Stanford University

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY: This proposal is devoted to the development, clinical implementation and evaluation of a novel, methodology for generating brain sodium images of unparalleled resolution and signal-to-noise-ratio. This methodology is a result or recent developments in Bayesian image reconstruction and manifold mapping techniques that together make it possible to robustly utilize anatomical information from high-resolution brain scans to guide the reduction of partial voluming effects from lower resolution, lower signal-to-noise ratio (SNR) brain scans. Though such “constrained reconstruction” schemes have been long-advocated in the MRI imaging community, their computational demands, limited performance and heavy operator input had previously limited their evaluation and practical characterization thus rendering them impractical for use within the confines of a clinical environment. Recently, we have shown that the combined use of segmentation free Bayesian approaches together with manifold mapping techniques can be used to provide a fast framework for Anatomically Guided Reconstruction where the information form high-resolution brain scans is used, routinely, to improve spatial resolution and SNR of concurrently acquired brain PET scans leading to improved sensitivity for the detection of low-contrast lesions in the brain. Our initial experience with extensions of this approach for sodium MRI of the brain has produced images of spatial resolution and SNR that far exceed what had been previously achieved using high and ultra-high magnetic field strengths. Establishing the limits of this approach and characterizing its performance on clinically relevant cases is the thrust of this proposal.

NIH Research Projects · FY 2026 · 2022-05

Work in the last decade from many labs has underlined the critical importance of RNA-mediated cellular pathways, and clear connections of specific RNAs to human health. RNAs are increasingly viewed both as appealing therapeutic targets, and as therapeutic agents themselves. We hypothesize that obtaining a deeper and broader understanding about how ligands interact with the many RNA species of the cell will provide important new insights into RNA networks and functions, provide new understanding of how current drugs cause cellular toxicity, and lend novel insights into improving RNA therapies. We are convinced that the analysis of RNA interactions transcriptome-wide is essential to future biomedicine. Unfortunately, methods for assessing RNA interactions directly in the cell lag well behind those for protein and proteome analysis. Recent work from this laboratory has established numerous new molecular tools for analysis of biologically and clinically relevant RNAs. We developed the first high-yield reaction strategy for functionalizing RNA 2'-OH groups, establishing broad utility of acylimidazole reagents. We designed the cell-permeable and broadly used structure-mapping reagents NAI and NAI-N3 - now commercially available - and applying them with RNA Seq, we mapped folded structures of 16000 mRNAs in mammalian cells. We developed rapid and simple chemical approaches for functionalizing RNA with fluorescent labels, biotin, hydrophobic groups, crosslinkers, and caging groups. Further, we designed strategies for labeling RNA either broadly or at specific sites. Unlike recent enzymatic approaches for RNA labeling, our methods require no engineered structure or sequence, and thus can be employed rapidly and easily with native RNAs of any origin or length. The proposed project will consolidate our RNA work into a broad program that will develop a new set of RNA-reactive reagents and methods, and will apply them to provide specific, quantitative information about ligand interactions with the transcriptome. We will develop first-in-class methods for functionalizing native RNAs at specific sites, and novel strategies for controlling RNAs with red light. Combining our reactive acyl tools and methods with next-gen sequencing, we will pinpoint and quantify ligand binding sites in the whole transcriptome. These methodologies, together termed Reactivity-Based RNA Profiling (RBRP), will be applied to analyzing off-target RNA binding by known small-molecule drugs with clinically limiting toxicity, to profiling RNA interactions of endogenous secondary metabolites, and to the analysis of how modified bases in next-generation mRNA vaccines and therapeutics affect their structures and interactions in the cell. This work is significant because it seeks answers to system-wide clinically-relevant questions regarding RNA interactions. Further, it develops the 2'-OH group as a nearly universal handle for manipulation, conjugation, and study of RNAs, introducing enabling molecular technologies that will broadly benefit researchers in the fields of RNA biology and contribute to improving future RNA therapies.

NIH Research Projects · FY 2025 · 2022-05

Project Summary Babies are a highly vulnerable population for which the ability to diagnose atypical vs. typical visual cortex development and intervene early could not be of greater importance. However, how infant visual cortex develops microstructurally and functionally is largely unknown outside primary visual cortex (V1). The goal of this research is to fill glaring gaps in knowledge by: (i) Using innovative quantitative (qMRI), diffusion (dMRI), and functional (fMRI) magnetic resonance imaging, to longitudinally measure and examine the relationship between microstructural and functional development of the human visual system during the first two years of life (Aim 1). (ii) Using advanced histological and quantitative methods in pediatric samples of visual cortex, determine the biological underpinnings of microstructural development (Aim 2). Specifically, Aim 1a will use qMRI and dMRI to longitudinally measure the microstructural development of infant visual cortex from 0-24 months. We will: (i) test if development varies across visual areas, and (ii) examine if microstructural development is associated with tissue pruning or proliferation. Aim 1b will use dMRI and qMRI to longitudinally measure the microstructural development of the white matter tracts of the infant visual system from 0-24 months and examine the relation between white and gray matter development. Aim 1c will use fMRI to: (i) examine the development of cortical responses to visual contrast, which rely on processing in V1, and responses to visual categories, which rely on processing in high-level visual regions in ventral temporal cortex (VTC), and (ii) determine if and how functional development is related to microstructural development in the same infants. Aim 2 will augment and validate in vivo metrics using ex vivo histology in tissue samples of 0-24 months-olds. Aim 2a will use quantitative histology to (i) measure the development of cortical myelination in V1 and VTC, and (ii) relate histological data to in vivo metrics to determine which neuroimaging metrics are coupled with myelination. Aim 2b will: (i) quantify the densities of multiple cell types (all cells, neurons, astrocytes, and oligodendrocytes) in V1 and VTC, and (ii) test if development varies across cortical expanses and if it is tied to myelination. Aim 2c will use transcriptomic analyses to determine gene expression pathways and elucidate molecular signaling pathways associated with microstructural development in V1 and VTC. The proposed research is highly innovative and groundbreaking as it will provide the first combined in vivo and ex vivo measurements of the microstructural and functional development of human visual cortex in infancy. This research is important as it will not only fill significant gaps in knowledge but also will develop novel, cutting-edge, and open-source methodologies for quantitative measurements of cortical microstructure that are linked to biological mechanisms. Thus, the proposed research has broad societal and clinical impact as it will lead to development of non-invasive biomarkers to diagnosis atypical visual cortex development in infants, leading to early inventions and better life-long outcomes.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Osteoarthritis (OA) is a leading cause of pain and disability worldwide and results in a reduced quality of life due to pain from common tasks such as walking or climbing and descending stairs. A particular challenge of OA is detection of disease at an early and reversible stage before irreversible structural damage has occurred. However, current imaging methods are performed in a static fashion and have limited sensitivity to joint function or the tissue response to loading. There is a major need to evaluate the joint response from everyday loading tasks, and how this response is altered in OA. Further, as age is a key risk factor of osteoarthritis, which predominantly affects older adults, the role of aging in tissue changes and response to loading stress is of particular interest. PET-MRI offers simultaneous evaluation of multiple early markers of OA in all joint tissues. In particular, [18F]NaF uptake can quantitatively evaluate bone metabolism, including bone perfusion and deposition of [18F] ions into the bone matrix. Further, we have observed that joint loading acutely alters the bone physiology affecting [18F]NaF uptake. This suggests that [18F]NaF uptake may be sensitive to increases in the metabolic response of bone where there is breakdown of the whole-joint unit that results in focal increases in bone loading. This project aims to develop a novel imaging “stress test”, based on [18F]NaF PET-MRI, that is able to evaluate at a single time point, both the function of the whole-joint unit after physiological joint loading and multiple early markers of OA. Our Specific Aims are to (1) develop a rapid, quantitative, dose- and time-minimized [18F]NaF PET-MRI protocol to evaluate joint function from a stair ascent/descent exercise “stress” test ; (2) evaluate [18F]NaF PET-MRI imaging to detect and characterize differences in joint function associated with age and between sexes; and (3) evaluate tissue response to loading stresses measured with PET-MRI in early OA and whether abnormal response is predictive of OA disease progression. The innovation of this project is the development of novel PET and MRI methods to assess the whole-joint response to loading and evaluation of early degenerative disease changes affecting joint function and future OA progression. The significance of this work is an imaging method that is able to rapidly assess joint function in response to physiological loads, and evaluation of how this joint response evolves with age and between genders, as well as the role of altered joint physiology in OA. This may allow us to detect early joint dysfunction at a single time point, to not only study OA pathophysiology, risk factors, and phenotypes, but also to develop targeted interventions.

