treg induction and to advance the development of immunotherapeutic strategies to treat metastatic disease.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Obesity is the second leading cause of premature death. Consumption of ultra-processed foods is theorized to be a key cause of obesity. Ultra-processed foods are formulations of cheap industrial sources of dietary energy and nutrients plus additives such as fat, sugar, and flavors that enhance acceptability of the foods. A cross- over experiment with overweight adults found that ad lib access to an ultra-processed diet for 2-weeks resulted in increased caloric intake (508 kcal/day) and more weight gain versus ad lib access to a minimally-processed diet matched for presented calories, energy density, macronutrients, sugar, sodium, and fiber (Hall et al., 2019). The fact that ad lib access to ultra-processed foods resulted in a large increase in caloric intake and weight gain implies that ultra-processed foods may more effectively activate brain regions implicated in reward processing, attention/salience, and memory that influence eating behavior. However, no brain imaging study has experimentally tested whether ultra-processed foods are more effective in activating brain regions implicated in reward, attention, and memory than minimally-processed foods or experimentally investigated the relative role of the elevated caloric density versus the flavor enhancers of ultra-processed foods in driving greater activation of these brain regions. Preliminary data showed that tastes of ultra-processed high-calorie chocolate milkshake produced greater activation in regions implicated in reward valuation (caudate, nucleus accumbens), attention/salience (precuneus), and memory retrieval (medial temporal gyrus, dorsomedial prefrontal cortex) than tastes of ultra-processed low-calorie chocolate milkshake. Aim 1 is to test the hypothesis that tastes, anticipated tastes, and images of ultra-processed foods activate reward, attention, and memory regions more than tastes, anticipated tastes, and images of minimally-processed foods, and evaluate the relative role of the higher caloric content versus flavor enhancers in engaging these regions using a 2 x 2 experimental design. Aim 2 is to test the hypothesis that ultra-processed versus minimally-processed foods promote stronger learning of cues that predict tastes of ultra-processed foods (incentive sensitization), which is important because elevated reward region response to food cues/images increases risk for future weight gain (Demos et al., 2012; Stice et al., 2015; Yokum et al., 2014). Aim 3 is to test the hypothesis that participants who show greater activation in reward/attention/memory regions in response to ultra-processed foods will consume more ultra-processed foods ad lib and show greater future body fat gain, and to establish neural fingerprints that predict ad lib ultra-processed food intake and body fat gain. Aim 4 is to test the hypothesis that participants who show stronger reward cue learning in response to ultra-processed foods will consume more ultra-processed foods ad lib and show greater future body fat gain. Improved knowledge of neural factors that increase risk for body fat gain should guide the design of more effective obesity prevention programs and treatments, which is critical because current prevention programs and treatments have limited efficacy.

