Stanford University

NIH Research Projects · FY 2026 · 2017-09

Project Summary/Abstract Ferroptosis is a non-apoptotic cell death process that can suppress tumor formation and contribute to disease pathology. The long-term objective of this research is to understand the molecular regulation of ferroptosis. Ferroptosis is thought to occur through a stereotypical and invariant lipid-dependent mechanism. However, in new preliminary studies we have uncovered evidence for two different forms of ferroptosis defined by unique genetic regulation and metabolic alterations. We find that ferroptosis triggered by direct inactivation of the enzyme GPX4 requires the function of the lipid metabolic enzyme ACSL4. A second sub-type of ferroptosis, triggered by cystine deprivation, does not require ACSL4 or related lipid metabolic enzymes and instead relies on a novel set of genes. Guided by these new preliminary data, our central hypothesis is that ferroptosis can be executed by two fundamentally distinct mechanisms. We will test this hypothesis by pursuing three specific aims: (i) defining the role of ACSL4 and related lipid metabolic enzymes in promoting ferroptosis in response to direct GPX4 inhibition, (ii) identifying cellular and molecular features that distinguish the two ferroptosis mechanisms in vitro and in vivo, and (iii) defining genes and processes that regulate ACSL4-independent ferroptosis in response to cystine deprivation. These studies will be carried out using cutting-edge chemical, genetic, functional genomic, lipidomic, and imaging tools and methods. This research will advance our fundamental understanding of ferroptosis, with the ultimate goal of improving human health.

NIH Research Projects · FY 2025 · 2017-09

The Canary Center at Stanford for Cancer Early Detection (“Canary Center”) is a world-class facility with the mission to foster interdisciplinary research leading to the development of blood (and other minimally invasive) tests and molecular imaging approaches to detect, localize, and stratify early cancers by integrating research in in vivo and in vitro diagnostics. Embedded within this mission is the need to formally present new and innovative approaches to scientific communities at all levels. This competitive renewal application is for continued support of the Canary Cancer Research Education Summer Training (Canary CREST) Program which fulfills an educational mission by introducing students to research education and new career paths. The overall goal of the program is to train a new generation of interdisciplinary scientists in early cancer detection by offering an integrative hands-on research experience at an early stage of their scientific education. The Canary CREST Program is a 10-week instructional summer program for 30 undergraduate students in the biological, engineering, mathematical, and/or physical sciences, and offers a structured research experience with a focus on early cancer detection. The program will be administered by the Canary Center and brings together a multidisciplinary group of faculty whose research groups are dedicated to the field of early cancer detection using experimental and computational approaches in biochemistry, bioengineering, bioinformatics, molecular imaging, and cancer biology. Proposed Canary CREST Program activities include: (1) Mentor-directed research in one of six investigative areas, namely, development of devices for cancer diagnostics, cancer biomarker discovery and validation, cancer biology, molecular imaging of cancer, clinical imaging of cancer, and cancer bioinformatics; (2) Specially-designed classroom sessions to provide a conceptual framework of the field of early cancer detection; (3) Seminars in scientific research; (4) A comprehensive professional development component that includes career talks, student presentations, workshops on communication skills, career opportunities, and collaboration. The key aspect of this program is to offer participants the opportunity to conduct mentor-directed research while developing an understanding of hypothesis-driven studies with critical interpretation of results and analysis of data. The long-term objective of this educational research program is to support the growing need of specialized researchers who will have a significant impact in the rapidly-expanding area of cancer early detection.

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY (Overall) We describe here the vision and plan for the second five-year period of support for a NIDA Center of Excellence, Neural circuit dynamics of drug action. This Center is dedicated to the development, application, and dissemination of brainwide and cellular-resolution analyses of altered states elicited by drugs of abuse. Our science will focus on identifying the causal circuit-level actions of drugs of abuse in modulating behavior relevant to assessment of context, risk and reward. In a manner that brings together the collaborating groups of the Center, we focus on clinically significant drugs with different molecular profiles but shared significance for understanding behaviors and perceptions relevant to social and nonsocial risk and reward. Specific agents employed include methamphetamine, MDMA, and ketamine, in the setting of validated human and rodent social and nonsocial behaviors. We will develop the brainwide technologies and engage in extensive outreach, training, and education to broaden impact, with the NIDA IRP and beyond. The Center includes four Research Projects (1: led by Dr. Karl Deisseroth, focusing on methamphetamine, MDMA and ketamine action in the cortex and across the brains of mice; 2: led by Dr. Lisa Giocomo, focusing on methamphetamine and ketamine action in entorhinal cortex and hippocampal formation of mice; 3: led by Dr. Robert Malenka and Dr. Boris Heifets, focusing on methamphetamine and MDMA action across the brain of mice; and 4: led by Dr. Leanne Williams and Dr. Brian Knutson, focusing on human structural and functional imaging relevant to methamphetamine, ketamine, MDMA, and risk/reward relationships. Broad and diverse interactions amongthese groups and external collaborators will be further enriched by the Center’s vital Training Core for disseminating these techniques to advance drug abuse research, a Technology Core for developing the next- generation technologies suitable for application to drug abuse research, and an Administrative Core for orchestrating these important interactions. This approach to the NIDA Center will allow us to capitalize on the unique strengths of our team, crossing scales from molecules and synapses, to circuits and behavior, reaching the scope of the intact human brain as we identify relevant structure-activity relationships within animal and human nervous systems.