NIH Research Projects · FY 2025 · 2022-04

High dimensional atlas of circulating neutrophils as reporters of solid organ functional status Chronic solid organ diseases (CSODs) collectively account for the majority of deaths in the United States. A central goal in modern medicine is to improve our ability to predict and detect CSODs in order to initiate successful therapeutic interventions early or to install appropriate early preventive measures. Several approaches have been devised to facilitate early detection of disease, including genetic testing and screening modalities such as imaging and laboratory tests. However, considerable number of CSODs lay silent and escape even the most watchful clinical eyes, only appearing when it is too late to reverse the pathophysiology of the disease. The identification of a non-invasive, accessible, sensitive, and comprehensive reporter system that simultaneously appraises the status of many solid organs would widen the window of opportunity for therapeutic intervention before overt disease occurs. Cellular injury and damage, which precede all organ-based disease, trigger an immune response that may be transcriptionally encoded into surveilling immune cells. The blood circulation accesses all solid organs and therefore provides an excellent portal into organ status. Specifically, neutrophils, the most abundant immune cells in humans, infiltrates nearly all organs under homeostasis. Contrary to their reputation as mere non-specific anti-microbial combatants, neutrophils have evolved as heterogeneous, functionally versatile cells that participate in organ homeostasis and mediate CSODs. The advent of high-dimensional approaches such as single-cell cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) have revealed numerous previously unknown neutrophil subpopulations with distinct transcriptional features. Moreover, tissue-infiltrating neutrophils assume specific organ-defined signatures. Unique from other immune cells, neutrophils do not establish permanent residence in the tissues they sojourn. This feature coupled with the neutrophils' short half-life yet significant transcriptional malleability renders them excellent candidates to serve as sentinels and reporters of organ status. In short, neutrophils that have infiltrated organs potentially return into the systemic circulation with vital organ-specific codes that may predict homeostatic state compared to perturbed, diseased states. This proposal will capitalize on these features of neutrophils to create a comprehensive atlas of their transcriptomics signatures, such that any tissue-specific dysregulation would be detected as an alteration in these transcriptional signatures. If successful, we envision a clinical world where blood analysis of neutrophil transcriptomic features would unveil lurking disease far before symptoms develop, prompting early intervention.

