Stanford University

NIH Research Projects · FY 2024 · 2021-07

We seek to understand the regulatory mechanisms that control the movements of mitochondria in cells. A protein complex that includes milton, Miro, and the kinesin-1 heavy chain (KHC) is essential for mitochondrial transport in neurons. Miro resides at the central hub to allow multiple cellular signals to control mitochondrial motility. These signals include mitophagy, calcium, hypoxia, and nutrient availability etc. Additionally, and most relevant to this proposal, we have obtained exciting preliminary results demonstrating that a novel machinery spanning both the mitochondrial outer and inner membranes–the mitochondrial intermembrane space bridging (MIB) complex, stabilizes Miro and regulates mitochondrial motility. This new discovery and our past work raise a burning question at the intersection of fundamental cell biology and neurobiology: How these discrete Miro-pathways functionally coordinate or converge on mitochondrial integrity in response to various stimuli and stresses? In this proposal, we will probe these questions using classical cell biological and neurobiological approaches combined with a powerful genetic tool (fruit flies) for functional validation. Aim 1: To dissect the functional significance of the MIB-Miro complex. Aim 2: To determine the nature of Miro interaction with its binding partners. Aim 3: To explore the interplay of the regulatory signals and machineries of Miro.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY The epidermal growth factor (EGFR) oncogene is amplified and drives tumor growth in 55% of adult glioblastomas (GBMs). However, EGFR inhibitors have failed to demonstrate clinical benefit in GBM, presenting one of the most fundamental challenges facing the field of neuro-oncology. As highlighted by the National Cancer Institute’s recent think tank on progress in GBM, despite clear signals about the genomic underpinnings of GBM, including the high frequency of EGFR amplification, new drug development programs have stalled because of the high risk of clinical failures. Intra-tumoral genetic heterogeneity, and the poor brain-plasma ratios of many drug candidates, are thought to play a major role in clinical failure. Building on the team’s recent discoveries demonstrating that EGFR is amplified almost exclusively on extrachromosomal DNA particles (ecDNA), driving intra-tumoral genetic heterogeneity, accelerated tumor evolution, and EGFR inhibitor resistance, and their discovery of actionable metabolic dependencies that arise when EGFR becomes amplified, this proposal will identify proteins on which EGFR-amplified GBMs selectively depend for survival, even in highly heterogeneous tumors. This proposal integrates a hypothesis-driven approach with unbiased discovery using activity-based protein profiling (ABPP). In clinically relevant patient-derived models of GBM, this proposal takes a chemistry- first approach to discover both actionable dependencies that arise when EGFR is amplified and ligands that engage these proteins, which can be made to be highly brain-penetrant. By deploying fully functionalized (FF) small-molecule libraries to enable direct progression from phenotypic screening to target identification in living GBM cells, including in patient-derived GBMs with amplified EGFR, this proposal is poised to inform actionable therapeutic targets for patients in vivo. The proposed integrated approach provides a rapid route towards initiating new drug development that directly addresses the fundamental challenges of GBM.

NIH Research Projects · FY 2025 · 2021-06

Rheumatoid arthritis (RA) is an autoimmune synovitis that affects 0.5% of the world population. RA is characterized by intermittent flares of clinical arthritis that is thought to be mediated in part by anti- citrullinated protein autoimmune responses. The best established environmental risk factors for developing RA include cigarette smoking and periodontal disease, suggesting oral mucosa is a critical site for disease initiation. Nevertheless, the mechanisms by which these environmental exposures lead to RA development and progression remain poorly understood. We have established a clinical and technical protocol for repeated home finger stick blood collection in RA patients to allow for longitudinal RNA sequencing (RNAseq). Using this novel approach, we recently discovered bacteria characteristic of human oral mucosa in the blood of anti-CCP+ RA patients, followed by activation of a signature B cell immune response 3 weeks later, and then clinical flare of disease activity 2 weeks after that. We also investigated B cell responses to these pathogens. We demonstrated elevations in IgA blood plasmablasts both in pre-clinical RA as well as in established RA, with the continual re-activation of a distinct set of IgA/IgG plasmablast clonal families in established RA suggesting a persistent mucosally-driven germinal center reaction. We demonstrate that the recombinant Mabs encoded by the persistently reactivated IgA/G plamablast clonal families encode antibodies that react with both human citrullinated antigens and citrullinated isoforms of oral bacteria identified in the blood of patients antecedent to flares. We anticipate that RA plasmablast Mabs with distinct specificities, either alone or in immune complexes, mediate activation of distinct cellular responses that promote synovitis and tissue destruction in RA. This R01 proposal will test the hypothesis that mucosal breaks trigger plasmablast responses that encode anti-bacterial antibodies that cross-react with host citrullinated antigens. We further hypothesize that mucosal bacteria-induced ACPA activate cellular responses, including macrophage TNF production, NETosis and osteoclast activation, which promote synovitis and joint tissue destruction in RA. Aim 1 will identify the antibody repertoires responsive to pre-flare bacteremia in two independent cohorts of RA patients. Aim 2 will characterize RA plasmablast IgA/G Mabs and sera for reactivity to citrullinated isoforms of bacterial species derived from subgingival collections. Aim 3 will characterize periodontitis tissue for evidence of RA-related autoimmunity. Aim 4 will determine the mechanisms by which cross reactive Mabs, either alone or in immune complexes, mediate arthritis. Success of this proposal would demonstrate that citrullinated periodontal bacteria and mucosal breaks play a key role in mediating RA flare, findings that could lead to development of new diagnostic and therapeutic approaches.

NIH Research Projects · FY 2025 · 2021-06

SUMMARY The overarching goal of this project is to understand how immune cells impact the brain during aging, with the objective of restoring old brain function. The brain has long been considered an ‘immuno-privileged organ’. However, recent studies have shown that immune cells infiltrate the brain in neurodegenerative diseases such as Alzheimer’s disease and during aging. A key remaining challenge is to understand how immune cells impact the brain during aging, and could this knowledge be used to restore functionality of old brain and treat neurodegenerative diseases? The subventricular zone (SVZ) of the adult brain provides a great paradigm to address this question, as this regenerative region of the brain contains many different cell types – neural stem cells (NSCs), endothelial cells, microglia – and exhibits clear functional decline during aging. To gain a single cell understanding of the changes that occur with age in neurogenic niches, we recently performed single cell RNA-sequencing of young and old neurogenic niches in mice. This analysis revealed a striking infiltration of cytotoxic T cells only in the old neurogenic niche, which was confirmed by immunofluorescence. Surprisingly, we found that T cells from old SVZs are clonally expanded and secrete interferon g (IFNg), suggesting that they have encountered specific antigens. We also showed that T cells can impair NSC proliferation both in co-cultures and in vivo. Based on these data, our specific hypothesis is that T cell clonal expansion in old brains drives the deterioration of the neurogenic niche with age, and that preventing this T cell expansion restores function to old neurogenic regions. Probing this idea would be critical to counter the decline in brain function during aging and neurodegenerative diseases, such as Alzheimer’s disease. To test our hypothesis, we propose the following experiments: 1. To determine how T cells infiltrate neurogenic niches in old individuals; 2. To understand the functional impact of T cells on old neurogenic niches; 3. To examine the interaction between immune cells and neurogenic niches in young, old, and rejuvenated individuals. Completion of these aims will provide unique mechanistic insights into the regulation of T cell and other immune cells during aging in regenerative niches of the brain. This work should also give a fundamental understanding of the mechanistic impact of the interferon response and T cell cytotoxicity on different cell types in the brain. Knowledge from our study should pave the way for building transformative strategies, including new immunotherapies, for the restoration of a pristine tissue, which will be a critical step for improving brain function during aging and age-related diseases such as Alzheimer’s disease.

