Stanford University

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Heart failure (HF) is the leading diagnosis of hospital admission in the US. Despite significant advances in medical therapies during the last three decades, HF remains one of the leading causes of mortality and morbidity worldwide. Cardiac tissue from patients with cardiomyopathy exhibits mitochondrial structural abnormalities. Current guideline-directed medical therapy (GDMT) in HF, including beta-blockers and renin– angiotensin-aldosterone (RAS) antagonism, reduces cardiac workload and helps in cardiac remodeling, but their impact on the mitochondria is not known. New therapies are needed. Stem cell-derived mitochondria- based technologies offer a novel approach to myocardial protection and repair, and their potential interaction with current gold-standard therapy also needs to be studied for successful translation. This proposal presents a study using both GDMT and mitochondria-rich extracellular vesicles (M-EVs) to transfer their mitochondria into injured cardiomyocytes and myocardium to improve viability and function in HF. In my first aim, I will determine the mechanism of the protective effects of M-EVs in combination with GDMT by adapting an innovative induced cardiomyocyte (iCM) model of hypoxic injury. To test this, iCMs will be treated with combinations of beta blockers, RAS antagonists, M-EVs, and combinations of GDMT and M-EVs. iCM function and viability will then be rigorously assessed. In the second aim, the functional benefits of the M-EVs and GDMT will be studied in a mouse model. I will study the effects of combined therapy on mitochondrial augmentation and biogenesis by looking at proteomic and metabolomic profiles of the injured myocardium in relation to a key regulator, PGC-1α. This proposed study will improve our understanding of the interaction of mitochondria-based therapies with existing heart failure therapies and give critical insight into the future development of much-needed novel therapies.

NIH Research Projects · FY 2025 · 2024-09

Project Summary / Abstract The typhoidal Salmonellas (TS), primarily Salmonella Typhi and Paratyphi A, are a major cause of global morbidity and mortality, particularly in Asia where widespread antimicrobial resistance is an increasing concern. The Indo-Pacific is endemic for TS and vulnerable to these infections due to suboptimal surveillance networks, insufficient sanitation and hygiene infrastructure, and lack of support for interventions to reduce TS burden (vaccine only targets S. Typhi, is not widely accessible, and indirect protection may be modest). This region is at increased risk for TS due to disproportionate susceptibility to hydrologic events (drought, flooding) associated with extreme weather. Predicting TS transmission and evaluating interventions to reduce TS risk in the setting of extreme weather is critical. Water, sanitation, and hygiene (WASH) improvements decrease TS burden through reduced fecal contamination of the environment. However, trials of WASH interventions often target the household-level (e.g., behavioral interventions, kitchen water filters) and are limited in reproducibility and generalizability, frequently using non-specific surrogate markers of human health. Revitalizing Informal Settlements and their Environments (RISE) is a cluster-randomized controlled trial of community-level WASH infrastructure upgrades currently underway in 24 informal urban settlements in Indonesia and Fiji, two countries with high TS risk. RISE provides an ideal opportunity to study targeted solutions to reduce TS burden and evaluate their impact. Utilizing RISE-collected samples, the research detailed in this proposal uses: Aim 1) novel serosurveillance techniques to estimate “seroincidence” as a surrogate for TS incidence and Aim 2) genomic methods to identify TS in human and environmental microbiomes. To inform improved future interventions under different scenarios, these data will be used to: Aim 3) establish compartmental modeling approaches to predict and assess the impact of WASH interventions on TS transmission and ultimately disease reduction. The candidate is an infectious diseases fellow with a background in molecular biology, genomics, computational biology, and global health research. The candidate’s primary career goal is to be an independent NIH-funded physician-scientist committed to developing, assessing, and implementing interventions aimed at controlling TS and other infectious diseases that disproportionately impact populations in resource-constrained settings. To achieve independence, the candidate requires targeted mentoring and additional training in: 1) laboratory methods and field research management, 2) advanced bioinformatics analysis and infectious diseases modeling, and 3) research communication and impact. The proposed research and training plans will be supervised by primary mentor Dr. Luby, a global health clinical research expert and PI of RISE. Dr. Andrews (seroepidemiology, modeling) will serve as co-mentor, providing additional focused expertise. The candidate will also meet quarterly with advisors Dr. Charles (laboratory methods, seroepidemiology), Dr. Bhatt (genomics, bioinformatics), and Dr. Baiocchi (statistical design and analysis).