NIH Research Projects · FY 2026 · 2022-04

Project Summary America currently faces a dire epidemic of substance use disorders. In 2019, 20.4 million Americans aged 12 or older had a substance use disorder. Approximately 1.6 million suffer from opioid use disorder, which causes more than 136 overdose deaths per day on average. Stimulant abuse is also a significant contributor to this social crisis' extension and severity, with 1 million suffering from methamphetamine use disorder (MUD) and another 1 million from cocaine use disorder. Methamphetamine and cocaine are currently major causes of overdose deaths behind opioids for illicit drugs. Methadone and buprenorphine are widely used treatments for opioid addiction, but there is currently no approved pharmacological treatment for stimulant use disorder (SUD). Thus, there is an urgent need to develop pharmacological interventions for SUD. Medical marijuana has been associated with reduced opioid prescription rates and deaths, and preclinical studies show that cannabinoids affect addictive-like behavior. These effects are believed to be mediated by cannabinoid receptors in the brain. The cannabinoid receptors 1 and 2 (CB1R and CB2R, respectively) are part of the endocannabinoid system and play a crucial role in modulating dopamine (DA) levels in the central nervous system (CNS). Marijuana and nonselective cannabinoids such as THC can activate both CB1R and CB2R. While extensive effort has been invested in understanding the role of CB1R in this DA system, the addictive and psychoactive properties of cannabinoids are also associated with the activation of CB1R. The recent discovery of inhibitory CB2 receptors in the ventral tegmental area (VTA) DA neurons and their inhibitory role in the dopaminergic circuit, leading to reduced DA release in nucleus accumbens (NAc), as well as a receptor modulating glial and microglia function in the CNS, has created a new opportunity for targeting this pathway for therapeutic development. With support from a U18 NIDA pilot grant for the past year, we have successfully identified a lead series of novel chemical entities (NCEs) that display functional selectivity for cyclase over arrestin (i.e., biased) and are also very selective for CB2R with minimal activity for CB1R. Our lead candidate has good oral absorption, brain penetration and, in animal models, reverses the addictive behavior of mice. This proposal aims to further optimize our lead compound as a preclinical/clinical candidate for the treatment of MUD. While displaying excellent selectivity for CB2R over CB1R, our lead compound has residual arrestin activity and needs further PK optimization. We have put together a panel of multidisciplinary experts covering CNS pharmacology, medicinal chemistry, biology, and preclinical and clinical addiction medicine and psychiatry to further support the development of this class of compounds with the help of the Blueprint Neurotherapeutics Network (BPN) before testing in humans.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT Behavioral obesity treatments can produce clinically significant weight loss but are often too costly or intensive to be implemented on a large scale. Standalone digital health interventions offer greater scalability than traditional in-person approaches, but produce only modest weight loss. To maximize efficacy, it is vital to determine the “active ingredients” of an intervention and eliminate the ineffective, or even detrimental, ones. Self-monitoring is a core component of behavioral obesity treatment that can be delivered via digital tools, yet little is known about the unique and combined impact of different self-monitoring strategies. The K23 candidate, Dr. Michele Patel, will address this gap by applying an innovative framework – the Multiphase Optimization Strategy (MOST) – to identify the most potent combination of digital self-monitoring strategies for weight loss. As the first part of this programmatic line of research, Dr. Patel will conduct a 6-month optimization trial that randomizes 176 adults with overweight/obesity to 0-3 self-monitoring components (tracking dietary intake, physical activity, and/or body weight) using a full factorial design. This study will leverage existing commercial platforms for self-monitoring, including a mobile app, wearable activity monitor, and wireless electronic scale. All participants will also receive an empirically- and theory-informed core weight loss intervention that includes goal setting, weekly tailored feedback, action plans, and behavioral skills training – components that enhance engagement and are well-supported by prior research. Aim 1a will examine the optimal combination of self-monitoring strategies that maximizes 6-month weight loss while Aim 1b will examine self-monitoring engagement and its association with weight loss. Aim 2 will evaluate barriers to and facilitators of engaging in these self-monitoring strategies, which will be assessed via semi-structured qualitative interviews with 40 trial participants. Aim 3 will assess a novel, interactive recruitment strategy via an embedded trial. Together, results will inform an R01 grant that evaluates the newly optimized intervention in an RCT. Building on Dr. Patel’s background in clinical trial methodology and behavioral obesity treatment, the proposed career development award will provide substantive training in 1) MOST and factorial designs; 2) qualitative and mixed methods research; 3) innovative recruitment and retention strategies; and 4) preparation for the transition into independent research. To facilitate successful completion of these goals, Stanford University’s outstanding environment for interdisciplinary research will be coupled with a highly-qualified, well- rounded mentorship team comprised of Primary Mentor Dr. Abby King, Co-mentors Dr. Gary Bennett and Dr. Lisa Rosas, and Consultants Mr. John Gallis (biostatistician) and Dr. Linda Collins (developer of MOST). This K23 will position Dr. Patel to become a leader in optimizing digital interventions for weight loss and will launch her career as an independent investigator dedicated to treating obesity through innovative solutions.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract All blood cell types are derived from a single hematopoietic stem cell (HSC) precursor. HSCs must balance expansion through self-renewing symmetric cell division with differentiation of progeny cells through asymmetric cell division (ACD) to produce a functional blood system. When ACD is disrupted, profound blood disorders occur often characterized by disproportionate production of specific blood cell types or uncontrolled expansion of blood progenitors. The goal of the proposed project is to understand how ACD influences developmental hematopoiesis and blood cell fate choice. Aim 1 will characterize how ACD occurs by identifying molecular players using RNAi-based depletion in hematopoietic progenitors and immunofluorescence techniques. Aim 2 will determine how ACD influences lineage choice during homeostasis and stress by monitoring blood cell fate upon disruption of ACD during homeostasis and after stressors such as oxygen deprivation or injury. Ultimately, these experiments will help elucidate a spatial and mechanistic model for ACD in hematopoiesis while establishing a Drosophila model that will help accelerate advancements in mammalian ACD hematopoiesis work. My career goal is to become a successful independent researcher at a top-tier academic institution. I aim to lead a research program that investigates how extrinsic cues from the niche and microenvironment influence cell polarity and cell division to impact HSC self-renewal and blood cell lineage choice. To achieve these goals, I will utilize techniques and training in basic cell biology and cytoskeleton research combined with genetic dissection of hematopoiesis and advanced imaging techniques. I will pursue the proposed training under the mentorship of Dr. Utpal Banerjee who has extensive experience using genetic techniques to dissect the molecular underpinnings of hematopoiesis using Drosophila as a model system. Additionally, my advisory committee will provide key support and resources to help me translate my findings to mammalian systems and develop cutting edge imaging techniques to monitor the blood system in real-time during homeostasis and stress. I will present my work and disseminate my findings at national and international conferences to help advance my research, build collaborations, and establish myself in the hematopoiesis field. This project will facilitate my transition to an independent research position by helping me establish the feasibility of using Drosophila to study ACD during hematopoiesis and a foundation of preliminary work to build upon. UCLA is an excellent place to pursue this work as it provides a rich landscape of resources, research collaborations, and professional development opportunities to help me advance my career. As a junior faculty, I will take part in training activities focused on leadership skills, grant writing, publishing in high impact journals, laboratory management, and mentoring to help me develop the skills needed to run a successful research team.

NIH Research Projects · FY 2025 · 2022-04

Project Summary / Abstract This application is in response to a specific request for proposals to develop immune cell engineering towards the treatment of type 1 diabetes (T1D). We propose to evaluate genetic engineering approaches in immune T regulatory cells (Treg) by integrating chimeric antigen receptor (CAR) proteins that are engineered with an external targeting domain (scFv) and internal stimulatory domain. This approach has revolutionized cancer cell therapy with heightened specificity and effectiveness of T cell action. We and others have found evidence that CAR Treg can help mediate immune protection of islets and may even act upon islets themselves to reduce stress and cell death. We propose to (a) determine how enhanced targeting and activation of Treg to human islets might improve islet function and local immune modulation to protect islets, (b) evaluate a method for enhanced islet targeting through the development and testing of a dual-targeting CAR system that exploits downstream T cell receptor signaling molecules that have not been previously evaluated and (c) determine how enhanced targeting and activation of Treg to human monocytes might result in more immunoregulatory monocytes that could help to alter response to islet autoantigens and prevent immune destruction. One very important component of our proposal is that we suspect that CAR Treg from patients with T1D may not function as well as CAR Treg from normal individuals. In fact, there is no data about this available. We think that some CAR Treg from some T1D patients might be more cytotoxic, less effective or more inflammatory and we propose to evaluate if this is true statistically and also to develop an approach to introduce and overexpress a set of genes known to be important for Treg function as a way of making sure that all Treg in all cases will exert effects that are wanted. We postulate that developing these Treg methods will produce novel clinical strategies to prevent T1D in high risk patients and to suppress autoimmunity and preserve β-cell mass in patients with recent-onset T1D.