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY The continuing discoveries of RNAs and their critical roles in cellular and viral machinery are inspiring novel antibacterial, antitumor, antiviral, and genome-editing therapies based on disabling, manipulating, and repurposing the RNAs involved. Unfortunately, our poor biophysical understanding of `how RNAs work' is slowing the development of these potentially life-saving efforts. A critical bottleneck has been the inapplicability of crystallography, NMR, phylogenetic analysis, and biochemical methods to determine the partly ordered conformations of non-coding RNAs in all their functional states. To address this bottleneck, we bring together biophysical modeling, electron microscopy, high throughput biochemical/sequencing experiments, machine learning, wet-lab- integrated crowdsourcing, and a wide collaborative network. Current projects that exemplify our approach involve the COVID-19 pandemic. With our Ribosolve hybrid structure determination pipeline, we are discovering that numerous segments of the SARS-CoV-2 RNA genome form well-defined 3D structures whose targeting by antisense oligonucleotides inhibits viral replication. In the OpenVaccine challenge, we are developing highly structured COVID-19 mRNA vaccines with sufficient in vitro stability to enable world-wide shipping of mRNA in prefilled syringes. This COVID-19 research has benefited from our agile approach and the flexibility allowed by MIRA support; many of the computational and experimental methods we use now did not exist before the pandemic. Because RNA is so fundamental to life, tackling many of science's further `big questions' in human disease could be accelerated if we could visualize and design any RNA. My lab seeks to create the RNA computational and experimental foundation needed to get all of us there in upcoming years.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY/ABSTRACT Plant natural products (NPs) are a critical source of clinically approved drugs and dietary nutrients, yet very few complete biosynthetic pathways have been characterized. As a consequence, many complex plant natural product scaffolds are currently still isolated from the producing plant or plant cell culture and then converted to a clinically-used drug by semisynthetic routes (e.g. etoposide, digoxin, morphine, vinblastine, and paclitaxel – all on the 2015 WHO list of essential medicines). Lack of information regarding their biosynthetic pathways severely limits the use of promising new approaches to produce plant molecules in heterologous hosts (e.g. yeast strains that make artemisinin), as well as the intriguing possibility of engineering the biosynthetic pathways to access analogs and non-natural derivatives with greater efficacy. Even less is known about pathways that could be the target of engineering or breeding efforts in edible plants to improve nutrient content. Given the critical role of plant natural products in human health and utility of biosynthetic genes, we propose here the development and application of a broadly generalizable platform to accelerate the discovery and engineering of key plant natural product pathways. One of the most challenging steps limiting the discovery of plant pathways to date is the identification of candidate biosynthetic genes. Here we propose three complementary approaches for pathway elucidation that we anticipate will enable access to small molecules with diverse biological activities relevant to human health: (1) comparative transcriptomics for branching families of plant natural products, (2) gene-to-metabolite correlation to uncover pathways that require whole-plant coordination for biosynthesis, and (3) gene-centric discovery targeting privileged pathway enzymes. These approaches have recently enabled the discovery of an 8-gene pathway to colchicine alkaloids, and engineering of this pathway into a heterologous production host. In this proposal we have prioritized pathway for clinically used NPs (homoharringtonine [Synribo] and galantamine [Razadyne]), molecules with immune modulatory activity in edible plants (tomato glycoalkaloids), and clinical candidates whose assessment would be enabled by access to the native compound or analogs (limonoids, huperzines, and indolizidine alkaloids). These compounds represent a diverse set of NP classes and will be used to demonstrate the broad utility of our discovery approach. A major outcome of this work will be sets of biosynthetic genes that can be used to engineer heterologous hosts to make plant NPs and analogs with potent biological activity of relevance to human health.

NIH Research Projects · FY 2024 · 2017-07

The All of Us Research Program has an explicit goal to enroll at least one million people from all communities and to leverage these data to learn about and improve the health of all of us. Our project aims to inform the All of Us Research Program about participant input and concerns to inform recruitment and retention, advise and enhance program improvement, and train/mentor biomedical scientists to help the All of Us Research Program realize its ambitious goals. Our Stanford University team holds deep expertise in conducting community-engaged research; operationalizing community input into program improvements; leading digital enrollment and retention with large, national, longitudinal cohorts; and mentoring investigators towards successfully completing research outputs (i.e., manuscripts, conference presentations). We will leverage this expertise to support the All of Us Research Program.

NIH Research Projects · FY 2025 · 2017-07

Our studies pursue exciting new data supporting the hypothesis that the circulating monocyte (MON) and its derivatives, the MON-derived dendritic cell (MO-DC) and the MON-derived macrophage (MO-MØ) orchestrate a chronic innate immune response that underlies the progressive occlusive vascular pathology in pulmonary arterial hypertension (PAH). In Aim 1, we relate a reduction in BMPR2, the most frequently mutated gene in PAH, to an increase in the retroviral element HERV-K in MON. We extend new findings in MON suggesting that reduced BMPR2, through a DNA repair response, increases phosphorylated KAP1, resulting in transcriptional activation of XIST, the lncRNA that inactivates the X chromosome. We elucidate whether the increase in XIST competitively recruits the deacetylase SPEN away from HERV-K, resulting in an increase in HERV-K expression and consequent viral innate immune interferon (IFN)-STAT1 signaling. We determine whether this is a feature of MON in all forms of PAH, particularly in females, and persists in PAH MO-DC and MO-MØ. We investigate whether the propensity to apoptosis in PAH MON is related to IFN-STAT1 signaling and results in differentiation of surviving MON that invade pulmonary arteries to become pro-inflammatory MO-DC and MO-MØ. In Aim 2, we add MON differentiated from induced pluripotent stem cells (iPSC) (iMON) to novel bio-fabricated vascular tubes perfused under physiologic or high shear stress and populated with iPSC-differentiated endothelial cells (iEC) lining the lumen surrounded by circumferentially arranged iPSC-differentiated smooth muscle cells (iSMC) derived from PAH patients carrying a BMPR2 mutation or from control subjects. Single cell RNA Seq is applied to find mechanisms explaining (i) differentiation of iMON to iMO-DC and iMO-MØ, (ii) iMO-DC induced iSMC proliferation, and (iii) iMO-MØ impaired phagocytosis. Monocytes from athymic rats treated with SUGEN 5416 where females develop more severe PAH than males, and Bmpr2+/- rats treated with 5-lipoxygenase that develop PAH with no gender bias will be assessed for the innate immune response observed in human PAH MON. Two strategies will be used to trace MON differentiation to MO-DC and MO-MØ during the evolution of PAH in the rats and we will determine whether blocking IFN prevents pulmonary vascular remodeling and PAH. In Aim 3 CRISPR inhibitory technology is applied in which iPSC expressing dCAS9KRAB are differentiated to iMON and transduced with guides targeting a reduction in 9 genes expressed in MON and mutant in PAH. We will determine whether these cells share a common pathway of gene dysregulation. Bio-fabricated vascular tubes populated with iEC and iSMC with a reduction in these PAH genes are used to determine the contribution of mutant vascular cells to iMON differentiation to iMO-DC and iMO-MØ. Genome wide CRISPR screens are applied to find regulators of HERV-K expression and agents that improve PAH MØ phagocytosis. By uncovering the mechanism causing an innate immune response in MON, MO-DC and MO-MØ, we aim to find new ways to treat PAH and conditions associated with a protracted innate immune response, e.g., related to IFN-STAT1 signaling.