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Since the 1990s, pediatric oncology has made sustained strides in understanding the biology that drives children’s cancers, but these understandings have not yielded significant therapeutic gains. Approximately 20% of children diagnosed with cancer today are not cured with current standard regimens and cancer remains the leading cause of disease related death in children in the US and other high-income countries. Nearly all current standard treatments are cytotoxic regimens, which are associated with shortened lifespans, high rates of severe late effects and second malignancies and diminished quality of life. There is an urgent need to develop safer and more effective, targeted therapeutics for treatment of children’s cancers. During the last three decades, the Mackall lab has made sustained contributions to a growing body of evidence demonstrating that immunotherapies can selectively target even the most aggressive cancers, raising the prospect that immunotherapies specifically designed for children’s cancers could improve cure rates and diminish toxicity. This program will build upon deep understandings of fundamental cellular and molecular immunology in the Mackall lab and recent progress in defining mechanisms of immune evasion and resistance to current immunotherapies to create next generation immunotherapies for children’s cancer that manifest enhanced safety and potency and potentially enhance access to patients. We will leverage a surfeit of emerging synthetic biology and protein engineering platforms emanating from scientific labs at Stanford University which have been, and continue to be, integrated into the Mackall laboratory. This work will occur within the enabling ecosystem provided by the Stanford Center for Cancer Cell Therapy, which is led by the PI and supports infrastructure to enable rapid forward and reverse translation of promising therapeutics. Areas of focus will include, but are not limited to, developing new approaches to overcome the suppressive tumor microenvironment in pediatric solid tumors, optimizing multi-specific CAR T cells, using combinatorial engineering to create uber potent T cells while enhancing understanding of toxicities that may limit application of these enhancements. We also will enter the promising new arena of in vivo gene delivery to engineer immune populations without the need for cumbersome and expensive ex vivo manufacturing. We have already demonstrated expertise in utilizing CRISPR/Cas9 for screens and therapeutic manipulation and are routinely employing synthetic biology technologies including CRISPR/Cas13 to regulate RNA, base editing to knockout genes without double strand breaks, mutagenesis combined with yeast display to create proteins with unique and specific properties, drug regulatable platforms to control protein expression and viral free cell engineering. Biopharma does not prioritize development of therapeutics for children’s cancers, and thus this Program addresses a critical unmet need to leverage the latest advances to develop more effective and less toxic immunotherapies for children’s cancers.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Myofascial pain syndrome (MPS) is one of the most common forms of acute and chronic musculoskeletal pain, a common cause for opioid use, and affects 10-15% of patients seen in general medical clinics. Central to this syndrome are myofascial trigger points (MTrPs), hard, palpable, discrete, and localized nodules that produce referred pain and local tenderness at the site upon compression. Despite its prevalence and clinical significance, the pathophysiology of MPS is not well understood. The subjective nature of current diagnostic methods and a lack of objective markers of MPS, hinders the precision of diagnosis and treatment. There is therefore a clinical need for improved diagnostic tools sensitive to the complex multifactorial (compositional, vascular and neurogenic) factors of MPS, that can unravel the intricate mechanisms of MPS and enhance patient care. Imaging offers objective measures of multiple disease features to improve the diagnosis and assessment of MTrPs and MPS. MRI, with its excellent soft-tissue contrast, can provide detailed anatomical information of skeletal muscle and fascia. Further, quantitative methods can probe muscle microstructure [Diffusion Tensor Imaging (DTI) and Diffusion Kurtosis Imaging (DKI)], microcirculatory velocity [Intravoxel Incoherent Motion (IVIM)], local contraction (dynamic Diffusion Weighted Imaging), muscle and fascial fibrosis and densification [Ultra-Short Echo Time (UTE) MRI]. Synergistically, PET imaging, with its sensitivity to functional and metabolic process, provides a tool for assessment of inflammatory processes including neurogenic inflammation. This work aims to develop PET and MRI methods to identify novel imaging biomarkers that can diagnose and characterize MTrPs in MPS. Our Specific Aims are (1) develop clinically-translatable [18F]FDG PET-MRI imaging markers that can reflect disease and pain mechanisms and characterize MTrPs in MPS; (2) evaluate whether PET and MRI biomarkers are able to differentiate the microstructural, compositional, functional and metabolic changes in MPS patients from normal myofascial and neurogenic features in age and sex matched controls as between MPS patients with pain in their upper back muscles (Trapezius, Rhomboid Major an Minor, and Levator Scapulae) and the same muscles on their non-painful contralateral side. If successful, based on an receiver operator characteristics (ROC) area under the curve (AUC) of 0.7, we will (3) evaluate our imaging approaches in a single-blind randomized clinical trial of patients being treated with ultrasound guided muscle anesthetic injections and a sham injection to evaluate treatment response and differences between treatment groups. The significance of our work is the development of novel biomarkers that can objectively diagnosis and characterize tissue level changes in MTrPs and MPS . Our key innovation is the development of PET and MRI tools to assess specific mechanisms of theorized MPS and MTrPs pathophysiology as well as mechanisms of pain generation in MPS. Our investigative team includes experts in novel imaging techniques, clinical assessment and treatment of pain, and clinical studies evaluating both imaging markers and treatment response.