NIH Research Projects · FY 2026 · 2022-04

Basic science, translational, and clinical research in Otolaryngology – Head and Neck Surgery is improving the way we diagnose and treat patients. A critical component of these research areas is the partnership among clinicians, clinician-scientists, and basic scientists. Herein, we propose to enhance a research training program designed to cultivate clinician-scientists who are interested in studying hearing, balance, taste, smell, voice, speech, and language. Our research training program is designed to provide residents and medical students with intense research experiences, a structured didactic program, and close mentorship and guidance. Trainees will be ingrained with the philosophy that research is collaborative between MD and PhD scientists. Our short-term goals are to recruit and train promising scientists studying communication health and diseases, who will make and share innovative discoveries. The ultimate long-term goal of our training program is two-fold: our graduates will 1) become independent NIH-funded investigators in faculty positions in academic departments, and 2) improve human health by advancing our field via scientific discovery that is translated to clinical care.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY Islet transplantation is a β-cell replacement therapy used to treat diabetic patients who lack the ability to secrete insulin. The conventional site for islet transplantation is the liver, however, this is far from optimal given that islets are subjected to hypoxia, toxic metabolites from the liver, a pro-inflammatory environment and an instant blood- mediated inflammatory reaction (IBMIR); together, this results in up to 60-70% of islets being immediately lost following transplantation. Furthermore, given that islet transplantation does not require the creation of a surgical vascular anastomosis, islets therefore need to build and secure a dedicated blood supply, which takes at least 3 weeks. In the interim, islets have to survive by relying on the diffusion of oxygen and nutrients (such as essential amino acids like glutamine and alanine) from the microenvironment of the transplantation site, which results in them enduring significant stress and bioenergetic depletion. Accordingly, we have identified several critical problems in the transplantation process which we have addressed with our innovative and clinically translatable solution that will maintain islet health and survival, during, and following, their transplantation. Recently, we developed and validated a novel collagen based cryogel 3D matrix that incorporates an oxygen generator to address the problem of insufficient oxygen which causes islet hypoxia. In Aim 1, we will functionalize this bioscaffold platform with a nutrient generator in the form of a mesoporous silica nanoparticle that releases amino acids. The release of both oxygen and amino acids to islets using these technologies will be modulated to ensure it is continuous over 3-weeks. Given isolated islets are stressed and exhibit exhaustion, which is further exacerbated following their transplantation, in Aim 2 we will aim to re-energize islets and restore their bioenergetic potential immediately after isolation using bone marrow derived mesenchymal cells (BM-MSCs); these cells can transfer their healthy mitochondria to islets via tunneling nanotubes (TNTs) and this can be potentiated when BM-MSCs are in close proximity to islets – hence, we will activate our bioscaffold platform by pre-seeding it with BM-MSCs. In Aim 3, we will then test the ability of our optimized “active” bioscaffold to restore glycemic control in diabetic animal models at 2 extra-vascular sites of transplantation (i.e. the omentum and the subcutaneous space) given this will mitigate the IBMIR normally encountered by islets following their delivery into the liver via the portal vein. At each of these sites, we will examine whether our active bioscaffold elicits an inflammatory response and foreign body reaction in the short term, and fibrosis/encapsulation in the long term; we expect these responses to be minimal given our bioscaffolds are made from collagen and they contain BM- MSCs that have potent anti-inflammatory, immunomodulatory and anti-fibrotic effects via their paracrine ability to release cytokines and extracellular vesicles. This data will pave the way for future clinical trials with our novel platform which can be scaled and produced conforming to GMP guidelines.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT Cellular ATP demand and mitochondrial ATP synthesis are tightly linked. ATP synthesis, in turn, depends on (1) NADH generation in the TCA cycle and (2) recycling of the adenine nucleotide pool. When ATP is depleted and AMP rises, the cell increases energy generation and curtails energy expenditure until homeostasis is restored. In the context of mitochondrial pathology, the system becomes unhinged… Reticular Dysgenesis (RD) is a rare hematologic disease, caused by biallelic loss-of-function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the phosphorylation of AMP to ADP in the inter-membrane space to generate substrate for ATP synthesis1. RD patients suffer from severe congenital neutropenia, lymphopenia, and die early in life unless cured by hematopoietic stem cell transplantation5. We have developed a novel biallelic CRISPR-knockout model of AK2 in primary human hematopoietic stem and progenitor cells to precisely mimic the failure of human myelopoiesis in culture and after transplantation into mice. Using broad metabolomic profiling, our preliminary studies revealed that AK2-deficient myeloid progenitors exhibit a high NADH/NAD+ ratio and NAD+ depletion, consistent with reductive stress. In addition, AK2-deficient myeloid progenitors displayed a decrease in mitochondrial metabolites, including TCA cycle intermediaries and aspartate, while lipid carnitines were increased, and lipid droplets were found in the cytoplasm. We also detected highly elevated levels of the purine intermediate inosine monophosphate (IMP) and a decrease in rRNA and ribosome subunits. Interestingly, our studies suggest the high IMP stems from deamination of AMP, rather than a block in purine de novo synthesis. Taken together, these observations raise the possibility that AK2 deficiency causes mitochondrial reductive stress, curtailing TCA cycle activity and diverting carbon and electron pools into lipid synthesis while counteracting the accumulation of AMP. These findings led us to hypothesize that AK2 deficiency causes two interconnected but distinct pathologies: I. Reductive stress redirecting energy metabolism into lipid storage rather than OXPHOS; II. Accumulation of AMP and IMP, leading to defects in nucleotide metabolism. Our proposed studies will test if failure of myelopoiesis is primarily a result of reductive stress and impaired energy utilization, versus impaired purine metabolism, or both. We will determine if myelopoiesis can be rescued by correcting the NADH/NAD+ ratio or nucleotide pools. Lastly, we will validate our findings in an in vivo model of RD and investigate if different compensatory mechanisms in different blood lineages result in the RD phenotype. We use RD as a model to dissect escape mechanisms at the juncture of energy metabolism, redox stress, and nucleotide homeostasis. These insights will advance therapies for mitigating reductive stress and using cell type-specific manipulation of purine metabolism as a strategy for immunosuppression and cancer therapy.

NIH Research Projects · FY 2026 · 2022-04

Project Summary The overall objective of this K24 application is to expand my current patient-oriented research (POR) and mentoring to integrate patient engagement from research to dissemination of care to improve the biobehavioral care of youth with chronic MSK pain. This award will provide the crucial protected time to enhance my quality and quantity of mentoring of junior investigators focused on POR including current K awardees, future T-32 fellows, and clinical fellows (physician and psychology). My currently supported POR encompasses translating targeted biopsychosocial assessments into mechanistically informed treatment approaches for optimal clinical care, coupled with pain neuroscience psychology that leverages experimental and neuroimaging methods to gain a mechanistic understanding of cognitive and affective processes that coalesce with function in children with chronic pain and their parents. All projects leverage the ubiquity of digital health to enhance patient access and reach. Our lab has world-leading expertise in theory- and mechanism-oriented research for patients and their families. The new direction of research to be supported through this award focuses on gaining new expertise in scientifically rigorous qualitative and codesign methods to partner with patients and their families across all stages of research. This application proposes two studies with significant mentoring opportunities within my long- term research objective to improve the health and well-being of youth with chronic pain. Study 1 characterizes the lived experience of youth with musculoskeletal (MSK) pain seeking, completing, and reflecting on pain care. Study 2 aims to codesign an optimization of our evidence-based graded exposure treatment for adolescents with chronic MSK pain and their parents, GET Living. The K24 builds on my previous research, leverages existing cohorts and data, adds new training, and solidifies collaborations with leaders in patient participatory research and qualitative data analysis to provide effective mentorship to the next generation of POR pediatric pain scientists. With support from the K24 award, I will enhance my mentoring capacity and I will work to enhance my career development with activities relevant to my mentees, my research, and my professional development. Specifically, I plan to deepen my understanding of qualitative data methods and analysis and patient participatory research methods. Moreover, I will dedicate time to ensure continued training in legal and ethical issues associated with research on human subjects and clinical trials and strengthen my professional leadership skills. The proposed studies provide the ideal context for enhancing mentorship opportunities via hands-on training and scientific development in the biobehavioral aspects of pediatric chronic pain applying patient-centered methods. With dedicated time and training from the K24, I will be better equipped to mentor the next generation of POR pediatric pain scientists and weave a patient-centered approach into all research from inception to execution.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Genome-wide association studies have identified thousands of noncoding genetic variants associated with common vascular diseases and traits. Each of these associations could point to a gene and vascular cell type to teach us about mechanisms of disease. Yet, it has been difficult to connect these noncoding variants to their molecular functions, in large part because they can act via long-range 3D contacts to regulate distant genes. To connect these variants to target genes, we will need to answer: How do cell-type and disease-specific features of the 3D genome impact gene expression in vascular cells? Our recent work suggests a new strategy to systematically map and computationally predict how the 3D genome connects vascular disease variants to their target genes. By collecting data on thousands of CRISPR perturbations of regulatory elements, we developed the Activity-by-Contact Model to describe how 3D features of the genome control enhancer-promoter regulation. By analyzing 3D contacts in human vascular cells in vitro, we connected one vascular disease variant to a target gene, endothelin-1, located >600 Kb away. These results provide a predictive framework to understand how 3D contacts impact gene expression, and reveal a strategy to systematically connect variants to function by mapping the 3D genome in vivo. We propose to map, model, and manipulate 3D enhancer-promoter contacts in vascular cells to connect risk variants for vascular diseases to target genes and cell types. We will: (1) generate a resource of genome-wide 3D contact maps in primary human vascular cells; (2) dissect how 3D contacts guide enhancers to target genes using combinatorial CRISPR perturbations; and (3) computationally and experimentally link vascular disease GWAS variants to effects on 3D contacts and gene expression. Our team includes experts in human genetics, vascular biology, genome engineering, 3D genome mapping, and computational genomics to map 3D contacts to identify targets for atherosclerosis. The environments at Stanford University, the Broad Institute, and Baylor College of Medicine are ideal for supporting these innovative and cross-collaborative studies. This study will provide a resource for studying genetic variants that influence vascular biology, illuminate a mechanism by the 3D genome regulates gene expression, and demonstrate a general strategy to identify biological mechanisms that influence risk for common vascular diseases and traits.