NIH Research Projects · FY 2026 · 2024-03

Project Summary: Chronic inflammation is a rising health issue that affects millions of people worldwide. Emerging evidence shows that dysbiosis of the gut microbiota can lead to changes in the production of microbial-derived metabolites (MDMs), which contribute to inflammation and the onset of inflammatory diseases, such as cancer, non-alcoholic fatty liver disease, and inflammatory bowel syndrome. MDMs are absorbed into host circulation through the gut and regulate immune responses. A key immune cell type at the interface of host-microbe interactions is macrophages, which maintain tissue homeostasis, and are key contributors to chronic inflammation. To date, few MDMs with well-studied cellular and molecular mechanisms describing the interplay between microbes and our immune system are known. Using metabolomics data from a dietary intervention study that found distinct immunological trajectories based on microbiota diversity, as well as data from a 176 gut microbiota strain library we identified 18 human-immune associated MDMs. Here we aim to determine the regulatory role of MDMS on the host immune system through a direct effect on macrophages. The impact of these MDMs on host molecular pathways will be characterized using murine and human macrophage cells, NFκB reporter lines, CRISPR/Cas9 systems, GPCR reporter lines and in vivo using IBD murine models (Aim1). On the microbe end, I will determine the metabolic pathways responsible to produce these immune-associated MDMs and generate knock-out or knock-in bacterial strains. I will perform a series of highly-controlled mouse experiments to determine the specific microbial components responsible for MDMs immune modulating effects (Aim2). My previous experience in immune regulation and animal models make me well-equipped to analyze and perform these technical experiments, and the expertise in microbial genetics and metabolomics facilities honed by my mentoring team make Stanford University an ideal location to learn the skills I will need to perform these experiments. Completion of these aims will increase the understanding of the microbiota-host interaction during times of homeostasis and acute or chronic inflammation, which will pave the way for the development of precision therapeutic strategies to reduce chronic inflammation and prevent acute disease.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Hearing loss is a prevalent condition affecting millions of individuals worldwide, primarily caused by the irreversible loss of auditory hair cells. While non-mammalian species possess the remarkable ability to regenerate hair cells, mammals, including humans, lack this regenerative capacity. Therefore, understanding the regenerative potential of hair cells in mammals is crucial for developing effective therapies to restore hearing. The objective of this project is to decipher the mechanisms for inducing S-phase entry in adult mammalian supporting cells and investigate their regenerative potential for sensory hair cell regeneration in the auditory system. In AIM 1, the study will align transcriptomic and epigenetic data at single-cell resolution to establish a baseline profile of supporting cells before, during, and after hair cell loss. The analysis will identify the molecular signatures associated with the quiescent state of supporting cells in mammals and explore the influence of epigenetic factors on the regenerative incapability of supporting cells and their differentiation into functional hair cells. Comparative analysis with chicken datasets will refine strategies for inducing cell cycle re-entry in supporting cells. AIM 2 focuses on developing a strategy to specifically target adult supporting cells after acute and chronic hair cell damage. The study will employ an inducible gene expression strategy in adult supporting cells using adeno- associated virus (AAV)-mediated gene transfer. AIM 3 investigates the possibility to induce division in mature supporting cells and hair cell fate specification using a co-misexpression approach with multiple transcription factors. The project will utilize innovative techniques such as multi-omics low-depth single-cell RNA-seq and single- nucleus ATAC-seq to characterize supporting cells at different time points post-hair cell insult. The employment of Fbxo2VHC/WT mice and inducible gene expression strategies using AAV-mediated gene transfer will ensure accurate representation and temporal control of gene expression in supporting cells. The use of various controls and statistical methods will ensure robust data analysis and mitigate technical biases. The outcomes of this research will shed light on the molecular and epigenetic identity of supporting cells, identify potential targets for therapeutic interventions, and refine strategies for inducing cell cycle re-entry and hair cell regeneration. Ultimately, this project aims to contribute to the development of regenerative therapies for hearing loss, providing hope for millions of individuals worldwide.