NIH Research Projects · FY 2025 · 2024-09

Concerns about deteriorating mental health, substance use disorders, early development of chronic conditions, exposure to economic shocks, and to violence---causes that may all lead to premature mortality among adolescents and young adults in the United States, have been rising in recent public discourse. Despite these concerns, and the importance of this age group for the society’s future well-being, research on the trends, variation, and above all distal causes of mortality in this age group has been limited when compared to evidence on the causes and consequences of mortality among infants and older adults. This dearth of evidence likely stems from the absence of data sets with wide population coverage for this age group that links mortality—a very rare outcome—with anything beyond the basic demographic information about the individuals. In this project, we propose to take advantage of extensive infrastructure for linking US administrative data that has been developed by the U.S. Census Bureau, to construct a new database that links administrative birth and death dates for 10 to 25 year olds, with data on their demographics (race, ethnicity, sex, age, disability status), education, familial circumstances, parental income, access to public health insurance, healthcare providers, and local geographies, covering the time period from 2005 to the 2025. Using this new database, we will document how youth mortality evolved over time and space in the Unites States. We will also describe mortality inequalities across different subpopulations based on a rich set of demographic and socio-economic characteristics of adolescents, young adults, their families, and communities. Finally, we aim to use natural experiments and statistical techniques of causal inference to improve our understanding of the underlying distal causes of observed mortality patterns.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Bronchopulmonary dysplasia (BPD), or infant chronic lung disease, is among the most devastating complications of preterm birth. BPD affects half of surviving extremely preterm infants, is associated with life- long deficits in health and cognition, and carries enormous societal burden and cost. Strikingly, there are no therapies shown to improve outcomes for infants with BPD. Our research seeks to resolve this care gap. Gastroesophageal reflux disease (GERD) is diagnosed in >40% of infants with BPD. Through aspiration and neurogenic mechanisms, GERD exacerbates lung disease in BPD by induction of bronchospasm, hypoxemia, airway injury, infection, and chronic lung inflammation. Unfortunately, there are no proven safe and effective ways to treat GERD in infants. Acid suppression and GI promotility drugs are ineffective and carry significant risks. Surgical fundoplication is invasive and often inappropriate for infants with unstable lung disease. Conversely, transpyloric tube feeding is easily initiated and has been shown to reduce aerodigestive sequelae of GER in older children and adults. Unfortunately, the safety and efficacy of transpyloric feeding in BPD is uncertain and the limited infant data are conflicting. Our preliminary data show variable contribution of GERD to lung disease in BPD and significant heterogeneity in response to transpyloric feeding. In a recent randomized trial of alternating 4d periods of transpyloric and gastric feeding in 15 infants with BPD, we showed that transpyloric feeding reduced hypoxemia and FiO2 need in some infants but worsened these in others. These findings demand identification of evidence-based means to individualize feeding route selection and GERD management in preterm infants with BPD. A key first step towards achieving this goal is to determine whether transpyloric feeding safely and effectively reduces GER in infants. To do so, we propose a randomized trial that will compare moderate duration (2wk) transpyloric vs. gastric tube feeding in very preterm infants (n=60) with grade 2-3 BPD. Serial gold-standard esophageal pH-impedance testing will be used to objectively define pre-trial GER and in-study treatment response. Motivated by our prior data suggesting heterogeneity of treatment effects, we will determine whether the tolerability and physiologic efficacy of transpyloric feeding varies by pre-trial GER severity. Common GER and lung aspiration biomarkers will be measured and compared to objective pH/MII and clinical outcome data. The results of this study will immediately inform evidence-based GER diagnosis and feeding practices in BPD and establish the foundation required to conduct a definitive, multicenter trial of prolonged transpyloric feeding in chronically tube fed preterm infants with grade 2-3 BPD who are at high risk for GER-induced lung injury.