NIH Research Projects · FY 2026 · 2022-04

Project Summary Gliomas comprise the most common form of brain cancer. In adults and children, high-grade gliomas are the leading cause of brain cancer-related death, whereas the neurotoxicity associated with the treatment of pediatric low-grade gliomas (LGGs) frequently results in long-term neurocognitive sequelae. For these reasons, there is a pressing need to better define the mechanisms that underlie glioma development and progression relevant to improving treatment and reducing lifelong neurotoxicity. This is especially important for children with the Neurofibromatosis type 1 (NF1) cancer predisposition who develop low-grade optic pathway gliomas (OPGs) that impair vision. These NF1-OPGs form during early childhood (mean age, 4.5 years), where they are localized to the optic nerve and/or chiasm containing the axons of retinal ganglion cells (RGCs) - the neuronal subtype responsible for transmitting light-induced signals from the retina to the brain. Given the intimate relationship between these tumors and the optic nerve, a collaborative venture between the Monje and Gutmann laboratories resulted in the identification of a key regulatory role for neurons in NF1-OPG biology using authenticated preclinical Nf1 optic glioma mouse strains that histologically resemble their human counterparts. In these studies, we found that decreasing retinal ganglion cell (RGC) neuronal activity prior to tumor formation prevents OPG initiation, while reduced RGC neuronal activity attenuates established OPG growth. In addition, Nf1 mutant (similar to patients with NF1), but not wild-type (normal), optic nerves exhibit increased neuroligin-3 expression and secretion in response to RGC activity, which is controlled by ADAM10 cleavage. Moreover, neuroligin-3 (Nlgn3) is a potent growth factor for Nf1-deficient OPG cells in vitro and genetic loss of neuroligin-3 in Nf1 optic pathway glioma mice blocks tumor formation in vivo. Lastly, inhibition of neuroligin-3 shedding using ADAM10 inhibitors reduces Nf1-OPG growth. Based on these exciting preliminary data, we hypothesize that Nf1 mutation in RGC neurons promotes dysregulated neuroligin-3 signaling that drives the initiation and maintenance of Nf1 optic glioma. In this collaborative R01 proposal, we aim to elucidate the intersection between cell-intrinsic vulnerability (NF1 tumor suppressor loss) and paracrine influences from neurons in the tumor microenvironment relevant to understanding the pathogenesis of these common brain tumors in children with NF1.

NIH Research Projects · FY 2026 · 2022-04

Glioblastoma (GBM) is the most aggressive type of primary malignant brain tumor in adults. GBM has a bleak prognosis of approximately 12-15 months, despite continuing advances to the standard of care. Immunotherapy has demonstrated the significant potential to boost immune responses against many cancer types; inhibitors of checkpoint molecules, such as CTLA-4 and PD-1, have been used to treat many solid tumors with varying success. While it has demonstrated efficacy in treating some tumors with an inflammatory milieu and high degree of infiltration by anti-tumor T cells, it has shown little to no response in treating tumors such as GBM. This tumor type is characterized by a particularly immunosuppressive microenvironment with a notable paucity of T cells. Targeted approaches designed to reduce immunosuppression in the tumor and increase anti-tumor T cell activity are crucial to successfully treat GBM. Recent preclinical data from our laboratory and preliminary findings from a Phase I clinical trial have shown promising signs of efficacy with co-blockade of PD-1 and the alternative checkpoint LAG-3. We have observed long-term survivors and radiographic responses in trial patients. We have noted improved T cell responses against the tumor and a reduction in myeloid-derived suppressor cells (MDSCs), following dual therapy of patients. We propose to study the mechanism by which the immune system is enhanced against GBM via PD-1/LAG-3 blockade. We hypothesize that dual therapy recruits cells of the myeloid compartment in order to boost anti-cancer T cell activity, and reduces the presence of immunosuppressive myeloid cells in the tumor itself. To test our hypothesis, we will investigate the: i) priming of T cells by antigen presenting myeloid cells in response to dual therapy in murine models of GBM, ii) role of soluble product generated from cleavage of surface LAG-3 molecule in inducing myeloid-mediated immunosuppression in the tumor (using both murine models and patient samples), and iii) expansion of anti-tumor T cells and reduction in MDSCs levels in patients following dual therapy. We will correlate our findings with overall survival and progression free survival of the trial patients. We expect that the data generated from these studies will provide novel insights into a previously unexplored mechanism by which dual immune checkpoint blockade therapy can modulate the myeloid response against tumors. The knowledge obtained from this study will contribute to improving the design of future therapeutic strategies to treat GBM patients.