NIH Research Projects · FY 2026 · 2024-03

Improving food security in the US is a fundamental strategy to promoting health for all populations. Food insecurity – the lack of consistent access to sufficient quantities of healthy food – leads to nutrition-sensitive chronic conditions like obesity, diabetes, and heart disease. To address the interrelated challenges of food insecurity and nutrition-sensitive chronic conditions, healthcare organizations are increasingly turning to novel intervention strategies. States have the ability to use Medicaid Section 1115 waivers to pilot coverage of nontraditional services; Georgia was the first to use it for nutrition-related purposes in 2010, and in 2016 Massachusetts was the first to apply it towards “Food is Medicine,” which is the provision for medically supportive food (tailored meals, produce) for patients with nutrition-sensitive conditions. California’s adoption in 2022 of the 1115 waiver, through the California Advancing and Innovating Medi-Cal (CalAIM) Medicaid section 1115/1915(b), provides a powerful, time-sensitive opportunity for a natural experiment to evaluate the impact of Food is Medicine. It is extremely critical to start the data collection as soon as possible to be able to complete follow-up data collection before CalAim expires in 2026. Programs such as Recipe 4 Health (Alameda County) offer Food is Medicine to Medi-Cal beneficiaries, offering produce prescriptions, group and individual health coaching, and the necessary coordination to facilitate the integration of health care providers with farms providing produce. We will use a quasi-experimental design to evaluate the impact of Food is Medicine on key markers of cardiometabolic health (e.g. glycemic control and cholesterol) among patients with obesity using two strategies. For strategy 1, we will utilize available Body Mass Index and laboratory data in the electronic health record (EHR) to compare participants to controls identified with propensity score matching. Strategy 2 will include a smaller sample of 240 Food is Medicine participants (compared to 240 controls) with comprehensive assessments at baseline and 6 months. With this subsample, lab draws testing at patient homes will support timely collection of key laboratory biomarkers (HbA1c and cholesterol), and telephone surveys will allow for assessment of food security and high-quality dietary intake data (24-hour recall) not feasible to gather in the course of usual clinical care. We are uniquely positioned to conduct this study as we have already built bi-directional community partnerships and infrastructure to share this EHR data. Successful completion of these aims will represent a large, generalizable, and rigorous assessment of Food is Medicine, with direct relevance to patients, the healthcare system, and policy makers.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY: This application seeks a career development award for an academic vitreoretinal surgeon with an interest in high myopia, a condition which predisposes patients to potentially blinding complications including retinal tears (RTs) and rhegmatogenous retinal detachments (RRDs). This proposal is a 5-year curriculum and research plan to transition Dr. Cassie Ludwig to independence. The candidate is an accomplished early career physician-scientist who will undergo all training and execute the research noted herein during this period. Myopia affects one third of the world's population today and has been predicted to affect 50% of the world's population by 2050.1,2 Worse, this prediction is likely an underestimate as myopigenic behaviors have been further compounded by the COVID pandemic and digital remote learning.3–8 This increasing prevalence has significant consequences as each diopter of myopia increases the risk of retinal tears and detachments, myopic macular degeneration, choroidal neovascularization, myopic traction maculopathy, strabismus, glaucoma, and cataracts. Slowing myopia progression even minimally can help prevent blindness. Using combined data from five large population-based studies, Bullimore et al. found that slowing myopia by one diopter should reduce the likelihood of a patient developing an RRD by 30%.2 Electronic health records (EHRs) and ophthalmic imaging databases contain enormous quantities of systemic and ocular data generated by clinical practice which can be used to better understand the relationship between systemic and ophthalmic risk factors, myopia and RTs and RRDs. EHR and imaging data can be fused into predictive models that employ machine learning to risk-stratify patients. In this proposal, Dr. Ludwig aims to achieve the following: 1. Develop and validate a structured EHR deep learning framework to predict RT and RRD risk in myopes and non-myopes 2. Develop and validate an unstructured EHR transformer-based deep learning model to predict RT and RRD risk in myopes and non-myopes, and 3. Develop and validate an ultra-widefield photography convolutional neural network (CNN)-based deep learning model to predict RT and RRD risk in myopes and non-myopes. The central hypothesis is that modeling of attributes from EHR data and images can predict risk of RTs and RRDs. The principal investigator, Cassie A. Ludwig, MD, MS, will perform this research as part of a larger effort to obtain additional training and mentorship in biomedical informatics, artificial intelligence, biodesign, and myopia. Dr. Ludwig’s career development plan includes a PhD program with didactic coursework, conferences, workshops, and frequent communication and interaction with a network of mentors with an impressive abundance of their own NIH funding and prior mentorship experiences. This experience will guide Dr. Ludwig into a career as an independent clinician-scientist with expertise in artificial intelligence and a focus on myopia and its sequelae.