NIH Research Projects · FY 2026 · 2017-06

Project Summary (30 lines) With its revised title “Emergent Properties of Complex Systems: From Atoms to Macromolecules; from Humans to Societies” this proposal has been broadened by adding data-analysis & simulation on a problem of grave current concern: namely how an air-borne virus like SARS-2-CoV spread in human population. Getting involved by accident, I became fascinated with how the numbers of daily cases & deaths group with time and what is the physical mechanism that make the data follow the Gompertz function. Michael Levitt, the Principal Investigator has a long career of independent scientific research that started in 1967 when he was one of the first to work in computational biology. His early work set up the conceptual, theoretical and computational framework for protein and DNA structure refinement, structure analysis and macromolecular simulations. He makes computer codes available and has been productive, scientifically rigorous and impactful for half a century. This approaches is continued here by a PI committed to mentoring young scientists as well as engaging in sustained research-community service and public outreach. 1. Protein Structure Refinement with Deep Equivariant Networks. We propose to use Deep Learning technique to refine models of proteins. We anticipate that such an approach, combined with the power of modern neural net architectures and computational performance of hardware will enable efficient sampling of the protein conformational space near the native state and will systematically provide structures with accuracy useful for drug development purposes. 2. Functional Dynamics of Ribosome. Our experience with structure curation will lead to a useful computer package for others. Our work on Ribosome dynamics will provide a model of how peptides such as SecM can stall the ribosome. Structures sampled from our MD simulations could also be used as potential targets for drug discovery. 3. Epidemic Analysis, Curve-Fitting and Simulation. Applied to SARS-Cov-2 and COVID-19, we show that viral spread follows the Gompertz growth function rather than commonly assumed Logistics or Exponential functions. This means that the population transmitting the infection is not uniform. Network simulation of viral spread shows that only when the connection network is scale-free does the simulated epidemic follow the Gompertz function. We will model a physical system with scale-free connectivity using molecular dynamics to simulate a 2D gas of particles with a wide range of masses. This novel multi- disciplinary approach may also apply to future respiratory viruses to enable better control of their spread. Studying biomedically significant systems in collaboration with experimental colleagues will reveal fascinating details of biology in action. We expect this work will help elucidate the relationship between underlying structure and function in complex systems, extending from macromolecular machines to human societies.

NIH Research Projects · FY 2026 · 2017-03

Project Summary Tuberculosis remains one of the leading causes of death worldwide, despite the widespread availability of effective treatment and prevention measures. Prisons harbor among the highest rates of tuberculosis worldwide, and, in many regions, tuberculosis burden in correctional settings is growing. The WHO now recommends active case finding for tuberculosis in prisons and other high burden settings, but there is a dearth of evidence for how to accurate and efficiently identify cases early to reduce transmission. To address these gaps, we propose to a prospect cohort study among incarcerated individuals in high tuberculosis burden prisons in Central Western Brazil to: 1) evaluate the use of portable, digital x-rays with automated interpretation for intensive case finding; 2) determine whether genomic data can be used to track transmission rates at the population level; and 3) use mathematical models to identify effective, scalable strategies for tuberculosis case finding and prevention in prisons. We will test the hypotheses that: 1) x-ray with automated interpretation can achieve WHO target product profile thresholds for accuracy as a screening test among incarcerated individuals, even among those with asymptomatic or early disease; 2) emerging phylodynamic methods can be used to monitor trends in transmission; and 3) serial screening combined with preventive therapy would be a cost-effective and impactful approach for tuberculosis control in high-burden prisons globally. Overall, this project will address critical gaps in tuberculosis diagnosis and prevention among a large, underserved, high-risk population, to protect their health and the health of their communities.

NIH Research Projects · FY 2025 · 2017-03

This project focuses on understanding a fundamental cellular mechanism underlying a range of important physiological signaling in humans including the control of feeding and obesity. The mechanism uses an ancient cellular signaling organelle, the primary cilium, to control responses to satiety signals generated following feeding. Bardet-Biedl syndrome (BBS) is a rare human syndrome called a ciliopathy because of mutations in genes encoding components of the primary cilium. Patients with BBS have inherited mutations in genes linked to a complex called the BBSome, discovered in our laboratory, that fail to present receptors critical to limit feeding after a meal. Our work has found that cilia also control adipogenesis via the de novo generation of new fat cells and the secretion of insulin and glucagon in pancreatic islet cells. We have focused on mechanisms of ciliary signaling and trafficking, enabled by the use of affinity purification/mass spectrometry to identify new components of the ciliary machinery. These studies have been initiated by using the ciliopathy disease genes as bait proteins to find new components and cell biological pathways linked to ciliary traffic and signaling. A number of these newly discovered components are themselves mutated in human pedigrees linked to obesity. In particular, a ciliary structure called the distal appendage serves as a critical gate for entry of ciliary receptors. We find that mutations in components of the distal appendage are linked to monogenic obesity syndromes. As monogenic obesity syndromes are rare, the lab has shifted to systematically surveying public data for over 750,000 patients in Genome Wide Association Studies (GWAS) for genes found to be altered in patients with high Body Mass Index (BMI) (a key measure of obesity) and diabetes. We have discovered 100s if not 1000s of candidates for a substantially broader list of candidates for obesity drivers linked to cilia in nonconsanguineous populations. In Aim 1 of this proposal, we will further explore the mechanisms by which the distal appendage is assembled and how that organizes trafficking into the cilium. In Aim 2, we will examine how the distal appendage traffics receptors and generates signals in the cell. In Aim 3, we will explore a new factor of the distal appendage, called CCDC92, which potentially controls signaling via proteolytic destruction of ciliary signaling regulators. In each Aim, we will use genetic lesions derived from patients with high BMI which we find have screened for defects in ciliary trafficking or signaling. Our goals are to continue to explain obesity lesions to allow accurate assessment of a patient’s genetic obesity drivers, to identify additional druggable targets for obesity and diabetes therapeutics, and to communicate these findings to the public to help predict dietary susceptibilities based on molecular genetic profiles. By identifying signaling pathways defective in obesity and diabetes, we can identify targets to protect or restore these tissues and molecular profiles of patients to facilitate patient selection for treatments to improve obesity and metabolic disease.