NIH Research Projects · FY 2026 · 2022-03

OVERALL SUMMARY The Cyclin D-Cdk4/6-Rb-E2F pathway integrates external and internal signals to control cell cycle progression at the G1/S transition of the cell cycle. Alterations in the Cyclin D-Cdk4/6-Rb-E2F pathway are found in the vast majority of human cancers. These alterations are thought to increase the proliferative potential of cancer cells. For example, the functional inactivation of the retinoblastoma (RB1) tumor suppressor or the amplification of Cyclin D genes is a recurrent event in the development of a wide range of human cancers. In the simple consensus model, the retinoblastoma protein Rb inhibits cell proliferation at the G1/S transition of the cell cycle by binding and inhibiting E2F transcription factors. In response to cell growth and proliferative signals, Rb is phosphorylated and inactivated in normal cells by a series of Cyclin-dependent kinase complexes (first Cyclin D-Cdk4/6 and then Cyclin E/A-Cdk2). Phosphorylation of Rb results in the dissociation of Rb from E2F transcription factors thereby causing transcription of genes important for DNA synthesis and other key aspects of cell cycle progression. Thus, cancer cells with constitutively inactive Rb are thought to acquire an increased proliferative potential. Knowledge of the Cyclin D-Cdk4/6-Rb-E2F pathway in normal and cancer cells has led to the development of specific Cdk4/6 inhibitors that have been approved for the treatment of breast cancer and are in clinical trials for several other cancer types. In this paradigm, inhibition of Cdk4/6 results in decreased Rb phosphorylation, which activates Rb’s cell cycle inhibitory function. However, many tumors do not respond to these inhibitors or do so only transiently. Recent observations in patients and pre-clinical models indicate that our understanding of the Rb pathway is not as complete as we previously thought. This may explain the variable results of Cdk4/6 inhibitors in the clinic. The overall goal of this proposal is to gain a deeper structural, molecular, and cellular understanding of the Rb pathway with the ultimate goal to help design new and improved therapeutic strategies targeting this pathway in a broad range of cancer patients. Our first goal is to determine the core mechanisms regulating the Cyclin D-Cdk4/6-Rb-E2F pathway, including how Cyclin D-Cdk4/6 phosphorylates Rb and how previously unknown post-translational modifications regulate Rb and E2F activities. Our second goal is to identify and investigate new functions of Rb pathway components, including new targets of Cyclin D- Cdk4/6 kinases, new functions for Rb and its family members p107 and p130, and new regulatory mechanisms controlling the concentration and activity of E2F transcription factors. Our third goal is to initiate the development of strategies that target the Rb pathway in innovative ways, including molecules that inhibit Cyclin D-Rb association, stimulate the tumor suppressor activity of p107 and p130, and manipulate E2F stability. These goals will be achieved in three inter-related Projects via a comprehensive, synergistic and multi-disciplinary approach. Ultimately, the information gained from these studies may provide new ways to target the Cyclin D-Cdk4/6-Rb- E2F pathway to improve cancer therapy.

NIH Research Projects · FY 2025 · 2022-03

Project Summary/Abstract The intestinal microbiota plays a major role in human health and is therefore the focus of significant interest as a target for therapeutic interventions. However, our understanding at the mechanistic level of the ecological and evolutionary forces shaping this diverse and densely colonized ecosystem is still tenuous. Notably, although we know that horizontal gene transfer is pervasive in the gut community, we understand only superficially the different roles of the majority of these exchanged genes and how this repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying community resiliency. This question is particularly intriguing for the Bacteroidales, the most abundant Gram-negative gut microbiome members, which can stably colonize for decades. This proposal will investigate the importance of biofilm formation by the Bacteroidales for community ecology and resilience, focusing on the conjugative megaplasmid pMMCAT which enables biofilm formation in the strains that acquire it. This plasmid is exceptional because of the high frequency of intrapersonal transfer to multiple Bacteroidales species and its ubiquity, with conserved architecture, across global human populations. Some studies suggest that mucosal biofilms in healthy humans are rare, but there is little information about other locations or unattached biofilms. I hypothesize that this megaplasmid, shared among many species in a community, enables the formation of multi-species biofilms and plays a role in community cooperation, notably through increasing community resilience. To test this hypothesis, or otherwise understand alternative roles of pMMCAT, I will systematically characterize the phenotypes conferred by this plasmid in culture and in gnotobiotic mice in a single strain (aim 1) or in different Bacteroidales consortia where some strains carry it (aim 2). To understand the ecological role of pMMCAT, I will evaluate if its conferred phenotypes are synergized or inhibited by the co-resident strains and whether all, only some, or none of the other strains benefit. To visualize biofilms in the colon prior to and following a stress pulse, I will use two different methods to preserve the spatial structure of the gut community and use a biofilm matrix-specific stain. I will subsequently examine the evolutionary dynamics of pMMCAT transfer in these consortia (aim 3). To this end, I will directly quantify and visualize plasmid transfer in culture and in a gnotobiotic mouse. I will also quantify the cost of carrying pMMCAT and expressing biofilm formation genes. I will evaluate the impact of the introduction of a cheater strain that doesn’t pay this fitness cost of producing the biofilm matrix public good. Finally, I will track the evolution of pMMCAT over the course of twelve years in four human volunteers previously found to have pMMCAT-harboring strains. This dissection of the dynamics and mechanisms underlying plasmid-encoded biofilm formation in the gut will improve our current understanding of intestinal ecology and its recent changes in human populations, and will provide one more stepping stone towards targeted microbiome interventions for health benefits.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Enteroviruses are the leading cause of viral meningitis in children and recent outbreaks of emerging non-polio enteroviruses (NPEVs) have been associated with a polio-like paralysis named acute flaccid myelitis (AFM). Discovery and characterization of cellular components that are critical for neuropathogenesis hold promise for revealing new approaches to treat enterovirus disease. In recent years, multiple receptors have been identified for EV-A71 and EV-D68, NPEVs, which are most commonly associated with AFM. Using unbiased genome- scale screens, we have identified the phospholipase PLA2G16 as an entry factor acting immediately downstream of receptor engagement following NPEV infection. How the multiple receptors and PLA2G16 work together to enable infection in cell types relevant for neuropathogenesis is, however, largely unknown. Infection of cell types present in the central nervous system is critical for developing severe neurological forms of disease following infection with NPEVs. Although mouse models have been widely used to gain insights into enterovirus infection processes, genetic and physiological differences between human and rodents limit their translational potential. Moreover, species incompatibilities in host factor interactions of these human enteroviruses necessitate overexpression of human receptors, mouse-adapted strains or neonatal infections. In work that forms a foundation for this proposal, we have developed from pluripotent stem cells human spinal cord organoids that recapitulate some of the cell diversity of the human spinal cord. Importantly, we have pioneered an approach to functionally connect motor neurons in spinal cord organoids with human skeletal muscle and cortical neurons in a preparation we named assembloids. These motor assembloids form functional neuro-muscular junctions and can control muscle contraction. Here, we propose to systematically study the role of known host factors in cell lines derived from neural tissue on EV-A71 and EV-D68, discover novel host factors by performing unbiased genome-scale genetic screens in neural cell lines, and compare cell lineage tropism and effect on neuronal function during enterovirus infections of cortico-motor assembloids. Our results will reveal the role and relative contribution of a distinct set of critical receptors and broad-acting host factors to infection by multiple enteroviruses, discover and provide details on the molecular mechanism of novel host factors in neural cell types, and leverage a unique neural organoid system to uncover the specific tropism and functional effect on human neural-muscular circuits during infections with the paralytic enteroviruses EV-D68 and EV-A71.