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Endometriosis, a debilitating chronic disease, affects around 10% of reproductive age women worldwide, severely impacting quality-of-life. Unfortunately, there is often a decade-long delay from first symptom onset to definitive diagnosis, underscoring the urgent need for improved earlier diagnostics. Evidence suggests that local endometrial immune microenvironment (EIM) dysregulation may play a key factor in endometriosis pathobiology. Even though uterine NK (uNK) cells are the predominant cytolytic lymphocyte in the EIM, they have been surprisingly understudied in endometriosis. The objective of this proposal is to gain a better understanding of the contribution of uNK cells to endometriosis and to identify non-invasive menstrual blood (MB) biomarkers that can be leveraged for diagnosis. I hypothesize that defective cytolysis of endometrial stromal cells by dysregulated uNK cells contributes to an abnormal tissue environment in endometriosis. The work in this proposal will be completed at Stanford University School of Medicine in Dr. Gaudilliere’s laboratory (primary mentor) with guidance from an interdisciplinary mentoring team at UCSF (Dr. Giudice, Dr. Sirota) and Duke University (Dr. Coyne). A rigorous research training plan is proposed that harnesses cutting-edge high-dimensional single-cell suspension and spatial immune profiling, sparse machine learning methods, and ex vivo assembloid modeling. Aim 1 (K99) will characterize the inhibitory/activating receptor repertoire and functional capacity of uNK cells of women with and without endometriosis (scRNAseq and mass cytometry). In addition, examining the spatial organization of the EIM will provide crucial insight into microenvironmental interactions that underlie immune responses in healthy and diseased endometrium (imaging mass cytometry). This aim builds on my current expertise in human reproductive immunology and multi-parameter approaches and is a continuation of prior work showing that uNK cells predominate in the EIM and exhibit a tissue-specific receptor profile. In Aim 2, an innovative MB immunoassay will be established on the mass cytometry platform (K99) by discerning the similarities and disparities between biopsy and MB-derived uNK cells. In the R00 phase, high-dimensional predictive modeling will identify uNK cell features in MB that accurately classify endometriosis versus control. In Aim 3, through Dr. Coyne’s training (K99), endometrial assembloids derived from primary MB-derived endometrial cells will be established to replicate the EIM ex vivo. In the R00 phase, uNK cells will be incorporated into this assembloid model to further investigate uNK-mediated mechanisms in abnormal endometrial tissue. In summary, the proposed studies will uncover fundamental uNK cell mechanisms that contribute to endometriosis pathobiology and establish a foundation for developing non-invasive diagnostics. The training, approach, and results generated will offer a unique framework for future research on various uterine and reproductive disorders, aiding my career development and enabling me to become an independent academic translational scientist in reproductive immunology, bridging systems immunology, assembloid modelling, and computational biology.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Exercise represents one of the most powerful and beneficial interventions for health and wellness, though patients with and without chronic disease struggle to exercise enough to garner its benefits. One sought after approach to overcoming these challenges is to provide the benefits of exercise pharmacologically, a so-called "exercise-in-a-pill" solution. This involves identifying biomolecular mechanisms responsible for exercise benefits and engaging them pharmacologically using small molecule agents. To accelerate discovery of exercise mimetic drugs, this project synergizes existing data from two National Institutes of Health Common Fund projects. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) provides a map of the biomolecular response to exercise, while the Library of Integrated Network-Based Cellular Signatures (LINGS) Program provides a map of the biomolecular response to small molecule exposure. The investigators hypothesize that biomolecular exercise pathways and small molecule drug candidates from these two resources can be matched by their shared biomolecular "signatures". The maps are linked by matching exercise-induced changes in biomolecular expression (i.e., a gene expression "signature" of exercise) from MoTrPAC to similar expression changes induced by small molecules found in LINGS. By linking these two data sets, the project will create a detailed, browsable, and interactive resource for identifying potential exercise mimetics. Aim 1 seeks to identify biomolecular signatures of exercise training from MoTrPAC by analyzing publicly released multi-omic response data from MoTrPAC's exercise training studies in young adult rats. Specific objectives include: 1) identifying biomolecular "signatures" through gene set enrichment analysis, network clustering, and gene regulatory networks; 2) evaluating the validity and reliability of these signatures across tissues, sexes, and timepoints in the rat study; and 3) generating an annotated database of signatures for alignment with LINGS. Aim 2 integrates MoTrPAC and LINGS biomolecular signatures in