NIH Research Projects · FY 2024 · 2017-02

PROJECT SUMMARY The Human RegulomeDB project provides an essential resource that facilitates medical research and exploratory investigations of gene regulation. The majority of sequence variation identified in genome sequencing projects and disease association studies (GWAS) lie within the 98% of the human genome that is non-exomic. RegulomeDB is a unique web accessible resource that provides integrated knowledge of the wealth of existing information concerning regulatory elements that lie within non-exomic regions. The unique feature of this resource is its ability to comprehensively annotate, integrate and display the experimentally defined functional and biochemical regulatory elements of the human genome. Information generated from individual laboratories and consortia concerning potential regulatory regions such as that affecting gene expression, transcription factor binding, chromatin modification and DNA methylation will be collected from the literature, and integrated into a common database and displayed at nucleotide resolution. The information can be readily accessed via a web accessible interface and related to sequence variations identified from large scale projects (e.g. db SNPs, 1000 genome project, GWAS studies). Researchers will be able to compare variants identified from personal genomes and large scale sequencing projects as well as GWAS studies to the wealth of information in RegulomeDB, and thereby rapidly gain knowledge of non-exomic information. Given the wealth of DNA sequencing project that are emerging, we expect this unique resource to have wide impact in the biomedical community.

NIH Research Projects · FY 2026 · 2017-02

PROJECT SUMMARY/ABSTRACT Pregnancy is a unique period in which the inherent biological complexity of any single human organism is exponentially amplified by an intimate interaction between a rapidly developing fetus and an adult mother who exhibits remarkable physiological adaptations over the nine months of pregnancy. Importantly, the biological interests of the two organisms are not always congruent, reflecting conflicting metabolic interests and limited supplies. Furthermore, maternal-fetal interaction does not occur through a passive sieve, but is actively and dynamically orchestrated by the placenta, an organ with its own set of physiological needs. It is therefore apparent that any disruption of the homeostatic equilibrium among the mother, placenta, fetus or their environment may manifest as a clinical disease that challenges maternal physiology (e.g., preeclampsia) or fetal development (e.g., fetal growth restriction), or may lead to premature termination of the pregnancy (e.g., preterm birth). The intact function of the placenta includes a set of signals that are generated by placental trophoblasts and communicated to the maternal and/or the fetal compartments. These signals include hormones (proteins, glycoproteins, steroid hormones) and growth factors, which have a paracrine and endocrine effect on maternal and, possibly, fetal tissues. Our new line of research centers on nanovesicle (exosome)-based communication. These exosomes are produced in human trophoblasts and harbor signals that are germane to pregnancy health. Among these signals are placenta-specific microRNAs (miRNAs) that, we recently showed, confer viral resistance to recipient cells. These miRNAs may also impact local placental biological processes, such as trophoblast migration and invasion. While the placenta produces an abundant number of exosomes, their target tissues are currently unknown. Moreover, the mechanisms by which placental exosomes deliver their cargo to target cells and the regulation of their intracellular function have not been hitherto investigated. We therefore seek to test the hypothesis that human trophoblastic exosomes use specific uptake mechanisms to target maternal tissues, locally and distantly, and impact cell function. We will test our hypothesis using human trophoblasts and exosomes derived from pregnant women. For those experiments that cannot be performed in humans, we will use mice that have been validated as appropriately modeling the human processes under study. Ultimately, our data will illuminate previously unknown mechanisms of crucial, exosome-based communication between the feto-placental and maternal compartments. Further, as placental exosomes are accessible via the blood, data generated by our investigation will introduce new means to investigate the human placenta, and may promote the use of exosomes as part of the diagnostics of placental dysfunction and indicate new avenues for nanoparticle-based therapeutics.

NIH Research Projects · FY 2025 · 2017-02

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by abnormally elevated pulmonary pressures and right ventricular (RV) failure. Inappropriate angiogenesis is a key pathological feature of PAH associated with endothelial dysfunction and progressive loss of pulmonary and RV microvessels. Angiogenesis is the process by which new vessels arise from existing vessels and is mainly driven by VEGF signaling. In response to VEGF-A, endothelial cells differentiate into tip cells, highly motile cells that direct vessel sprouting and elongation. Our previous R01 was built on the hypothesis that tip cell formation by PMVECs requires crosstalk between the VEGF and the Wnt/planar cell polarity (Wnt/PCP), a pathway responsible for coordinating cell movements during tissue morphogenesis. We demonstrated that Wnt/PCP activation in PMVECs is driven by the interaction between the ligand Wnt7a and ROR2, a tyrosine kinase receptor that phosphorylates residues in the endothelial VEGFR2 cytoplasmic domain to augment VEGF signaling output. We found that, compared to healthy donors, tip cell formation and angiogenesis in response to VEGF-A was significantly reduced in pulmonary microvascular endothelial cells (PMVEC) from PAH patients. Most importantly, supplementation with recombinant Wnt7a or restoration of ROR2 expression in PAH PMVECs results in recovery of the VEGF-A response, leading us to conclude that Wnt7a/ROR2 signaling is required for appropriate VEGF signaling activation and angiogenic response in PMVECs. This renewal will focus on elucidating the transcriptional and epigenetic mechanisms that regulate Wnt7a/ROR2 expression in healthy and PAH PMVECs (Aim 1), how interaction between ROR2 and integrins is required to establish a functional lung endothelial barrier (Aim 2), and the critical role of Wnt7a/ROR2 as a key pro- angiogenic mechanism that supports compensatory angiogenesis during RV adaptation to PAH (Aim 3). The studies in this renewal will confirm the role of Wnt7a/ROR2 signaling as a master regulator of cardiopulmonary angiogenesis and demonstrate the therapeutic potential of restoring Wnt7a/ROR2 signaling to prevent small vessel loss and improve RV function in PAH.