NIH Research Projects · FY 2025 · 2021-06

Abstract Groundbreaking work within the NIH BRAIN Initiative has revealed many new types of neurons and their genetic signatures. The dividends from this research will include sophisticated tools allowing selective genetic access to these cell-types, such as for imaging, optogenetic or tracing studies. To complement these powerful genetic tools, it will be equally important to have new imaging techniques that can reveal how multiple neuron- types work together in the live brain to support information-processing and construct different brain states. To address this challenge, Stanford University and The John B. Pierce Laboratory at Yale University will create optical techniques for imaging the concurrent voltage dynamics of up to 4 separate neuron-types in behaving animals. First, we will combine machine learning methods and an automated, high-throughput protein screening platform to engineer 4 different categories of genetically encoded fluorescent optical indicators of neuronal transmembrane voltage. We will then innovate several types of optical instruments tailored to work in conjunction with the new voltage indicators. These instruments will enable unprecedented studies of voltage rhythms and spiking dynamics in 2–4 genetically identified neuron-types in superficial and deep brain areas of awake behaving animals. One instrument will allow us to track the concurrent, population voltage oscillations of 2 neuron-types in freely behaving rodents. Another instrument, an optical mesoscope, will enable imaging studies of voltage waves and oscillations across the entire neocortical surface of behaving mice. A third device will be a high-speed miniature microscope for tracking neural dynamics at single cell-, single spike-resolution in freely behaving mice. Lastly, we will develop the capability to image with millisecond-scale precision the simultaneous spiking dynamics of 4 targeted neuron-types in either cortical or deep brain areas. Five external beta-tester labs will evaluate all these innovations in live mice and flies and provide critical user-feedback. If our work succeeds, it will be a ‘game-changer’ for studies of brain dynamics, yielding vital knowledge about how different neuron-types synergize their dynamics to shape animal behavior and the brain’s global states in health and disease. To facilitate this outcome, we plan a 5-fold strategy for resource sharing: (i) All voltage-indicator constructs, viral vectors, transgenic flies, software and screening data will be deposited at public repositories for open distribution; (ii) All instrument designs will be published in extensive detail to facilitate replication; (iii) Our novel imaging devices will be integrated into an existing NIH-supported, publicly accessible facility for brain-imaging in rodents; (iv) In project years 2–4, we will conduct 4 training workshops for 40 visiting scientists per year (120 in total) to learn the new technologies firsthand. These visitors will also provide extensive user-feedback; (v) We will license our imaging instruments for commercial distribution. Overall, we expect our project will lead to major conceptual advances in brain science and multiple new technologies that will reshape the practice of mammalian brain imaging.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Alcohol consumption and mortality due to alcohol-associated liver disease (ALD) are increasing in the United States, and ALD is now the leading cause of liver transplantation. The natural history of ALD is distinct from other etiologies of liver disease, with more advanced disease at the time of presentation but also opportunity for hepatic recovery. Accurate prediction of outcome remains challenging. The proposed research will establish a prospective registry and biorepository that will evaluate the outcomes of patients with ALD and alcohol use disorder who are hospitalized with acute hepatic decompensation. This study will take a multifaceted approach, considering the biologic and psychosocial influences on clinical outcomes. Patients will be recruited during hospitalization and followed for 2 years in the outpatient clinics, with longitudinal measures of alcohol use patterns, serum biomarkers, radiographic features, nutrition, frailty/sarcopenia, and non-invasive assessments of fibrosis, inflammation, and steatosis. Specific Aims: (1) Characterize the cohort and optimize retention procedures using frequent communication, collateral contacts, and technology-based resources, (2) Identify distinct trajectories of ALD using latent class trajectory analysis and the predictors of trajectory classification, and (3) Develop a patient-specific risk prediction model to predict hepatic recovery after an acute hepatic decompensation. These project aims will identify distinct phenotypes and predictors of outcome in ALD and improve prognostication to better target interventions and allocate resources. The principal investigator (PI) is a hepatologist and clinical researcher at Stanford University, with a long-term vision of improving care for patients with ALD. Her experience with outcomes research and her Master’s in Clinical Research and Epidemiology have prepared her well to execute the project aims. The proposed research and career development plan are well-supported by a multi-disciplinary mentorship team and the institution. The PI will acquire advanced skills in longitudinal data analysis and machine learning, as well as content expertise in alcohol research, which will allow her to apply advanced statistical techniques to optimize prediction of outcome in ALD in a clinically relevant context. In addition, she will have a well-characterized ALD registry and biorepository to serve as a platform for future translational and multicenter studies. This award will provide the PI with the protected time, mentorship, training, and research experience to develop an independent research career in ALD and improve our understanding of this serious and prevalent condition.