a cloud-based infrastructure that matches MoTrPAC signatures with those available from LINGS. This infrastructure will be designed to 1) organize, browse, and fiter the MoTrPAC signatures database; 2) query existing cloud-based applications developed by LINGS for engagement with their library of data; and 3) deliver these results in a visually informative and interactive web application to maximize the user's ability to gain novel insights. Future efforts will expand the infrastructure by adding new MoTrPAC data and annotating exercise signatures with knowledge from other Common Fund datasets. Additionally, the project aims to leverage results to identify and prioritize pathways related to insulin resistance and other diseases for experimentation in model systems. This comprehensive approach seeks to accelerate the identification of exercise mimetics and provide valuable resources to translational researchers and stakeholders in the pharmaceutical pipeline.

NIH Research Projects · FY 2025 · 2024-09

Project Summary In mammals, 5-methylcytosine is the most common form of DNA methylation, and the level of methylation of some specific CpG sites shows a strong correlation with age. These correlations can be used to build machine learning-based models that can accurately predict the age of biological samples. Because these models can quantify age with very high accuracy, researchers have termed them epigenetic aging clocks (e.g., Horvath’s pan-tissue epigenetic clock and Hannum’s blood-based epigenetic clock). However, the reliability of existing epigenetic clocks is limited, as they are built based on pure correlations, and it is unclear whether age- associated methylation changes are causal to aging-related phenotypes. A new generation of epigenetic clocks built on causal information will be more reliable and can enable the possibility of large-scale screening of anti-aging interventions. For the F99 phase of this proposal, I performed epigenome-wide Mendelian Randomization to identify CpGs potentially causal to aging-related traits. This causal information was then incorporated into epigenetic clock models to build causality-informed aging clocks, which are shown to separate age-related damage from adaptation, namely DamAge and AdaptAge. I also built ClockBase, a database that contains over 300,000 experimental samples from GEO with the epigenetic age pre-calculated. I plan to further standardize the sample information using large language models and apply the causality-informed biomarkers to screen for anti-aging interventions. In the K00 phase, I will use the protein language model and protein design tool to expand the universe of anti- aging interventions. Specifically, I will study the protein structural features across mammalian species with various lifespans to understand which features are associated with longevity. Then, I can incorporate this information into protein design and optimize existing proteins to support a longer lifespan. This proposal will advance our understanding of the molecular mechanisms underlying aging by incorporating causality into epigenetic clock models. By distinguishing between age-related damage and adaptation, we can develop more precise and informative aging biomarkers, which will have significant implications for aging research and potential clinical applications. The K00 phase of the project will pioneer the application of protein language models and protein design tools in aging research. Ultimately, it could pave the way for a completely new branch of aging research – treating aging through the gradual redesign of the proteome.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Aging is associated with dramatically increased susceptibility to infectious diseases like influenza. Although vaccination is the primary strategy for minimizing flu deaths, older adults have consistently impaired vaccine responses. Preliminary analyses of tonsil T cells from older vs younger adults reveal that CXCL13 is significantly decreased in old follicular helper T cells as compared to young. Further preliminary experiments in tonsil organoids reveal a role for CXCL13 in memory CD4+ T cell homeostasis. While CXCL13 is expressed by follicular helper T cells in humans, mouse T cells lack CXCL13 expression entirely. The Research Strategy proposed here will leverage human immune organoid technology, developed in sponsor Dr. Mark Davis’ lab, to define the role of CXCL13 as an aging-associated defect in memory T cell homeostasis. This work will reveal the mechanism of CXCL13-mediated maintenance of memory T cells (Aim 1) and define the identity of the T cell pool that is maintained by CXCL13 (Aim 2). In Aim 1, the applicant Dr. Casey Beppler will use CRISPR- engineered CXCL13-KO tonsil organoids, specific cell type depletions, and single cell RNA sequencing to reveal the cellular and molecular mediators of CXCL13-mediated T cell homeostasis. In Aim 2, Dr. Beppler will integrate cellular phenotyping of human tonsil and spleen T cells across adult lifespan with spheromer staining of antigen-specific T cells (also developed in the Davis