NIH Research Projects · FY 2026 · 2017-01

Building on the extensive genome wide association studies (GWAS) of coronary artery disease (CAD), and single cell characterization of atherosclerosis, we have shown that the smooth muscle cell (SMC) lineage harbors much of the risk for vascular disease. Further, these data indicate that SMC can assume two disease- related transition states linked to disease risk. We identified TCF21 as a protective CAD GWAS gene and showed that it regulates a disease-related transition of medial SMC to a fibroblast-like phenotype, producing cells we term “fibromyocytes” (FMC). Further, we and others have found that SMC can also transition to a second phenotype, characterized by expression of genes known for their role in endochondral bone formation and intimal vascular calcification. Mouse genetic models have indicated that this chondrogenic process, which gives rise to cells we term “chondromyocytes” (CMC), is promoted by CAD GWAS causal factors Pdgfd and Twist1, and actively inhibited by protective TCF21 target CAD GWAS signaling molecules ZEB2 and SMAD3. Further, recent transcriptomic and epigenomic (multi-ome) single cell studies indicate that FMC and CMC are the end products of independent trajectories that SMC can differentially traverse in the disease setting. We thus hypothesize that CAD protective GWAS genes TCF21 and ZEB2 promote disease protective FMC and inhibit disease promoting CMC trajectories through regulation of critical enhancers and their regulatory transcription factors (TFs) that underlie the fundamental mechanisms of CAD genetic risk in the SMC lineage. Algorithms that link disease variation and disease transcriptomic data at the whole genome level show that disease genetic risk resides in the cell state changes from SMC to FMC and SMC to CMC. We now propose to investigate the enhancers, related TFs and their target genes (regulons) that mediate these phenotype transitions, through the following Aims. In Aim 1, we will perform time-course studies with mouse disease models targeted for disease inhibiting CAD associated genes Tcf21 and Zeb2 to map the cis- and trans-acting factors and disease-related regulons that determine the disease trajectories and directionality of disease risk. Findings from these studies will be mapped onto multi-omic single cell assay data of human coronary lesions to establish that orthologous enhancers and molecular SMC mechanisms mediate phenotype transitions in human disease. In Aim 2, we will address the hypothesis that over-expression of Tcf21 in medial SMC will promote increased transition to the FMC phenotype, as indicated by epigenetic and gene regulatory network modules, as well as increased fibrous cap formation and additional risk protective features. In Aim 3, CRISPRi PerturbSeq experiments in human coronary artery smooth muscle cells will validate the results from previous Aims, and further characterize downstream enhancers, related TFs, and regulatory networks. The proposed studies will identify cellular and molecular mechanisms that mediate SMC transitions to FMC and CMC, and the relationship of these transitions to vascular calcification and disease risk.

NIH Research Projects · FY 2025 · 2017-01

Project Summary IgE antibodies and receptors are important mediators of the allergic cascade, but may also provide distinct functions in anti-tumor immunotherapy. IgE engages its high affinity IgE Fc receptor (FceRI) on mast cells and basophils as well as a low affinity receptor (FceRII/CD23) expressed on B cells and epithelial cells. IgE binding to FceRI sensitizes allergic effector cells to allergens and is very stable, with the complexes persisting for months even in the presence of potent anti-IgE antibodies. We have developed approaches to rapidly destabilizing IgE:FceRI complexes and desensitizing allergic effector cells using hybrid, bispecific IgG/DARPin molecules that recognize two distinct epitopes on the IgE-Fc. Here in Aim 1 we will explore alternative approaches to target and remove IgE:FceRI complexes from cell surfaces using novel bispecific anti-IgE antibodies. In addition, we will use two anti-IgE antibodies that we have produced to gain greater insights into the structure and dynamics of intact IgE. The IgE interaction with CD23 also mediates important biological effects in B cells, together with CD21. Here, in Aim 2, we will use yeast display to evolve higher affinity variants of CD23 for its ligands to enable structural studies of these complexes and to probe CD23-dependent regulation of IgE production by B cells. Finally, in Aim 3, we seek to isolate and study new anti-FceRIa antibodies using yeast display, to develop novel reagents for the FceRI-directed modulation of allergic effector cell activities.

NIH Research Projects · FY 2026 · 2016-12

ABSTRACT Lack of physical activity (PA) has been identified as the fourth leading risk factor for global mortality and a major contributor to disability from non-communicable diseases (NCDs) such as metabolic disorders (e.g., diabetes), cardiovascular disease, neurological diseases, and cancer. Conversely, PA has multiple physiological benefits and effectively prevents and treats NCDs. Moreover, regular PA also contributes to overall physical and mental wellbeing. Despite indisputable evidence that regular PA has a profound beneficial impact, the molecular mechanisms by which PA promotes human health remain poorly understood. Identification of these mechanisms holds great promise for discovery of novel therapeutic targets for disease prevention and treatment, and improvement of classical drug treatment with PA. In this context, high throughput ‘omics’ technologies have the unique ability to reveal the biological networks affected by PA and increase our understanding of how PA works at a molecular level. When complemented with genome data, there is an opportunity to understand how genetic variation leads to phenotype variation in PA and associated health benefits. The overall goal of the Molecular Transducers of Physical Activity Consortium (MoTrPAC) is to understand the biological processes and pathways through which physical activity influences health and disease outcomes. MoTrPAC has been developing a molecular map of transducers that underlie the effects of physical activity in humans through a variety of omics technologies, namely genomics, epigenomics, transcriptomics, metabolomics, proteomics, and lipidomics. This data are subsequently provided to the research community through the MoTrPAC Data Hub. To date, 966 individuals have had whole genome sequencing (WGS) data produced and there is a critical need to complete WGS on 1188 additional participants and to complete transcriptomics past Tranche 5 on at least 2200 more samples. Generating these data will capture essential genetic and molecular variability and make use of valuable specimens provided to MoTrPAC participants. Such data will significantly advance the outcomes and investment in MoTrPAC as more complete accounting of genetic variability can be connected to molecular and phenotype variability for analysis of trial outcomes. Through this data we will be able to combine genome and molecular omics data to expand understanding of how genetic variation impacts exercise molecular responses and shares impacts with a broad range of health-related phenotypes. We expect this data will maximize the return on investment in the MoTrPAC human clinical trial.