NIH Research Projects · FY 2025 · 2021-06

The placenta is essential for fetal development and growth, maternal homeostasis, and broadly, pregnancy health. Yet, our ability to non-invasively probe placental health during human pregnancy is hampered by its deep intrauterine location and its highly vascular composition, rendering the placenta largely inaccessibly for safe and dynamic investigation. Whereas placental research has been advanced by cell culture, ex vivo systems, animal models, and postpartum analyses, these indirect approaches provide ex post facto information about placental health. Placental imaging has revolutionized the field of placental medicine, but resolution at the molecular, cellular, or metabolic level remains limited. To address these challenges, we and others have focused on the release of extracellular vesicles (EVs) from placental trophoblasts, which, in humans, are directly bathed in maternal blood. We focused on exosomes (now termed small EVs or sEVs), microvesicles, and apoptotic blebs, which are continuously and abundantly released from trophoblasts into the maternal circulation and are accessible throughout pregnancy by peripheral blood tests. Among these EVs, we focus mainly on placental sEVs, which harbor messages that are seldom expressed by any other cell types and execute unique placental biological functions, such as an antiviral response. While informative, recent data indicate that sEVs are not a uniform population of vesicles, but comprise several subgroups, defined as large sEVs, small sEVs, and exomeres. In addition to their size, these sEV subtypes are characterized by distinctive cargo. Although the recent discovery of sEV subpopulations has excited researchers due to their potential to revolutionize the field of non-invasive diagnostics, sEV subpopulations have yet to be utilized in clinical settings. This is largely due to the difficulties associated with separation and isolation the nano-sized sEV subpopulations. Our group has now developed advanced acoustofluidic technologies designed to effectively, reproducibly, and rapidly isolate sEVs from blood. We show that we can separate placental sEVs into their specific subpopulations, which has not been previously accomplished. Our proposed investigation therefore focuses on the production of human placental sEV subpopulations, along with their RNA and proteome cargo. We posit that, by profiling these analytes from sEV subpopulations, we can illuminate a unique landscape of bioactive molecules that are relevant to placental health. To reduce data complexity, we propose a machine learning pipeline that will be used to probe the sub-sEV spectra during normal and pathological pregnancies. Further, we will improve our ability to purify sEV subpopulations from lipoproteins, and generate a single, integrated device that can reliably separate vesicles in real time across human gestation. We believe that our automated acoustofluidic approach to separating sEV subpopulations in a high-yield, biocompatible manner is critical to unlocking the clinical utility of sEVs. Insights gained from our investigation will improve non-invasive diagnostics during pregnancy and may uncover new targets for personalized placental therapeutics.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY The optics of the eye suffer from imperfections (wavefront aberrations) that blur retinal images. This blur limits our ability to diagnose eye disease before irreversible damage and vision loss take place. Adaptive optics (AO) ophthalmoscopy can correct for this blur, which is unique to each eye and retinal location. Current AO ophthalmoscopes can only capture images of very small retinal areas because they cannot correct for the variation of the wavefront aberrations across the retina. This limitation impedes the use of this technology for detecting early signs of disease, which in turn, enable the early treatment that would mitigate irreversible vision loss. Here we propose to first measure how wavefront aberrations change across the retina in a human subject population to improve the design and operation of all future AO ophthalmoscopes. We also propose two novel methods for measuring and correcting wavefront aberrations that vary across the retina to increase the field of view of these instruments. First, we propose to measure aberrations sequentially at various points in the field of view and then deliver appropriate sequential wavefront corrections at each retinal location. This low-cost, low complexity approach could be used to double the retinal coverage of current AO scanning ophthalmoscopes. The second approach uses two wavefront correctors, the optimal axial positions of which will be determined by using the data on wavefront aberration changes with retinal location and wavelength across the population. This more complex approach aims to at least quadruple retinal coverage. The resulting increase in the field of view will be a major step towards translating AO ophthalmoscopy into a practical clinical tool for improving the diagnosis and treatment of eye diseases.

NIH Research Projects · FY 2025 · 2021-06

Project Summary / Abstract: This MIRA proposal merges two distinct projects supported by R01GM128142, “The role of membrane curvature in surface nanotopography-induced cell functions”, and R01GM125737, “Developing nanoscale electrophysiology sensors for robust intracellular recording”. While the two projects focus on different biological questions, the unifying theme is to develop nanoscale probes to elucidate the cellular machinery in the intricate environment of living cells. In this proposal, we discuss topics along the lines of the parent grants, focusing on the significance of the biological problems, our recent and evolving results, and directions for the future. For the first project, the long-term goal is to understand how membrane curvature regulates biochemical signals that are transmitted through the cell-matrix interface. At the cell-matrix interface, where the cells make physical contact with extracellular matrices, the membrane may be locally deformed by matrix topography or mechanical forces. As it remains a challenge to manipulate nanoscale membrane curvature in live cells, our current understanding of how local membrane curvature affects signal transmission is limited. We propose to use nanotechnology-based precision engineering to control interface membrane curvature in live cells. We seek to understand how cellular processes are affected by membrane curvature and the underlying molecular mechanisms. The knowledge gained will help us understanding how cells interact with extracellular matrix and also help us designing biomaterials for better integration with cells. For the second project, we are developing vertical nanoelectrodes into a robust and easy-to-use electrophysiology tool that can reliably achieve parallel intracellular recording of cardiomyocytes with minimal perturbation. Simultaneous nanoelectrode and patch clamp recordings on same cells confirmed that nanoelectrodes accurately record action potential waveforms for classification and characterization of stem-cell-derived cardiomyocytes. These nanoelectrodes will enable us to understand how in vitro interventions accelerate the maturation of stem-cell-derived cardiomyocyte. Furthermore, nanoelectrodes provide an ideal tool for monitoring the generation and resealing of membrane pores on cardiomyocytes that are prone to membrane rupture due to their large size and strong mechanical contraction. We will use nanoelectrode to investigate how proteins participate in the membrane resealing process. We hope to achieve a broad impact by combining the development of new tools with applications to specific biological systems.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract: Glaucoma is the leading cause of irreversible blindness, affecting over 60 million people worldwide. Glaucoma patients vary widely in their presentation, with some retaining long-term disease stability, and others progressing quickly to vision loss. If glaucoma patients at highest risk of progression could be identified early, clinicians could better personalize their treatment approaches. Many clinical factors that affect glaucoma progression, such as intraocular pressure, treatment history, and medication adherence, are documented within the free-text notes of the electronic health records (EHR) and are not in large-scale administrative claims databases. Recent advances in artificial intelligence (AI) and natural language processing (NLP) have enabled the integration of the rich and complex EHR data into highly accurate predictive algorithms for health outcomes in medicine and surgery. We hypothesize that we can extend these AI and NLP techniques to build predictive algorithms for glaucoma progression that outperform traditional models reliant on only administrative features. The goal of this project is to build and evaluate predictive algorithms for glaucoma progression using large-scale EHR data, while developing Dr Wang's expertise in AI and NLP, advancing her career as an independent clinician scientist. Aim 1 focuses on using the structured clinical data within the EHR, which are numeric or coded and readily machine-readable, to build baseline machine learning models predicting glaucoma progression requiring surgery. Aim 2 focuses on using and augmenting clinical named entity recognition tools to integrate information from EHR free text into AI models predicting glaucoma progression to surgery. Aim 3 focuses on understanding, explaining, and evaluating the performance of AI algorithms in a real-world prospective setting, by evaluating their performance on key subpopulations, their reliance on key features, and investigating potential areas of bias in a new cohort of glaucoma patients. This proposal is innovative in developing AI-based predictive algorithms for glaucoma progression using numeric and textual clinical data uniquely available in the EHR. The tools and methods Dr Wang will build and evaluate will substantially impact the ophthalmology field by enabling evidence-based tailoring of treatment approaches to patients' unique clinical characteristics, a step towards precision medicine. Furthermore, the careful evaluation of AI predictive algorithms on a new cohort of patients will provide insights into their performance on key subpopulations and reliance on key features, which is critical to advancing our understanding of possible limitations of deploying AI in the clinical workflow. Dr. Wang's career and research will advance under the primary mentorship of Dr. Tina Hernandez-Boussard, a national leader in informatics and expert in using NLP on EHR to improve patient care. Her outstanding Advisory Committee, including clinician-investigators Drs. Pershing, Stein, Chang, and Goldberg, will ensure Dr. Wang's success in becoming an independent clinician-investigator integrating ophthalmology and informatics.