NIH Research Projects · FY 2025 · 2022-03

Project Summary/Abstract Pulmonary hypertension (PH) is a fatal disease of the pulmonary arteries with few supportive therapies and no cure. In PH, occlusive `neointimal lesions' grow within small pulmonary arteries and narrow vessel lumens, increasing pulmonary vascular resistance, ultimately resulting in right heart failure and death. Available PH therapies are vasodilators that do not target neointimal growth and neither prevent progression nor reverse disease. Understanding the biology of neointimal lesion growth – which cells are responsible for lesion expansion, and the pathways that control their proliferation – is key to the development of more effective therapies for pulmonary hypertension. In preliminary studies we find that proliferating neointima cells are adjacent to artery endothelial cells and have distinct gene expression that distinguishes them from cells located away from the endothelium. We hypothesize that a molecularly defined subset of neointimal cells located adjacent to the endothelium is the proliferating fraction that expands neointimal lesions, and that signals from the endothelium regulate lesion growth. Specific Aims: (1) Using proliferation tracking, genetic lineage tracing and ablation, identify and molecularly characterize the subset of neointimal cells whose proliferation is responsible for lesion growth. (2) Through bioinformatic analysis of single cell transcription in neointima and artery endothelial cells from mouse and human, identify candidate signals driving neointimal proliferation in PH and the core disease mechanisms shared between mouse and human. (3) Test the ability of a key candidate driver of neointimal proliferation for a role in lesion growth in multiple PH models. Here, by utilizing cutting edge technologies, genetics, and single cell approaches across multiple model systems, these experiments will provide a granular understanding of the cells and signals driving neointimal lesion expansion, adding substantially to the current knowledge surrounding the pathology of vascular remodeling in PH, findings we hope will ultimately lead to neointima-blocking treatment options.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY/ABSTRACT Salivation is a critical physiological activity that aids digestion, maintains oral health, and supports functions such as speech, swallowing and taste sensation. Salivary gland dysfunction results from ageing, diseases such as Sjögren syndrome and from radiotherapy for head and neck cancers. Therapeutic irradiation causes permanent damage to salivary glands, highlighting their poor regenerative ability. One potential obstacle to recovery of salivation may be that damage, particularly during radiation therapy, is inflicted not only on saliva-generating epithelial cells but also on supporting mesenchymal cells. Indeed, preservation or restoration of mesenchymal cell function may constitute an ideal therapeutic target, as an optimized mesenchymal microenvironment may augment the function and regenerative capacity of residual salivary gland epithelial cells and their progenitors. A knowledge of how mesenchymal cells function during homeostasis and contribute to regeneration after injury thus may provide a new approach to activate mechanisms that protect salivary glands and enhance their repair. In many organs the mesenchymal expression of signals that provide regenerative feedback to the epithelium during homeostasis and injury repair is induced by expression of a Hedgehog (Hh) protein signal from the epithelium. Using mouse genetic models, cell lineage tracing, and single-cell transcriptomics, we have discovered that Desert hedgehog (DHH), the least studied of the three mammalian Hh family members, drives an epithelial-mesenchymal feedback (EMF) circuit in the major adult salivary glands, and that activity of this circuit is crucial for salivary gland maintenance and for regeneration after radiation injury. Importantly, although DHH expression in cells of the salivary gland epithelium is essential for regeneration, our findings also highlight a vital role for mesenchymal response to this signal for execution of the regenerative program. Here we propose to elucidate the role of Hh signaling in salivary gland homeostasis and regeneration by characterizing at a single cell level the transcriptomic and epigenetic consequences of EMF circuit activity, and to assess the conservation of DHH-driven EMF circuitry in human salivary glands. With the goal of manipulating Hh pathway activity for protection from or enhancement of tissue repair after radiation injury, we have developed a conformation-specific nanobody against the Hh receptor Patched1 that activates Hh pathway response. This nanobody can be targeted to specific cell and tissue types, thus mitigating potential adverse effects arising from systemic Hh pathway activation. With this agent, we will test the possibility that precise, tissue-targeted activation of the Hh pathway can effectively enhance endogenous reparative mechanisms for salivary gland protection from and restoration after injury from irradiation like that administered in head and neck cancer therapy.

NIH Research Projects · FY 2026 · 2022-03

ABSTRACT – Overall Component In the current proposal we will use a systems vaccinology approach to address two fundamental issues in vaccinology. The first issue concerns the immunology of COVID-19 vaccines, which utilize novel platforms (mRNA) or adjuvants (Matrix M used in the Novavax vaccine). The second issue is related to the role of the microbiome on vaccine immunity. With respect to the first issue, despite the rapid development of COVID- 19 vaccines, there is a paucity of understanding about the mechanisms by which they induce innate and adaptive responses. Furthermore, the nature of the immune response induced by mRNA vaccines in special populations such as those with serious allergic disease is unknown. Interestingly, there have been reports of rare but severe allergic reactions to vaccination, in individuals with an atopic background. Therefore, we will assess immunity to the BNT162b2 vaccine atopic versus healthy subjects. In the case of the Matrix-M adjuvanted recombinant COVID-19 vaccine developed by Novavax, there is a paucity of understanding of immune mechanisms stimulated by the saponin-based Matrix-M adjuvant. We will analyze samples collected from a Novavax sponsored clinical trial in South Africa. The second theme of the proposal is focused on the impact of the microbiome on immunity to vaccination in healthy adults. Our recent work involving antibiotics driven ablation of the microbiota has highlighted an important role for the microbiome in modulating immune responses to vaccination with the seasonal influenza vaccine. However, the immune response against seasonal influenza vaccine in adults represents a recall response, because of prior exposure to influenza. The impact of the microbiome on a primary immune response, such as the response to rabies vaccination, is unknown. These two issues will be addressed in the following highly collaborative projects and cores: Project 1 (PI Pulendran) will utilize a multi-omics approach to define innate responses driving adaptive immunity immunity to vaccination. The signatures identified in this project will be correlated with antigen-specific T and B cell responses assessed in Projects 2 (PI Davis) and 3 (PI Boyd), respectively. Project 2 will perform an in-depth analysis of the dynamics of the antigen-specific T cell responses to vaccination. Project 3 (PI Boyd; Co-I Nadeau) will perform an in-depth analysis of the dynamics of the antigen-specific B cell responses to vaccination. The three projects will be assisted by 4 cores. The Administrative Core will support the coordination efforts across the HIPC-Stanford Center. The Clinical Core (PI Nadeau) will ensure a standardized approach in the recruitment and clinical characterization of human subjects in all studies; the Data Management and Analysis Core (PI Khatri) will provide bioinformatics expertise, and the Human Immune Monitoring Core (PI Holden) will support the projects by providing immune monitoring assays.