NIH Research Projects · FY 2025 · 2016-12

Abstract Few interventions have been shown to be as beneficial to human health as physical exercise, yet we remain largely ignorant of the mechanisms by which those potent effects are transduced. The Molecular Transducers of Physical Activity Consortium examines the response to acute and chronic exercise at multiple scales and in multiple tissues across thousands of humans and in animal models. The studies of the Consortium combine state of the art phenotyping with molecular omics approaches. Building on our long history of analytical innovation in high throughput biology and experience in the analysis of perhaps the largest multi-omic study funded to date, the Stanford MoTrPAC Bioinformatics Center provides core compute, storage and analytic expertise to the MoTrPAC investigators. In this administrative supplement, the MoTrPAC BIC proposes to continue its collaboration as part of the CFDE; to contribute to data organization to enhance MoTrPAC dataset FAIRness; to regular interact with other CFDE entities; and to advance the mission of the Common Fund Data Ecosystem through outreach and dissemination activities. We propose to interface and collaborate with the Common Fund Data Ecosystem to improve the interaction of MoTrPAC data with other Common Fund data resources. We remain focused on developing data standardizations and reproducible analysis pipelines for various ‘omes in collaboration with the CF DCCs that can be repurposed and customized by the scientific community; we will continue transcriptomic data harmonization in collaboration with several CFDE entities. We will continue to harmonize the data catalog of the MoTrPAC BIC with the CFDE data model and to record and analyze user experience and deploy tools and training for the research community to easily use existing datasets to address novel cross-cutting biological questions . We expect that with the above activities, we will contribute towards CFDE’s long term goal of developing and deploying resources and tools, training materials, empowering the research community to use CF data sets for novel scientific research, hypothesis generation, discovery, and validation, leading to new insights into health and disease.

NIH Research Projects · FY 2025 · 2016-09

The Stanford Molecular Imaging Scholars (SMIS) program is an integrated, 3-year cross- disciplinary postdoctoral training program at Stanford University that brings together 29 faculty mentors from 13 departments in the Schools of Medicine, Engineering, and Humanities and Sciences. Molecular imaging (MI), a noninvasive technique to visualize and quantify specific molecular and biochemical processes in living organisms, has revolutionized medicine and biomedical research, and continues to expand its applications in the detection, treatment, and management of cancer. SMIS faculty mentors, and extensive resources, provide a rich and diverse training environment spanning fields such as biology, physics, mathematics, biocomputation/biomedical informatics, engineering, chemistry, biochemistry, cancer biology, immunology, and medical sciences. The centerpiece of the SMIS program is the opportunity for trainees (with PhD, MD, or MD/PhD degrees) to conduct innovative molecular imaging research that is co-mentored by faculty in complementary research and clinical disciplines. SMIS trainees also engage in specialized coursework, seminars, national conferences, clinical rounds, including ethics training and the responsible conduct of research. The three-year program culminates with the preparation and review of a mock NIH grant proposal, in support of trainee transition to an independent career in cancer molecular imaging. Fifty-one trainees (7 current; 44 former) have entered the SMIS Program since its inception in 2006. Our graduates have moved on to faculty positions, other academic positions, or working in biotechnology/pharmaceutical research positions. The overall goal of the SMIS program is to continue to provide talented young investigators with the scientific and professional education/career development opportunities to become leaders in the field of molecular imaging of cancer.

NIH Research Projects · FY 2025 · 2016-08

PROJECT SUMMARY: Nearly 40 million people in the United States annually are provided anesthesia for surgery. Reactive aldehydes, produced during surgery, are toxic metabolites which drive cellular dysfunction and end organ damage. For Asian Americans that are descents from East Asia a genetic variant (present in nearly 540 million people in the world) severely limits reactive aldehyde metabolism. Besides inefficient reactive aldehyde metabolism, several other genetic differences occur for Asians within the lipid peroxidation pathway; a pathway which regulates damage that occurs during organ injury. As mortality following surgery is the third leading cause of death in the United States, with organ injury a major cause of this mortality, understanding how these genetic differences in Asians may alter lipid peroxidation-induced organ damage could unlock novel treatment strategies for all ethnicities to reduce organ injury occurring during surgery. For this MIRA program, we will study the genetic differences existing within the lipid peroxidation pathway for Asians, including the genetic variant which causes inefficient reactive aldehyde metabolism and the impact on organ injury. To carry out these studies, we generated tools to study reactive aldehydes in the basic science laboratory including a knock-in mouse model to reflect the human genetic variant that causes inefficient reactive aldehyde metabolism and sensitive assays to detect reactive aldehydes. We will use these tools to examine whether an analgesic given during surgery exacerbates cellular injury for rodents with inefficient aldehyde metabolism. Further, we will also study how reactive aldehydes may impact a transient receptor potential channel to trigger organ injury. We also expanded these aldehyde tools by developing non-invasive methods to assess reactive aldehyde levels in humans and methods to identify phenotypes for inefficient reactive aldehyde metabolism. We plan to use these tools in humans undergoing surgery to identify people with inefficient reactive aldehyde metabolism and monitor their aldehyde levels during surgery in real-time. Asian Americans are one of the fastest growing populations in the United States and are projected to reach nearly 34 million by the year 2050. Asian Americans will require specific anesthetic plans for surgeries due to genetic differences in the lipid peroxidation pathway, including genetics which cause inefficient reactive aldehyde metabolism. Providing precision medical care for people who require surgery with this genetic variant will ultimately reduce health care costs and improve quality of care for a large subset of Asian Americans. As we describe here, studying genetic differences can also provide insight into biological mechanisms and unlock novel strategies that can impact medical care for people of all ethnicities.