NIH Research Projects · FY 2025 · 2021-06

ABSTRACT – OVERALL COMPONENT The overall goal of this U19 project is the development of an HCV vaccine to prevent disease progression after virus exposure in a vaccinated host. Our overall approach is based on a growing body of data from studies in patients undergoing primary HCV infections indicating that a subset of these individuals generates immune responses that result in viral clearance and conferring immunity against reinfection. This naturally occurring protective immunity appears to include early and robust humoral and cellular responses during the acute phase of infection. Consequently, the early kinetics, strength, and quality (i.e., cross-protective capacity of both humoral and cellular responses against the diversity of the evolving HCV quasispecies) are critical for efficient clearance of repeated exposures to diverse HCV isolates/infections in at-risk individuals. Thus, a successful vaccine will need to induce robust and durable adaptive B and T cell immune responses in the vaccinated host. This Program is designed to achieve this goal by four complementary Projects and a Scientific Core. Project 1 focuses on a structure-guided approach to develop an immunogen that enhances the induction of broadly neutralizing antibodies (bNAbs) including those when combined lead to synergistic virus neutralization. This project will interact extensively with Project 4 on structural analysis of HCV envelop glycoproteins and on structural aspects of bNAbs and receptor binding to HCV particles. Project 2 will design HCV NS Mosaic antigens for T cell recognition of HCV genotypes and subtypes that cause most infections globally. Mosaic antigens are designed computationally by recombination of viral genomic sequences retrieved from databases. Project 3 takes a systems approach to evaluate short- and long-term B and T cell responses and innate responses to vaccination with vaccines that are formulated in Projects 1 and 3, and in combination with powerful adjuvants. These studies will be undertaken in non-human primates that will be executed in a Scientific Core. The Administrative Core will provide the operational support necessary to successfully achieved the goals we have laid out for each project and program as a whole. Taken together, the work proposed in these projects and scientific core are highly interdependent that will lead to a vaccine design capable to elicit protective B and T cell immunity against HCV. We expect that at the end of this program project, we will have a candidate vaccine to begin pre-clinical studies that will progress to Phase I/II clinical trial.

NIH Research Projects · FY 2025 · 2021-06

PROJECT DESCRIPTION Assembly-line polyketide synthases (PKSs) are enzyme machines that catalyze vectorial biosynthesis of a growing polyketide chain through a uniquely defined sequence of acyl carrier protein and ketosynthase domains involving alternating chain translocation and elongation reactions. Notwithstanding the discovery of >3000 naturally occurring assembly-line PKSs, we do not understand how they blend catalytic specificity with evolutionary flexibility. Our lab is motivated by the goal of understanding the enzymology and evolution of assembly-line PKSs while enhancing our ability to engineer known PKSs and decode “orphan” ones. Our Goals for the next five years are to: 1) Understand the chemical logic of vectorial biosynthesis by an assembly-line PKS: We will study: (i) the structural dynamics of individual PKS modules; (ii) how different conformations of a module enable its elementary reactions; and (iii) the extent to which transitions between successive reactions are coordinated across an assembly line. Our proposed mechanistic investigations will exploit: (i) our ability to functionally reconstitute PKSs in vitro; (ii) epitope-specific monoclonal antibodies to trap individual PKS modules in specific conformational or catalytic states; and (iii) advances in X-ray, SAXS, and cryoEM analysis of PKSs. 2) De-orphanize the nocardiosis-associated NOCAP synthase: We have de-orphanized the nonamodular NOCAP synthase found in isolates of Nocardia associated with nocardiosis. Now, we propose to solve the structure of the fully tailored natural product, and to elucidate its biological role in nocardiosis. This will require us to: (i) characterize a putatively doubly glycosylated polyketide product; (ii) establish a phenotypic assay for its bioactivity in a macrophage-like human cell line; and (ii) harness genome-wide CRISPR knockout and shRNA knockdown screens to gain insight into its mode of action. 3) Decipher the role of GRINS in the evolution of assembly-line PKSs: We have discovered a new genetic element, named GRINS (genetic repeats of intense nucleotide skews), that is widespread in assembly-line PKS genes. We hypothesize that GRINS play a major role in diversifying assembly-line PKSs. To test this hypothesis, we will: (i) identify candidate genes in Streptomyces that are involved in introducing nucleotide skews or enabling gene conversion; (ii) identify a bacterial host in which gene conversion is enabled by GRINS under laboratory conditions; and (iii) develop an experimental model for GRINS-based PKS engineering. The significance of our proposal is two-fold. On one hand, it offers the opportunity to break new ground in our understanding of the structure, mechanism, and evolution of assembly-line PKSs. On the other hand, it tests the extent to which our understanding of these remarkable megasynthases can be harnessed to discover novel bioactive polyketides from “orphan” assembly-line PKSs.

NIH Research Projects · FY 2025 · 2021-06

Colorectal cancer is the third most common malignancy in the world, with approximately 1.4 million new cases and 700,000 deaths each year. Global incidence rates are expected to escalate 60% by 2030 as Western diets and lifestyles become more common, and colorectal cancer is afflicting increasing numbers of young adults. Despite preventative screening and surveillance, approximately 20% of colorectal cancer patients have metastatic disease at the time of diagnosis, and 40-50% of early-stage patients will relapse after treatment. Unfortunately standard colorectal cancer therapies such as anti-mitotic agents, epidermal growth factor receptor antagonists, and angiogenesis inhibitors are largely ineffectual against late-stage disease. As a result, the 5-year survival rates for these patients is only 12%. It is now widely believed that eliminating cancer stem cells (CSCs) is the key to durable clinical responses, as these self-renewing cells drive tumor relapse, chemoresistance, and metastasis. Our project strives to achieve this goal by investigating and pharmacologically targeting metabolic pathways that are unique to colorectal cancer CSCs. Our work builds on recent reports that aldehyde dehydrogenase 1B1 (ALDH1B1) is expressed in intestinal stem cell and required for the growth of colon cancer-derived spheroid cultures and xenografts. Our findings support a role for ALDH1B1 in colorectal CSC maintenance, and we have developed the first known ALDH1B1-selective antagonists. We have also solved the first X-ray crystal structures of ALDH1B1 and ALDH1B1-inhibitor complexes, uncovering the molecular basis of antagonist action and gaining insights for further compound development. Our latest lead compounds can inhibit the viability of colorectal cancer spheroids, with minimal effects on adherent cultures or non- cancerous cells. In addition, our preliminary studies indicate that ALDH1B1 inhibitors can suppress the growth of colon cancer xenografts in mice. We are now investigating the mechanisms by which ALDH1B1 promotes colorectal cancer (Aim 1). We will explore the potential roles of this mitochondrial enzyme in colorectal CSC maintenance, chemoresistance, and invasiveness, using cell lines that are representative of various colorectal cancer subtypes. We will also determine whether oncogenic ALDH1B1 function involves the oxidation of retinal and/or lipid peroxidation products, and we will elucidate the ALDH1B1-dependent transcriptome. In parallel with these mechanistic studies, we will use medicinal chemistry, biochemical assays, and cellular models to develop ALDH1B1 inhibitors with optimized potency, selectivity, and pharmacological properties (Aim 2). We will then evaluate the activities of ALDH1B1 inhibitors in colorectal cancer xenograft models (Aim 3). Together, these investigations will deepen our understanding of ALDH1B1 function and colorectal CSC biology. They will also generate new chemical tools for studying ALDH1B1-dependent pathways, reveal the therapeutic potential of pharmacological ALDH1B1 inhibition, and provide valuable leads for the development of ALDH1B1-targeting drugs.