Lab) in CXCL13-KO tonsil organoids to determine the specific T cell population that is maintained by CXCL13. Although work proposed here will focus on the role of CXCL13 in secondary lymphoid organs, these findings are likely to reveal mechanisms at play in tertiary lymphoid structures across disease states from cancer to autoimmunity. The research and career development training plans are tailored to enable Dr. Beppler to gain new skills in modeling human lymphoid tissues with immune organoids and single cell RNA sequencing, as well as professional development skills in mentoring, oral presentation, and grant writing. Sponsor Dr. Mark Davis is a long-standing expert in the field of antigen- specific T cell responses and a leader in human systems immunology. Advisors will contribute with their complementary expertise: Dr. Purvesh Khatri (bioinformatics), Dr. Le Cong (CRISPR and gene editing), and Dr. Bali Pulendran (multi-omics analysis of human immune responses). The Stanford Institute for Immunity, Transplantation & Infection directed by Dr. Davis is an excellent environment for collaborative and cutting-edge research, supported by outstanding infrastructure (Stanford Center for Genomics, Human Immune Monitoring Center). In summary, the strong mentorship and training plan will prepare Dr. Beppler for her future independent career studying human T cell immunology. This project will improve our understanding of an aging-associated defect in human lymphoid tissues, thus providing the potential for future therapies to promote memory homeostasis and limiting the number of deaths to vaccine-preventable diseases.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Age related macular degeneration (AMD) is a leading cause of vision loss in individuals over 55 years of age and is increasing as the world population becomes older. Treatment options for early AMD are limited and, therefore, vision will become increasingly threatened in the elderly population. Evidence from observational studies suggest that whole foods, Mediterranean diets and low glycemic load (LG) diets are associated with less progression of AMD, suggesting a cost-effective approach to slow AMD progression. The goal of this project is to assess feasibility aims for dietary intervention in a non-diabetic AMD cohort with early stage disease and to plan a future large, multicenter study that will compare LG-Mediterranean dietary intervention to standard diet. Our aims are to: 1) demonstrate feasibility of behavioral intervention to change dietary intake in subjects with intermediate AMD from consuming a typical Western diet to a LG, Mediterranean diet; 2) assess the feasibility of obtaining sensitive retinal images and sdOCTs and to explore assays (metabolomic markers, complement factors, gut barrier dysfunction and dysbiosis, AGEs, and oxycholesterols (e.g. 7-ketocholesterol) for consideration as non-disease biomarkers for a future UG1; 3) develop a protocol for a future multicenter study and a Manual of Procedures. Our team is cross-disciplinary including experts in behavioral dietary intervention, endocrinologists, retinal specialists, clinical trial experts and leaders of large consortia of AMD intervention studies in the US and Europe. Innovative aspects of the planning study include: to assess the willingness of an elderly population without diabetes to adopt a dietary intervention; to use continuous glucose monitoring as a coaching tool for the cohort and to learn about the sensitivity of continuous glucose monitoring for the future multicenter study; to explore ophthalmologic and non-ophthalmologic biomarkers for the future study; and to perform feasibility studies on systemic biomarkers relevant to the pathophysiology of AMD and that may improve sensitivity of outcome measures in the future multicenter clinical trial.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ ABSTRACT The brain and spinal cord are filled with and surrounded by a complex fluid, the cerebrospinal fluid (CSF). CSF directly contacts brain progenitors to act as a stem cell niche that provides buoyancy, ionic and osmotic balance, and health- and growth- promoting factors. Pathological deviations in CSF volume and composition are associated with congenital, neuropsychiatric, infectious, and geriatric diseases, as well as injury. As the brain matures during development, CSF composition changes profoundly. We recently discovered that CSF ion concentrations also change dramatically across development, including a ~2.5-fold drop in CSF [K+] during the first postnatal week in rodents (from ~10 mM to ~3.2 mM). This large natural shift in CSF [K+] has the potential to affect key processes in brain development including progenitor maintenance, neurogenesis, and physiology. Our lab has the tools and expertise to directly control CSF [K+] and assess neurodevelopmental outcomes. Extracellular K+ is a fundamental signal for proliferation, survival, and cellular migration. K+ is also a key ion regulating cellular physiology, excitability, and ion co-transport. It is therefore crucial to understand how developmentally