NIH Research Projects · FY 2025 · 2016-08

The coordination of cellular function with the environment is essential for adaptation and survival. Dynamic nutrient environments are ubiquitous throughout nature and include competitive growth environments of proliferating microorganisms and tissue niches in multicellular organisms. Failure to adapt can lead to cell death, developmental defects, and disease. Adaptive cellular responses are often achieved by rapid inducible changes in gene expression programs. An ideal mechanism to achieve this is through modification of chromatin. Despite this knowledge, the mechanisms by which chromatin modification contributes to metabolic plasticity remain largely unexplored. As such, many broad biological questions remain unanswered: How do metabolic signaling pathways communicate with chromatin to regulate gene expression? How does the metabolic environment modify chromatin to facilitate adaptive gene expression and coordinate cell division? How do chromatin modifications influence energy metabolism plasticity during developmental programming? How is metabolic memory propagated? Our proposed research is significant because it will establish chromatin modifiers as necessary components of metabolic homeostasis, and serve as a platform to investigate epigenetic alterations and metabolic dysfunction in developmental abnormalities and disease states. Our broad research goal is to define the chromatin modification events that coordinate metabolic plasticity and are central to adaptive cellular responses. Our central hypothesis is that chromatin modifiers link nutrient sensing pathways to metabolic gene regulation required for proper fitness, proliferation, and development. We plan to investigate this hypothesis using innovative approaches that include metabolic-synchronization, as well as single-cell chromatin and metabolic profiling. We are ideally suited to carry out these studies, as our research was the first to demonstrate that a chromatin remodeling complex functions downstream of metabolic signaling pathways to regulate coordinate metabolism with cell division and developmental timing. Through achievement of our research goals we expect the following outcomes: Comprehensive determination of histone modifications that are in tune with energy metabolism pathways; determination of the relationship between nutrient sensing pathways and chromatin; characterization of the tissue-specific metabolic requirements during development; identification of novel chromatin-mediated mechanisms for metabolic memory and diversification. These investigations will greatly enhance our knowledge of metabolic plasticity mechanisms and how they contribute to cellular and organismal viability, development and disease.

NIH Research Projects · FY 2025 · 2016-07

Project Summary Signal transduction in development and disease (PI: Rohatgi) The goals of my research program are to uncover new regulatory mechanisms in cell-cell communication pathways, to understand how these mechanisms are damaged in disease states, and to devise new strategies to repair their function. Over the last 4.5 years, funding from the NIGMS has supported 23 publications across four different research areas in my laboratory: Hedgehog (Hh) signaling, WNT signaling, drug resistance mechanisms and intrinsically disordered proteins. Trainees involved in MIRA-supported research have won competitive fellowships (including a K99/R00 award from the NIGMS) and obtained independent group leader positions in both academia and industry. The next project period will tackle major unsolved problems in the vertebrate Hh and WNT signaling systems, two iconic cell-cell communication pathways that coordinate the construction of tissues during development and their subsequent maintenance throughout adult life. Despite the importance of these pathways in human diseases ranging from birth defects to cancer and degenerative conditions, many steps in Hh and WNT signaling remain poorly understood at the biochemical and cell biological level. In the Hh pathway, our focus is on understanding how a signal is detected at the cell surface and transmitted across the plasma membrane to transcriptional effectors in the cytoplasm. These signaling steps in the vertebrate Hh pathway depend on primary cilia, antenna-like organelles that project from the surfaces of most cells and are implicated in human birth defect syndromes called “ciliopathies.” Major questions under investigation include (1) how Patched 1 (PTCH1), the receptor for Hh ligands, regulates the function of Smoothened (SMO), the protein that transmits the signal across the membrane, (2) how SMO is activated at primary cilia and (3) how SMO signals to the Glioblastoma (GLI) family of transcription factors. Our MIRA- supported work has led to a new paradigm in transmembrane signaling: the use of cholesterol accessibility in the ciliary membrane as a second messenger to communicate the signal between PTCH1 and SMO. Our focus in the WNT pathway is on the multi-protein β-catenin destruction complex that suppresses WNT signaling by promoting the degradation of β-catenin. Defects in this complex drive the vast majority of colorectal cancer, a disease with an increasing burden (especially amongst people <50 years of age) predicted to cause over 1 million deaths yearly by 2030. Our emphasis is on uncovering differences in the genetic and biochemical requirements for oncogenic (mutation-driven) and physiological (ligand-driven) WNT signaling, since any successful anti-WNT drug will have to distinguish between the two to achieve an acceptable therapeutic index. Our work is supported by long-term collaborations and embraces a broad range of techniques that span structural biology, lipid biochemistry, CRISPR/Cas9-based genetic screens and microscopy. The successful completion of this project will provide a deep mechanistic understanding of these fundamental cell-cell communication systems and new strategies to monitor and modulate these pathways in human diseases.

NIH Research Projects · FY 2025 · 2016-07

Project summary/abstract This MIRA renewal grant proposal briefly summarizes the accomplishments over the past 4 years of support and outlines plans for continued support. The theme that unifies this research is the development and application of new physical methods that can impact the quantitative analysis of complex biological systems. The freedom to develop and broaden our research provided by the MIRA support has led to a significant evolution of the emphasis of part of our work on infectious diseases. Specifically, we will focus on the biomedically critical need to understand the origin(s) of antibiotic resistance using the TEM -lactamases as an initial target. Likewise, our efforts to develop novel ways to organize and manipulate biological membranes now focus on the mechanism of viral membrane fusion. While these two areas had completely separate origins in the parent R01’s that were merged in the MIRA, they have both provided rich areas for new and impactful research. My lab develops spectroscopic methods for probing protein-exerted electric fields which we use to obtain quantitative information on how electric fields contribute to catalysis at the active sites of enzymes. We led the development of vibrational Stark effect spectroscopy as a general approach to map these fields. Using this approach, we can, for the first time, quantify the electrostatic contribution to the catalytic proficiency of enzymes. Moving beyond ideal model enzymes, we will use this approach to provide a deeper understanding of the mechanism(s) by which TEM--lactamases evolve to cope with man-made antibiotics. By studying the connection between evolution and electric fields, we hope to develop general design principles for these enzymes and discover the physical origins of antibiotic resistance. We discovered that “split” GFPs can be photo-dissociated, and we study the underlying mechanism of this unusual process for optogenetic applications. This deeper view of strand photo-dissociation along with our work elucidating factors that control bond-specific photo-isomerization pathways are connected to our work on protein electrostatics and will provide a framework for understanding GFP’s electro-optic properties. Our lab pioneered the development of model membrane architectures, along with imaging and analytical methods that probe fundamental aspects of biological membrane organization and dynamics. Our current focus is the application of these architectures and novel single particle assays to characterize the elementary steps by which enveloped viruses, such as influenza A, fuse to target membranes. In parallel, we characterize the organization of lipids with high lateral resolution using imaging mass spectrometry. Recently we showed that atom recombination can be used to identify which lipids and proteins are in very close proximity (< 3nm) in biological membranes. This new approach addresses major challenges in membrane biophysics and structural biology where local organization is key to emergent function.