NIH Research Projects · FY 2025 · 2021-05

Up to 100 million Americans live with ongoing pain, costing $635 billion annually. In the United States, there are more than 200,000 people living with SLE, a chronic inflammatory rheumatic disease with multi-organ involvement that disproportionately affects females and racial minorities. Living with a chronic disease such as SLE confers multiple challenges. Pain is a frequent self-reported symptom in SLE and is often one of the first symptoms of the disease. Despite treatment advances, pain remains the most prominent, unaddressed patient complaint. The management of pain in SLE has recently become more challenging because of the alarming epidemic of addiction and mortality attributed to opioid misuse. An estimated 31-46% of patients with SLE use prescription opioids. In one study, 70% of individuals using opioids used them for ≥1 year, and 22% were taking ≥2 opioid medications at the same time. Patients with SLE are nearly twice as likely as the general population to have opioid-related overdose hospitalizations. However, efforts to mitigate opioid misuse cannot be achieved without a detailed understanding and sustained investment in clinical research on the underlying mechanisms that produce and maintain chronic pain. Characterizing the burden of chronic pain in SLE is challenging on at least two counts. First, we lack data on the prevalence and burden of chronic pain in SLE, partly due to the absence of reliable approaches to identify patients with clinically significant pain in electronic health records (EHR). Second, there is a critical need to understand the biopsychosocial mechanisms and correlates that drive the pain experience in SLE. In this mentored career development award (K01), Dr. Titilola Falasinnu will use computational methods to increase the understanding of the clinical management chronic pain in SLE using EHR. In Aim 1, Dr. Falasinnu will develop a computational chronic pain phenotyping algorithm using diagnostic codes, pain scores, narrative clinic notes and medications extracted from the EHRs of two large healthcare systems (n~2,400). She will then use the algorithm to estimate chronic pain prevalence in a population-based registry (n~76,000). In Aim 2, Dr. Falasinnu will comprehensively phenotype biopsychosocial correlates of chronic pain using an existing registry of ~500 patients with SLE attending a multi-disciplinary pain center. Throughout the award, Dr. Falasinnu will build on her doctoral training as an epidemiologist and biostatistician to develop new skills in biomedical informatics to conduct impactful pain medicine research. These skills will include working with EHR and registry data, machine learning and natural language processing, pain science, grant-writing, and scientific communication. Through coursework, clinical observation in pain medicine clinics, mentorship, and external conferences and workshops, Dr. Falasinnu will gain the skills needed to apply for her first R01 and pursue a career as a tenure-track principal investigator.

NIH Research Projects · FY 2025 · 2021-05

It is a nearly ubiquitous phenomenon – teens have a difficult time going to sleep early enough to get a full night of sleep. The resulting sleep curtailment is associated with a variety of negative consequences including depression, substance abuse, fatigue, poor academic or work performance, poor socialization, increased risk-taking behavior, and an increased risk for the development of diabetes and obesity. According to the Department of Health and Human Services in Healthy People 2020, fewer than one-third of all students in grades 9-12 get sufficient sleep – a number that has remained unchanged since 2009. This pervasive loss of sleep in adolescence is often debilitating for both the teen and the family. There are both biological (natural delay in circadian timing) and social causes, which are mutually reinforcing, for this delay in sleep timing. As a treatment for the biological component, bright light “phototherapy” is often prescribed for teens who wish to go to sleep earlier. This treatment often consists of 1-2 hours of bright light administered every day prior to desired wake time; this treatment is meant to advance the timing of the circadian clock to an earlier hour. This means that an adolescent who needs to wake up at 7AM for school would need instead to wake up at 5 or 6 AM and sit in front of bright lights for hours every day (the changes in circadian timing would revert without the daily light exposure), a difficult if not impossible set of instructions to follow. We have recently demonstrated that: (1) sequences of brief, millisecond light flashes are more potent than continuous light at changing the timing of the circadian clock, (2) exposure to such sequences of light during sleep impacts circadian timing without significantly interfering with sleep, and (3) at-home exposure to such sequences to teens increases self-reported nightly sleep by nearly one-hour per night. We hypothesize that such a light sequence can increase objective measures of sleep length, as well as improve mood and cognition in teens. In order to examine these hypotheses, adolescents enrolled full-time in high school will be recruited to take part in a 10-week, at-home placebo-controlled parallel group study, followed by a 10-week open-label study. For 8 of the 10 weeks of the parallel group study and during all 10 weeks of the open-label study, adolescents will be exposed to a sequence of flashes while they sleep. The quality and quantity of their objectively-determined sleep, measurements of mood from both the adolescent’s and the parent’s perspectives, and executive function, will be captured before, during, and at the end of the protocol. Results from this experiment could fundamentally change the manner in which delayed sleep is treated, significantly improving sleep in adolescents and simultaneously improving mood, academic performance, and family life. Results from this study will also be useful in understanding how light administration during sleep could be useful in the treatment of circadian-based sleep disorders, such as jet lag, shift work sleep disorder, and Advanced Sleep Wake Phase Disorder.

NIH Research Projects · FY 2025 · 2021-05

Abstract Although multiple pathways and targets have been proposed for OA treatment, the rate of drug failure in clinical trials has been astoundingly high. The reasons for the limited success include the late detection of the disease and a lack of understanding of the molecular heterogeneity between patients. In this proposal, we aim to capitalize on the newly developed single-cell proteomic technique, mass cytometry (CyTOF) that allows detection of 40-80 proteins simultaneously in single cells, with the aim of identifying the diverse cellular subpopulations in OA cartilage. Although cartilage is a relatively simple tissue, with a single cell type being encapsulated in its secreted extracellular matrix (ECM), the variable degree of degeneration associated with each OA patient suggests that understanding this tissue (and other joint tissues) at a single cell level can provide novel insights into both OA pathology and patient heterogeneity. This will compliment single-cell transcriptomic data, with the additional advantage that the proteomic snapshot can also identify active signaling pathways in the identified subpopulations. The single-cell proteomic approach is especially pertinent in robustly identifying rare cell populations that are difficult to discern from RNA-sequencing data. In this proposal, we will establish single cell profiles of a large cohort of OA cartilage samples using a refined panel of rare earth metal labeled antibodies in Aim1 to identify distinct subpopulations in OA cartilage. In aim 2, we will test if the modulation of two newly identified rare subpopulations would be therapeutic in a mouse model of post-traumatic OA as well as follow their dynamics with disease progression. In Aim 3, we will analyze how drug treatments affect the cartilage subpopulations and their crosstalk in different patients especially to discern between a uniform or heterogenous response among the patient cohort. Collectively, the proposed studies will be impactful in identifying novel regenerative and pathological cell populations in OA and testing the therapeutic potential of their modulation.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Recurrent pregnancy loss (RPL) affects up to 5% of couples, yet nearly half of cases remain unexplained by current testing recommendations. Euploid pregnancy loss, in the setting of unexplained RPL, is particularly frustrating for patients and providers because there is no clear explanation or any proven therapies to mitigate risk of subsequent miscarriages. As clinical presentation and subsequent pregnancy outcomes vary widely, this complex disorder will ultimately require a precision health approach. While more than 3000 human genes are conserved and likely essential for early development, remarkably little is known about their contribution to RPL and current genetic databases are essentially devoid of RPL entries. Moreover, there is currently no database that annotates phenotypes and genotypes of these essential genes. This proposal aims to define genetic determinants of RPL through clinical and molecular phenotyping and genomic sequencing of a large RPL cohort, combined with novel bioinformatics and machine learning approaches to derive predictive risk algorithms. A comprehensive approach to identify genomic markers of pregnancy loss by whole genome sequencing of well- characterized RPL trios (mother-father-pregnancy loss) will be undertaken in Aim 1. These genetics efforts will be paired in Aim 2 with metabolomic, lipidomic and single cell transcriptomic profiling preconception and in early pregnancy. Leveraged with innovative machine learning strategies in Aim 3, this approach will significantly advance understanding of the genetic underpinnings of unexplained RPL. A clinical ‘intolerome’ database will be constructed in Aim 4 to facilitate worldwide collaboration and curation of genotypes and associated phenotypes, making the genetics and omics data and results available to the public as well as other funded teams. This multidisciplinary team includes leaders in RPL, genetics, genomics, prenatal diagnosis, bioinformatics and machine learning at Stanford, UCSF and OHSU. Combined we have a substantial cohort of RPL patients that will serve as a robust recruitment source, along with a collaboration with the unique UK Pregnancy Baby BioBank of existing trios to accomplish project goals. The proposed study is anticipated to have significant clinical and research impact by identifying the genomic contribution to RPL in a large and well phenotyped cohort and building improved risk predictions based on machine learning incorporating clinical, genetic, and molecular data. This work will lay the foundation for precision medicine-based interventions for RPL couples who are difficult to diagnose and have few proven treatments.