dynamic CSF ions contribute to brain generation and maturation. A major tissue source of CSF ions is the choroid plexus. We found that choroid plexus-restricted knockdown or overexpression of the sodium- potassium-chloride cotransporter NKCC1 (Slc12a2) delays or accelerates the drop in CSF [K+], respectively. It is now possible to directly test hypotheses that stage-specific CSF ions support neural progenitors and immature neurons to drive long-term brain function. Here, we propose to answer fundamental, yet transformative questions of whether CSF ions are necessary and sufficient to support brain development. Here we adapt explant manipulation and in vivo AAV gene delivery to investigate how the higher [K+] that we observe in early CSF specifically supports early neurodevelopment (aim 1); how the lower [K+] that we observed in postnatal CSF specifically supports neural maturation (aim 2); and test whether the shift in CSF [K+] alters the Cl- and K+ shunting that occurs as part of the developmental GABA switch (aim 3). This multi-tiered approach will yield widely applicable information and tools for testing hypotheses of CSF ion function over development, and in health and disease. Each component builds on my unique expertise to facilitate a new research program investigating how CSF supports the maturation of neurons and circuits underlying psychiatric disease. This innovative research program will fundamentally change our understanding of brain development and reveal roles for CSF ions in supporting brain generation and physiology. The CSF is an accessible avenue for CNS surveillance or supplementation, even in humans (e.g. intranasal spray, intrathecal injections). Therefore, outcomes will guide efforts to harness CSF to provide a supportive developmental environment for the brain, reduce neurologic symptoms, and may provide therapeutic strategies for tackling a range of disease.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT There is considerable societal need to better understand neurobiological mechanisms, psychosocial processes, treatments, preventions, and policy in child and maternal pain. This application seeks funding for a T90/R90 institutional postdoctoral training program in clinical pain research, with a focus on maternal and childhood pain, at Stanford University. Our proposal describes a collaborative, interdisciplinary postdoctoral training program to bolster the clinical pain research workforce as outlined through the NIH HEAL Initiative Partnership to Advance Interdisciplinary (PAIN) Training in Clinical Pain Research. We venture to join with other T90/R90 postdoctoral training programs in clinical pain research to foster a valuable cohort experience for trainees to collaborate across institutions. This T90/R90 will reside within the Department of Anesthesiology, Perioperative, and Pain Medicine and be in partnership with the Maternal and Child Health Research Institute, the Department of Pediatrics, Psychiatry and Behavioral Sciences, and Biomedical Data Science at Stanford University School of Medicine. This program will provide mentorship to trainees to launch and maintain productive careers in the clinical pain research workforce. We propose training of five fellows per year who will spend 2-3 years cumulative time in research. Research opportunities are offered by the NIH- funded faculty mentors with proven records of success in the training of postdoctoral fellows, with various research programs ranging from data science, translational, clinical, and health services research. Core faculty research leaders will help match trainees to mentors within the HEAL clinical pain research content areas of: (1) bioinformatics, (2) pain across the lifespan, specifically child and maternal pain, (3) nonpharmacological (behavioral) interventions for pain, (4) prevention of the transition from acute to chronic pain and (5) health and wellness in the field of pain. Program mentors will provide training in clinical pain research, neuroscience, biomedical data science, behavioral health, epidemiology, maternal health, and health in children and adolescents. The training will (1) integrate the biopsychosocial model of pain, (2) use a team science approach through partnerships with psychiatry, pediatrics and biomedical data science, (3) center on whole person health, (4) incorporate patient participatory research methods to ensure lived experiences of pain inform the clinical research outputs, (5) provide advanced statistics and research methods, (6) involve grantsmanship skills, and (7) prepare for applying for and securing academic positions in clinical pain research. Lastly, the program will coordinate monthly webinars related to HEAL priority areas an annual Maternal and Child Pain School open to all cohort participants, and a robust didactic program to provide career development skills with seminars in stated training areas. Trainee progress will be monitored by the Assessment and Evaluation Team. The long-term goal of this T90/R90 is to train the next generation of interdisciplinary scientific leaders in maternal and/or child pain.