NIH Research Projects · FY 2026 · 2016-07

Project Summary: Overview: Neurosurgical resection, ablation and stimulation can be curative for patients with medically refractory epilepsy (MRE), however, successful treatment depends on precise localization of the seizure onset zone (SOZ). Stereo-electroencephalography (EEG) is the gold standard for determining the SOZ and involves implanting many intra-cranial depth electrodes to detect epileptiform activity. Unfortunately, there are often no visible lesions on brain MRI and only a crude spatial map from scalp-EEG to guide the stereo-EEG implantation, resulting in electrode positions that fail to definitively determine the SOZ. To meet the unmet clinical need for consistent and accurate stereo-EEG guidance we will develop innovative diffusion MRI (dMRI) encoding paradigms that provide specific sensitivity to epileptic microstructural pathology and validate with 3D histology and high-density scalp- and stereo-EEG. Relevance: There are ~400,000 patients with MRE and non-lesional MRI in the U.S. Given the lack of guidance and sparse sampling of stereo-EEG, identifying the seizure onset zone with confidence is extremely challenging for in these non-lesional patients. If we are successful in the proposed work, we will enable MRI identification of a probable lesion location for the majority of patients with MRE. In this way , we will increase their likelihood of a definitive stereo-EEG identification of the SOZ and enable a potentially curative neurosurgical treatment option that might not have been possible otherwise. Approach: Our approach is to: 1) Develop and validate diffusion MRI cortical fiber mapping for detection of the disrupted cortical architecture within focal cortical dysplasias. 2) Test whether diffusion MRI cortical fiber mapping can detect focal cortical dysplasias and 3) Test whether diffusion tractography and functional connectivity MRI can predict seizure propagation. Specifically, we will use these MRI connectivity to predict probable cortical nodes of the seizure propagation network, as well as, the latency and spatial propagation of epileptiform activity between stereo-EEG electrodes. Summary: The proposed neuroimaging methods will improve localization of the SOZ and propagation network enabling more patients with MRE to be treated more effectively with neurosurgery.

NIH Research Projects · FY 2025 · 2016-07

This competitive renewal Stanford T32 Training Grant Proposal combines pediatric and adult pulmonary programs in order to select the most promising Trainees and provide an optimally diverse interdisciplinary training experience centered on pulmonary biology. Dr. David Cornfield, the pediatric chief, Dr. Mark Nicolls, the adult chief share Program Director responsibilities; each bringing unique and complementary skills to the leadership position. In the first few years of this new T32 funded in 2016, we have accomplished our goal of creating a more integrated and cohesive academic culture. We describe the excellent progress of our first nine trainees. We have developed 38 Mentors from the Stanford biomedical community with a strong track record of training and academic productivity and 2 Junior Mentors. Based on the expertise of our Mentors, we have identified nine domains of excellence from which trainees can choose a primary focus for an individualized development plan. These research areas include: 1) Vascular Disease, 2) Stem Cells & Lung Development, 3) Genetics & Genomics, 4) Lung Injury & Repair, 5) Lung Immunology, 6) Lung Microbiome, 7) Lung Cancer and 8) Outcomes Research, and 9) Imaging. The grant proposal describes a process by which fellows are recruited to Stanford, exposed to research areas, introduced to Mentors, and move through a selective process designed to identify Trainees with the best chance of success in academic medicine for T32 support. We describe a well-supported and carefully constructed system of oversight to promote recruitment of qualified candidates. All T32 trainees will have an individualized development plan that includes a core curriculum and electives appropriate to their research domain. Through a longitudinal monthly T32 meeting lead by the PDs and quarterly informal meetings with T32 trainees, Mentors and PDs, Trainees will be immersed in a microculture of academic pulmonary medicine. A Scientific Oversight Committee identifies appropriate coursework and ensures ongoing and timely Trainee progress. We have assembled an experienced Internal Advisory Committee at Stanford and an acclaimed group of national leaders in pulmonary medicine for the External Advisory Committee. We will utilize regular feedback from these groups, the Mentors and Trainees to identify programmatic strengths and weaknesses and reassess our processes to adjust, adapt and improve the T32 fellowship. As a world university embedded in a vibrant local economy, Stanford is particularly well situated to develop and inspire the next generation of physician-scientists and clinical researchers.

NIH Research Projects · FY 2025 · 2016-07

In 2016, we established a T32 research training program at Stanford University to create independent ELSI scholars who can conduct rigorous research on ethical, legal, social or policy implications of genetics and genomics (ELSI). The overall goal of the program is to produce interdisciplinary researchers who can effectively identify and address the complex issues at the intersection of genetics, ethics, law, society and policy. The goal of this renewal is to train 7 more postdoctoral fellows and to continue our successful contribution to the excellence of ELSI research. Program: Interdisciplinary three-year postdoctoral training. A total of seven postdoctoral predoctoral fellows will have completed their training over the five-year award period. Trainees: PhDs and PhD candidates recruited from a wide range of related disciplines, including genetics, biological sciences and engineering, medicine, computer and information sciences, philosophy, health services research, anthropology, and other social sciences. Mentors: Multiple mentorship model, tailored to individual trainee needs and interests. Trainees are assigned a primary mentor responsible for overall development of the trainee’s plans, and secondary mentors assigned based on specific career, research methods, and topic area needs. Program Co-Directors: Mildred Cho, PhD, the current director of the research training program, will be joined by Holly Tabor, PhD as a Co-Director, bringing her experience in ELSI research and training. Program Faculty: 14 Program Faculty from 9 primary departments and centers, representing Schools of Medicine, Humanities and Sciences, Law, and Engineering who conduct ELSI-relevant research. Core Faculty members Holly Tabor, PhD David Magnus, PhD, Hank Greely, JD and Kelly Ormond, MS are experienced mentors in the program. Four new faculty were added, broadening the range of opportunities for ELSI research projects and methodological approaches for fellows. Education Program: Individualized training program for each trainee that includes core courses in bioethics, and human genetics, elective courses, and program-specific ELSI seminars, providing rich interdisciplinary interaction with faculty and trainees from this program and from other training programs. Career development opportunities include participation in research ethics and clinical ethics consultation, an award-winning grant writing academy and the opportunity to obtain a teaching certificate through Stanford University. Research Program: Mentored research by trainees will bring together faculty from varied disciplines to identify and address important and novel ELSI issues through empirical or normative research. Trainees have numerous opportunities to conduct research as part of ongoing ELSI projects as well as to develop new ideas, and to present their research at professional meetings and publish in peer-reviewed journals.