NIH Research Projects · FY 2025 · 2021-05

The breakthroughs in diagnosis of rare diseases made possible by genome sequencing (GS) are some of the most exciting in medicine today. Rare diseases affect nearly 30 million individuals in the United States, and two-thirds of those affected are children. Studies suggest that GS (including exome and whole genome sequencing) may be able to provide a diagnosis to up to 50 percent of patients previously undiagnosed after extensive clinical and genetic testing. However, the downstream benefits and costs of these tests for patients, families, and the healthcare system remain poorly defined and methodologically challenging to assess. In response to these challenges, leaders in health economics and policy have called for the development of new, interdisciplinary methods for defining and measuring the value of GS. In order to ensure these methods are not only accurate, but also ethical, it is critical to bring to the surface a consideration of the values and preferences of various stakeholders (i.e., patients, families, clinicians, payers) involved in the use of GS for diagnosis of rare diseases, and whose values are served by different methodological approaches to defining and measuring the value of GS. The goal of the proposed research is to examine both how we can, and how we should, define and measure the value of GS for pediatric patients with rare diseases. This proposal has three specific aims. Aim 1: to identify the range of potential downstream impacts of GS for pediatric patients undergoing GS for diagnosis of rare diseases and their families, using in-depth ethnographic methods to capture perspectives of diverse stakeholders. Aim Two: To build on Aim 1 to develop a framework mapping the range of costs and benefits of GS as they relate to a) diverse stakeholder perspectives on the value of GS for diagnosis of rare diseases; and b) relevant domains of health-related quality of life (HRQL) for pediatric patients with rare diseases. Aim Three: To build on Aim 2 to develop a preference-based measure for assessing the impact of GS on HRQL specifically for pediatric patients with rare genetic diseases for use in future economic evaluations of GS. If successful, the proposed research will provide essential and timely data to guide policy recommendations for effective, ethical, and equitable implementation of GS in clinical care. Dr. Halley will achieve these aims by drawing on her current skills in ethnography and health services research, as well as on additional training in biomedical ethics, genetic and genomic testing, and health economics, to be carried out at the Stanford Center for Biomedical Ethics. Dr. Halley is already an accomplished scholar with a track record of high-quality research. The proposed training and mentored research will provide her with the additional knowledge and skills necessary to become an independent, interdisciplinary researcher examining the ELSI of new genomic technologies, with a focus on the intersection of medical anthropology, biomedical ethics, and health economics.

NIH Research Projects · FY 2025 · 2021-05

Project Summary: Chronic pain is a major healthcare problem with an annual cost of above $600 billion. The quality of data available for treatments of chronic pain is not optimal. Generalizability of explanatory randomized controlled trial data is problematic as these trials exclude up to 90% of patients: leaving out real-world patients with serious medical and psychological comorbidities. Pragmatic trials embedded in patient care compare effectiveness of currently used treatments in real-world application leading to findings that generalize to broader range of patients. The changes in clinical practice and workflow necessary to integrate this type of research within patient care present pragmatic challenges. In this research, my overall objective is to overcome these challenges using an open-source learning health care system – CHOIR – developed by my mentor at the Stanford Pain Management Center. CHOIR is currently used to track patients’ clinical trajectory and treatment response across multiple academic sites resulting in over 25 publications characterizing chronic pain. Through our pilot studies, we have already developed a point-of-care randomization for CHOIR that facilitates integration of research and patient care by allowing the physicians to randomize patients during clinic visits. We have already demonstrated feasibility of the randomization and data collection platform in two ongoing pilot pragmatic clinical trials. We are proposing to better integrate pragmatic research within our clinical practice through conducting a randomized comparative effectiveness trial in 450 patients with chronic pain comparing effectiveness of anti- convulsants and anti-depressants (two most commonly used classes of medications for treatment of chronic pain). We will use the data available in CHOIR as well as the real-world data generated from this clinical trial to build, validate and test a model to predict what clinical characteristics can predict response to either of these classes of medications. The proposed study is the first step to use flexible point-of-care randomization to compare effectiveness of different treatments in different subgroups of patients whenever equipoise exists. Our prediction model will guide decision making process of clinicians choosing between these medications based on clinical characteristics of individual patients. Dr. Salmasi is a physician-scientist with clinical training in Pain Medicine as well as academic training in Clinical Research and Epidemiology. The detailed career development and research plan presented in this application will provide the required resources and mentorship for him to become an independent R01-funded expert in two domains critical to his long-term career goals: (1) advanced clinical trial design and conduct; and (2) advanced statistics and machine learning.