NIH Research Projects · FY 2026 · 2024-03

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by a poor overall prognosis, aggressive and early pattern of metastases, and a lack of treatments that lead to sustained disease control. There is a critical need for molecular targeted treatments for patients with TNBC that are more effective, have reduced side effects, and decrease the mortality associated with TNBC. Mucin-1 (MUC1) is overexpressed and underglycosylated in 94% of TNBC and the receptor density is extremely high. The combination of the modification of the glycosylation and overexpression allow tumor associated MUC1 distinguishable and targetable from normal tissue. Targeted radionuclide therapy (TRT) is a molecular targeted treatment that specifically uses radiolabeled moieties as biological targeting vectors intended to deliver localized cytotoxic radiation to cancer cells that overexpress specific receptors, without harming normal cells. We propose to combine the suitability of MUC1 as a therapeutic target in TNBC with the promising technique of TRT to develop a novel theranostic peptide for diagnostic imaging and TRT of TNBC. The aims of this proposal are to develop MUC1 targeted peptides that can successfully be applied for TRT, exhibiting properties suitable for clinical translation. The advantages of peptides over previously studied antibody radioisotope complexes include the superior pharmacokinetics, low immunogenicity, and ease of synthesis for widespread application. In Aim 1 we will determine the mechanism and specificity of peptide binding, optimize the incorporation of a metal chelator through various spacer moieties, and employ different cyclization strategies to improve peptide affinity and stability. In Aim 2 we will radiolabel three peptide sequences with promising binding affinities with gallium-68 and lutetium-177. We will complete a comprehensive analysis of binding and internalization in TNBC cells, and determine in vivo tumor targeting properties and biodsitribution in mouse models of TNBC. In Aim 3, we will advance the most promising peptide sequence, identified through quantitative metrics, to therapeutic studies and determine the ability of MUC1 TRT to treat TNBC in mouse models. We will additionally complete a preliminary safety assessment. Through these studies we will also understand any issues related to proceeding with clinical translation of the MUC1 theranostic peptide for application in TNBC. Given the mortality associated with TNBC and the critical need for targeted treatments, this MUC1 TRT will have high impact for the treatment and management of TNBC. We will provide proof of concept that MUC1 is a valid therapeutic target and that TRT can effectively treat TNBC. We intend for our results to lay the foundation for subsequent development of randomized, controlled clinical trials that test this promising treatment in patients with TNBC. If successful, this will be a significant step forward for reducing the mortality associated with TNBC.

NIH Research Projects · FY 2026 · 2024-03

Project summary Despite remarkable advances in single-cell profiling, machine learning and systems biology, our ability to exploit these measurements is limited by the lack of an appropriate framework to model and analyze them. In this application, I propose an organic synthesis of experimental technological development, mathematical modeling, and machine learning algorithm innovations to move beyond conventional descriptive and merely statistical analyses of single cells to mechanistic and predictive modeling of cell fate transition over time and space, and across transcriptomic, epigenetic and proteomic levels. Firstly, in order to unveil the regulatory networks that govern the maintenance of stem cells and progenitors, I will extend the dynamo framework that published recently to predict key regulators that stabilize or destabilize cells states, e.g. the hematopoietic stem cell state, via sensitivity analyses of the reconstructed vector field. In addition, I will build upon the current success of predicting a broad range of hematopoietic cell fate transitions with our least action path approach to extend it to study other biological systems, such as pancreatic endocrinogenesis. To validate these predictions, I will continue my ongoing collaboration with Dr. Vijay Sankran’s lab (co-mentor lab) to first implemented metabolic labeling based scRNA-seq with the 10x chromium system and integrate it with perturb-seq that championed by the Weissman lab (my mentor lab) to test the predicted factors’ efficacy in maintaining the HSC state. Second, I will develop new approaches to seamlessly integrate multi-omics and harmonize short-term RNA velocities with long-term lineage tracing. By doing so, we can enable even more accurate modeling of single cell fate transitions that consider lineage-resolved, epigenetic, proteomic kinetics, offered by cutting-edge single-cell genomic technologies and cutting-edge deep learning methods. Lastly, I will take advantage of my early access of mouse embryogenesis dataset profiled with the powerful Stereo-seq through my close collaboration with BGI research to build 3D in silico spatiotemporally models of mammalian organogenesis. I will also train myself to study other state-of-the-art in-situ sequencing approaches, for example the STAR-map method from my collaborator, Dr. Xiao Wang from Broad. Through the K99 phase of this proposed career development plan, I will develop new computational toolkits and further strengthen my experiment skills, both in human hematopoiesis, Perturb-seq and spatial transcriptomics. When combining these new skills with my rigorous training in systems biology, and single cell genomics, I will be better prepared to transition into an independent investigator in a top-tier research university. Undoubtedly, my research and career development during both K99 phase and my transition to R00 phase will be greatly facilitated thanks to the excellent research environment in Whitehead institute, Broad and Harvard stem cell institute. To sum up, my proposed study will pave the road to launch my future interdisciplinary team that aims at building mechanistic and predictive models of cell fate transitions with a focus in human hematopoiesis.