NIH Research Projects · FY 2026 · 2022-03

We seek to create a real-time ultrasound imaging tool for guiding interventions, with resolution that exceeds that obtained using CT but without the need for radiation or iodinated contrast agents. Advancements in medical imaging and device technology allow minimally-invasive procedures for the diagnosis and treatment of various disorders. Real-time ultrasound has become an integral aspect of many image-guided interventions. Advantages of US imaging include the low cost, lack of ionizing radiation and real-time visualization of anatomy and physiology. Our approach will be to: 1) create an extended aperture 2D transducer (512 by 16 elements) capable of imaging an extended azimuthal field of 9 cm with in-plane resolution of hundreds of microns (to provide a wide field of view at high resolution), 2) apply the 2D array to image multiple adjacent planes (to facilitate the view of biopsy needles or ablations), 3) achieve a 30 volume per second update rate by using plane wave transmissions to enhance contrast imaging modes and implement novel beam formation algorithms, 4) integrate methods for aberration correction, and 5) apply this technology in B-Mode, color Doppler, volumetric vector flow imaging and contrast imaging. The array will be realized using tiled modules that can be switched in a mode-dependent fashion to accomplish B-Mode imaging, color Doppler and contrast imaging. Over the past 4 years, Stanford and the University of Southern California have designed an adult extended-aperture abdominal-imaging system and demonstrated the improved spatial resolution, field of view and contrast that can be achieved. We exploit these tools here to develop a high-volume rate capability for monitoring liver interventions. Our aims to accomplish this are to: 1) Create and integrate tileable acoustic/electronic modules to implement signal buffering and multiplexing and create a large aperture array with elevational focusing. Utilizing newly designed Integrated Circuits (IC)’s and highly sensitive and wide-bandwidth single crystal transducer material, we will construct individual 2D array modules with co-integrated transducers and electronics. 2) Optimize the protocols for guiding biopsy and ablation in phantom and animal studies. A) Create software for imaging of small lesions and microwave ablation. We will implement singular value decomposition (SVD) based beam formation for aberration correction. B) Evaluate performance in phantoms and ex vivo tissue. C) Assess speed and accuracy of needle placements. D) Conduct contrast imaging and ablative studies in porcine liver in vivo. 3) Conduct diagnostic and interventional imaging studies as a proof of concept. A) Test the protocols to image the liver of adult volunteers and establish the signal to noise ratio in vivo as compared with phantoms. B) Assess 3D visualization of liver vasculature and lesions in patients referred for MR or CT imaging of a liver lesion. C) Compare the 3D visualization of ablated zones to contrast-enhanced CT (CECT) in patients that are referred for liver ablation.

NIH Research Projects · FY 2026 · 2022-03

The objective of the Stanford Cardiovascular Summer Research Training Program for Medical Students is to provide meaningful ten-week summer research experiences to a cohort of 15 first-year medical students from across the country in the field of cardiovascular science. Our long-term goal is to bolster the pool of clinician-scientists in our workforce by 1) providing opportunities to increase student representation from a broad range of backgrounds 2) exposing students to the landscape of 21st century translational medicine, and 3) providing students with a strong foundation in rigor, reproducibility and the responsible conduct of research. We will work toward the first of these goals by partnering directly with Meharry Medical College and the University of Puerto Rico to provide opportunities to highly-qualified medical students from their institutions to obtain research experiences with Stanford faculty. Our program is open to all, and applicants undergo a rigorous and unbiased application review process. After recruiting a broad and well-qualified cohort of medical students, we will provide our trainees with a comprehensive ten-week curriculum that will prepare students for careers as physician-scientists in cardiovascular medicine. 80% of trainee time will be devoted to a cardiovascular research project in the lab of a Stanford physician-scientist faculty mentor. The remaining 20% of their time will be spent in a comprehensive curriculum comprised of seminars in cardiovascular science, research training, physician-scientist career development opportunities and social events. To accomplish the second of our goals, preparing trainees for innovative research in translational medicine, students will be exposed to scientific opportunities that are especially enriched at Stanford. First, Stanford has a strong track record of pioneering next generation cardiovascular research in precision medicine. Second, Stanford is located in the Bay Area biotechnology hub and many of our faculty mentors can share their experiences on collaboration with industry. Third, Stanford faculty mentors lead numerous cardiovascular clinical trials that students will be able to learn from. Precision medicine, industry collaborations, and familiarity with clinical trials are all important components of the research endeavors for the next generation of physician-scientists. To accomplish our third goal of providing trainees with a strong foundation in rigor, reproducibility and the responsible conduct of research, we will provide a dedicated curriculum to address these topics under the guidance of leaders in the fields of research ethics and the philosophy of science. The program’s success and longevity will be monitored and improved upon through a robust plan for internal and external evaluation. The Program Directors and faculty mentors for this T35 have ongoing state-of-the-art cardiovascular research programs spanning basic, translational, clinical, and population level domains. Together they have the mentoring expertise and the available resources to support this T35 summer research program, which will train a qualified and committed cohort of medical students from a broad range of backgrounds to become responsible, collaborative, and innovative physician-scientists.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY/ABSTRACT As the dome-shaped, transparent outermost part of the eye, the cornea provides the majority of the focusing power for the visual pathway. When it is damaged due to severe injury or disease, scarring often ensues, resulting in reduced vision and, in many cases, blindness. In spite of the various types of corneal transplants that are available, there remains a major clinical need for new modalities to restore transparency to scarred corneas without donor tissue, which is in short supply worldwide. Corneal mesenchymal stromal cells (c- MSCs) have known therapeutic effects on corneal scarring and wound healing, but the optimal way to deliver their benefits to the eye have yet to be determined. We are developing Sutureless, Pro-regenerative, Anterior Additive Collagen gel KeratopLasty (SPAACKL), a procedure that removes and replaces blinding corneal scars with a transparent, stroma-like gel matrix containing c-MSCs. After removal of corneal scar tissue, the material is applied to the defect as a viscous liquid suspension of c-MSCs, forming a crosslinked, transparent cellularized stromal substitute within minutes that not only recreates the smooth surface necessary for clear vision but also promotes rapid re-epithelialization. This technology leverages a crosslinking technology known as copper-free click chemistry that is bio-orthogonal: it does not react with proteins, cells, or biologic systems of any kind. As such, it can be safely applied to a corneal wound and around c-MSCs without producing toxic side products, and without the need for light energy, catalysts or accelerators. Our central hypothesis is that bio-orthogonal crosslinking can improve the regenerative benefits of c-MSCs by preserving the bioactivity of its encapsulated cargo compared to less specific crosslinking chemistries that are used currently in corneal surgery. In preliminary work, we have demonstrated that bio-orthogonally crosslinked gels support the growth of encapsulated stromal cells and have demonstrated the regenerative capacity of these cell-matrix composites to support rapid, multi-layered epithelialization both ex vivo and in vivo. Motivated by this data, our first aim is to test the hypothesis that matrix stiffness, composition, and crosslinking chemistry influence c-MSCs’ viability and secretion of pro- regenerative factors. Our second aim is to test the hypothesis that encapsulated c-MSCs exert their pro- regenerative influence on the corneal epithelium primarily through paracrine signaling. Our third aim is to evaluate the ability of bio-orthogonally crosslinked hydrogels to deliver therapeutic cargo that enhances epithelial and stromal regeneration through an in vivo keratectomy model. This research will build the foundational data for eventual clinical translation of a new way to treat corneal blindness without the need for sutures, light energy, or cadaveric donor tissue, and has the potential to one day help patients avoid the need for a traditional corneal transplant.