NIH Research Projects · FY 2026 · 2021-05

PROJECT SUMMARY The O'Brien Lab generates new knowledge to address fundamental questions about how stem cells within our organs dynamically sense and respond to changing physiological demands. In the long-term, our discoveries help improve regenerative therapies for organs that become damaged, debilitated, or dysfunctional. Our dis- tinctive, holistic approach views stem cells as one component of the organ's cellular ecosystem, which also includes differentiated stem cell progeny and supporting cell types. To learn how organs stay healthy over a lifetime, we examine how cells in this dynamic ecosystem behave and communicate across time and space. When this exquisite, systems-level control breaks down, intestinal diseases can develop or worsen. Our re- search uses the intestine of the adult fruit fly: Its stem cells, genetic regulation, and digestive physiology are similar to human intestine, but its simpler anatomy and powerful genetic tools enable detailed study that would be infeasible or unethical in vertebrates. Adding to these strengths, our lab pioneered new methods to directly observe intestinal cells inside living flies at vivid resolution, opening the field to the study of live cell dynamics in their native environment. Ultimately, we want to understand how the intestine’s cellular ecosystem monitors the organ’s size, cell populations, and physiological state, and how it decodes this aggregate information to direct cell behaviors for optimal organ form and function. Over the next five years, our investigations of the gut’s cellular ecosystem will focus on three complementary areas. First, we will investigate how the gut main- tains homeostasis during periods of constant dietary load, precisely calibrating stem cell division to replace old cells that die and are shed. We previously discovered a mechanism that couples division and death at the or- gan-scale. Here we advance our understanding of this mechanism by uncovering how it operates live at the single-cell level, using a new protocol that precisely eliminates a selected cell to probe the live dynamics and control of individual cell replacement events. Second, we examine how the gut senses increased dietary load and expands its digestive capacity by increasing both its size and cell number. Although nutrient signals are important for growth, our new data demonstrates an orthogonal trigger: mechanical distension by ingested food. We will identify the mechanosensors that instigate stretch-induced gut growth, define the live, dynamic responses of stem cells and mature progeny, and determine how each cell type contributes to the growth re- sponse. Finally, we ask how the gut's supporting tissues—visceral muscle, enteric neurons, and tracheal vas- culature—coordinate single-cell replacement and stretch-induced growth, leveraging new orthogonal drivers to simultaneously manipulate distinct cell types within the same organ. Altogether, this work will provide a com- prehensive map of the single-cell dynamics and regulatory signals that orchestrate both homeostatic cell turno- ver and stretch-induced growth in this physiologically adaptive organ. The principles and mechanisms emerg- ing from these studies will establish foundational knowledge for next-generation regenerative therapies.

NIH Research Projects · FY 2026 · 2021-05

PROJECT SUMMARY This K24 award will support the candidate as a patient-oriented research mentor and investigator in pain and opioid science. Multiple high-quality datasets involving more than 3,000 participants from her federally funded randomized clinical trials will be leveraged to conduct hypothesis-driven secondary analyses that were beyond the scope of the original trials, thus capitalizing on existing investments for new knowledge. Secondary analyses will center on a new research direction characterizing High-Impact Chronic Pain (HICP), a national research priority, and will include traditional and advance analytics (i.e., machine learning approaches) applied to mentee-led projects. Below is the prime example for proposed secondary analyses in a national comparative effectiveness non- inferiority pain treatment trial (N=1,650) that is comparing two evidence-based behavioral pain treatments (online 1-session Empowered Relief vs. online 8-session cognitive behavioral therapy, CBT) at 3 and 6 months post-treatment. The candidate and her team will characterize long-term comparative effectiveness and heterogeneity of treatment effects for HICP vs. Lower-Impact Chronic Pain (LICP). The following proposed specific aims are outside the scope of any funded work. Specific Aim 1 -- Effectiveness and non-inferiority at 1 year: Determine the long-term (1-year) effectiveness of each treatment, and whether 1-session Empowered Relief (ER) is non-inferior to eight-session CBT on multi-primary pain outcomes (pain intensity and pain interference): H1a: ER and CBT will show baseline to 1 year reductions in pain intensity or pain interference. H1b: ER pain intensity or interference reductions will be non-inferior to CBT (non-inferiority margin = 4.5 T-score points). Specific Aim 2 -- Subgroup analysis: HICP vs. LICP: Characterize treatment outcomes between patients with HICP (baseline Pain Interference >65) and those with LICP (Pain Interference < 65). H2a: ER and CBT will show baseline to 1-year reductions in pain intensity or interference in HICP and LICP, with greater reductions for HICP due to their higher baseline impairment, thus more room to improve. H2b: ER will be non- inferior to CBT for reducing pain intensity or interference in HICP and LICP. Exploratory Aim 3 -- ML-based predictors of response: We will iteratively test established machine learning classifiers (e.g. random forests, support vector machines, and neural networks) to explore 1-year primary and secondary outcomes (sleep, depression, pain catastrophizing) to identify additional patterns and predictors of treatment response in patients with HICP and LICP. The candidate will complete coursework and readings in machine learning, will implement an informatics advisory committee comprised of local statistical and data science collaborators, and will gain further training in implementation science and research rigor and reproducibility to support enhanced mentoring capacity.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY The purpose of this project is to examine social factors in the long-term mental health of young adults. Depression, anxiety, and loneliness have steeply risen among college and university students in the last decades, creating an enormous public health burden. Mental health difficulties promise to intensify during and after the COVID-19 pandemic, making it especially urgent to examine and amplify sources of resilience among young adults. Decades of evidence demonstrate that social connectedness, in the form of subjective belonging, objective social ties, and supportive interpersonal interactions, bolster mental health in several key ways. We propose that connectedness early in college, and students’ ability to regulate their emotions through social interactions, could play a pivotal role in encouraging long-term mental health. Though foundational, past work is limited in its ability to test these predictions because it typically examines (i) dyadic relationships rather than broader networks, (ii) the effect of small numbers of social factors, independently, and (iii) short time spans. These limitations are especially relevant to undergraduate settings, as student social life is centered in broad communities on which individuals depend for social support. This project will merge tools from social psychology, network analysis, and neuroscience to provide a rich, precise, and longitudinal account of how social connectedness supports young adult mental health over time. Our team has mapped the social networks formed by a large (n > 850) cohort of incoming university students, and combined this with ecological momentary assessment of students’ interactions and indices of mental health. We have found novel evidence that (i) “social microclimates,” such as the empathy of a student’s neighbors, affect individual well being, (ii) students search their social networks for supportive peers when under stress, (iii) peer interactions mitigate stress over time, and (iv) lonely students under-perceive close social ties, and under-utilize social resources. Here, we will expand this work in several ways. First, we will incorporate a longitudinal approach: measuring students’ connectedness and well being over their college career. We will combine these data with cutting-edge predictive modeling to quantify how social ties formed early in college relate to well being in later years, as well as students’ subsequent “mental health trajectories.” Second, we will recruit a longitudinal replication cohort to establish the robustness of our effects. Third, we will build on previous neuroimaging work of our team to probe neural “signatures” of social connectedness and examine their relationship to other measures of connection, and to well being, over time. At the level of basic science, this project will represent a novel, naturalistic approach to the study of social factors in mental health, and produce a large-scale, multifaceted dataset, which will be made publicly available to facilitate the collaborative and cumulative study of social connection. At a translational level, the resulting data can pave the way for policies aimed at fostering stronger social ties—and mental health—among a broad swath of the population.

NIH Research Projects · FY 2026 · 2021-05

PROJECT SUMMARY/ABSTRACT Delirium occurs in up to 50% of patients after cardiac surgery and is associated with cognitive decline and Alzheimer’s disease and related dementias (ADRD). However, the underlying mechanisms for these complications are elusive. Further, the extent to which events in the early postoperative period increase risk for delirium, cognitive decline, and ADRD is unclear. The goal of this proposal is to examine cerebrovascular contributions to delirium / cognitive decline, with a focus on cerebral perfusion in the cardiac surgery intensive care unit (ICU). Given the wide variations in blood pressure in the ICU, coupled with the high prevalence of cerebrovascular disease, cerebral malperfusion in the ICU may contribute to delirium and cognitive decline. Current practice of targeting empiric mean arterial pressure (MAP) goals in the perioperative period may be inadequate for individual patients. Our group has championed a more personalized method based on cerebral autoregulation monitoring. Through the process of cerebral autoregulation, the brain is regulated to maintain a constant cerebral blood flow across a range of MAP. However, when MAP exceeds limits of autoregulation or when autoregulation is impaired, compensatory mechanisms fail and inadequate or excessive cerebral blood flow results. Our work in the cardiac surgery operating room has shown several results that emphasize the importance of individualizing blood pressure goals. First, the MAPs at the limits of autoregulation vary widely in patients, and both impaired autoregulation and MAP outside the limits of autoregulation are associated with organ injury. Second, in a recent trial, targeting MAP to be >lower limit of autoregulation during cardio- pulmonary bypass vs. usual care reduced delirium by 28% and improved memory scores at 1- and 12-months. To date, the majority of research has been conducted in the operating room during cardiopulmonary bypass. However, our preliminary data suggests that the early phase of ICU care may be equally important. In a small pilot study, we found that in the ICU, the extent of MAP outside the limits of autoregulation, as well as impaired autoregulation, were associated with delirium. Importantly, cognitive change was not assessed in this pilot and mechanisms for these findings are unclear. These results motivate the proposed observational study, which will examine whether (a) MAP outside the limits of autoregulation and (b) impaired autoregulation in the ICU are associated with delirium after cardiac surgery (Aim 1) and cognitive change from baseline at 1- and 12- months (Aim 2). In an exploratory mechanistic aim (Aim 3), we will characterize whether perioperative brain injury mediates or baseline neurodegeneration moderates the association of cerebral autoregulation characteristics and delirium and cognitive decline. The results of this study will more precisely characterize the role of cerebral malperfusion in the ICU with delirium and will identify mechanisms through which brain injury occurs. Promising results would also support a trial to target MAP in the ICU based on these methods. Although the cohort is only followed for one year, these results may also provide insight into potential mechanisms for longer-term cognitive decline and ADRD.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT One of the promising strategies to treat retinal diseases is to generate desired neuronal types, which are damaged or lost during disease, and develop cell replacement-based therapies. A comprehensive understanding of how distinct retinal types are formed during development can greatly inform these therapeutic strategies. While mitotic retinal progenitor cells (RPCs) are thought to be intrinsically different, the fates of retinal cells are not determined in RPCs. Many newly born postmitotic cells are still plastic. It is currently not clear how newly born postmitotic cells attain their final fate states. We propose to uncover the genes and pathways that regulate fate decisions in newly born postmitotic cells during development and determine whether they can also promote cell reprogramming in adult retina. We collected genes that are enriched in newly born postmitotic cells in the retina based on published single cell RNA-seq data, and developed novel methods to study their function specifically in newly born postmitotic cells in vivo by utilizing retroviral-based genetic approach and light sheet microscopy. A zinc finger transcription factor Myt1 (Myelin transcription factor 1) was found to be enriched in newly born postmitotic cells, but not RPCs, during development; it can promote neurogenesis, especially the formation of bipolar cells, while repressing glial fate in newly born postmitotic cells at neonatal stages. We hypothesize that Myt1 ensures the neuronal fates in newly born postmitotic cells and can contribute to neuronal reprogramming in mature retina. In the proposed studies, we will test this hypothesis through two aims. In Aim1, we will elucidate the function of Myt1 in fate determination, and test the hypothesis that Myt1 promotes bipolar cell fates by titrating down, but not completely shutting down Notch signaling pathway, and by actively repressing glial genes in newly born postmitotic cells. In Aim2, we will determine whether Myt1 together with Ascl1 and Brn2 can promote direct reprogramming of Müller glial cells into neurons in mature mouse retina. Taken together, this proposal aims to elucidate the plasticity of postmitotic cells in mammalian retina. We will focus on understanding how zinc finger transcription factor Myt1 promotes specific neuronal fates in newly born postmitotic cells during development and determining whether Myt1 can enhance neuronal reprogramming from glial cells in mature retina. This work will improve our understanding of basic biology and provide new candidate genes and possibilities for the regeneration of retinal neurons. Notably, mutations in human Myt1 gene are associated with Oculo-Auriculo-Vertebral Spectrum diseases, which are developmental disorders with ocular defects such as microphthalmia. Elucidating the function of Myt1 in retinal development can also shed light on the disease mechanisms.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract We propose to understand at cellular and circuit levels how Kiss1-expressing neurons in the anteroventral periventricular hypothalamus (AVPV) regulate female mating behavior. The ventromedial hypothalamus ventrolateralis (VMHvl) and AVPV have been shown to influence diverse female reproductive behaviors and physiology. We recently showed that presynaptic termini of progesterone receptor (PR)-expressing neurons of the VMHvl (Pvl) exhibit significant plasticity in the AVPV across the ovarian cycle. Optogenetic inhibition of this projection of Pvl neurons to the AVPV essentially eliminates female sexual behavior. In preliminary studies, we find that the subset of AVPV neurons expressing the neuropeptide Kisspeptin (Kiss1) are innervated by Pvl neurons, and that Kiss1+ AVPV (Kavpv) neurons are important for regulating female sexual behavior in vivo. Our proposed work is distinct from previous AVPV studies in that we will perform our unbiased circuit mapping, imaging, and functional studies focusing exclusively on Kavpv neurons. The AVPV is heterogeneous not only molecularly but also functionally, and brain-wide connections and behavioral contributions of distinct AVPV neuronal subtypes remain poorly understood. Moreover, and in contrast to prior work in this region, our studies will assess Kavpv neuronal connectivity and function across distinct phases of the female cycle, thereby shedding new light into how physiologically distinct hormonal states influence Kavpv neurons and behavior. In Aim 1, we will map the presynaptic inputs and postsynaptic projections of Kavpv neurons in an unbiased, brain- wide manner and validate the synaptic connectivity across the estrus cycle using electrophysiology and in vivo 2-photon imaging. In Aim 2, we will determine the activity patterns of Kavpv neurons in female during sexual and other social behaviors in freely moving animals. In Aim 3, we will test whether acute manipulation of Kavpv neurons is essential for and, even when females are in a hormonal state that renders them unreceptive, sufficient to induce female sexual behavior. The two PIs have complementary expertise for the proposed studies, and the team is therefore well suited for this project. In summary, if successful our studies will uncover mechanisms whereby an ovarian hormone sensitive hypothalamic circuit regulates female sexual and reproductive behaviors. Health Relatedness: It is well known that ovarian sex hormones can influence behavioral, cognitive, and emotive states in women. How these hormones regulate distinct behaviors and other states at the level of specific neurons and synapses is poorly understood. In addition, translational research has identified diverse neuro- psychiatric illnesses that are influenced by these hormones. Our basic research proposal, if successful, will provide new insights into how ovarian hormone sensitive hypothalamic pathways regulate social interactions in healthy animal models, and they have the potential to suggest new research avenues in translational work focused on ovarian sex hormone influenced neural circuits in disease states.