NIH Research Projects · FY 2025 · 2024-09

This proposal is for a new T32 predoctoral program to train the next generation of cancer researchers with expertise in examining complex factors influencing cancer risk. The rationale for this T32 is to integrate multiple fields of cancer research through cross-disciplinary education and professional development. The program utilizes the strengths of Stanford University and Silicon Valley to offer specialized training in cancer research through a combination of coursework, seminars, internships, and mentored projects. Coursework and seminars include core offerings developed for this program. Trainees will acquire skills in key cancer research disciplines, including epidemiology, biostatistics, genetics, omics, and environmental health. The program also introduces trainees to the multifactorial influences on health outcomes, spanning biological, social, and environmental domains. The T32 will emphasize both technical and interpersonal skills such as advanced research methods, data analysis, communication, teamwork, and leadership. Training will be grounded in the principles of responsible research conduct. Drs. John Witte and Melissa Bondy lead the program having overseen multiple previous training programs and with decades of successful experience training the future generation of cancer researchers. The T32 mentoring team is comprised of 26 outstanding faculty members with cancer-focused research projects across scientific disciplines. The program has extensive financial and administrative support for trainees from Stanford University and the Stanford Cancer Institute, rigorous student advising, and evaluation among the trainees, mentors, an internal executive committee, and an external advisory committee. By integrating multiple approaches, the T32 has a strong foundation in which trainees develop the skills vital for furthering their careers in research to decipher the causes of cancer and contribute to the development of more effective prevention and treatment strategies of this common, but complex disease.

NIH Research Projects · FY 2025 · 2024-09

Project Summary / Abstract Up to 80% of patients with end-stage kidney disease (ESKD) receiving dialysis have secondary hyperparathyroidism (SHPT), a condition of abnormal bone and mineral metabolism which is associated with higher mortality, cardiovascular events, fractures, and parathyroidectomy. Calcimimetics, a mainstay of treatment for SHPT, act by lowering parathyroid hormone (PTH) secretion; cinacalcet and etelcalcetide are the two FDA-approved calcimimetics available in the United States. Before 2018, over 20% of Medicare beneficiaries lacked access to these medications because they lack Medicare Part D (prescription drug coverage). In 2018, all dialysis patients suddenly gained access through a change in Medicare policy known as the Transitional Drug Add-On Payment Adjustment (TDAPA). Using administrative claims data from the United States Renal Data System (USRDS), which houses Medicare claims for all individuals on dialysis, this project will leverage the TDAPA policy as a natural experiment to evaluate its effect on calcimimetic prescriptions and subsequent patient morbidity and mortality. Aim 1 seeks to compare the prescribing patterns of calcimimetics for individuals on dialysis before and during TDAPA, using a longitudinal differences-in-differences analysis to estimate the effect of the TDAPA policy in patients without Medicare Part D coverage. Early studies of TDAPA suggest that calcimimetic prescription patterns varied widely from facility to facility. Sub-Aim 1a will study differences between cinacalcet and etelcalcetide use after implementation of TDAPA, focusing on comparing the facility characteristics associated with greater likelihood of etelcalcetide use. Aim 2 attempts to evaluate SHPT-associated outcomes and determine whether expanded access to calcimimetics ameliorated poor outcomes through a differences-in-differences analysis of TDAPA. Though SHPT is associated with morbidity and mortality, whether calcimimetics can improve such outcomes is less clear, owing to limitations of randomized-controlled trials. In this aim, the TDAPA policy will be used as a natural experiment to examine the causal, real-world effect of calcimimetics on SHPT-associated outcomes, with broader implications for examining how Medicare policies affect important outcomes for patients on dialysis. The proposed work will serve as a post-doctoral fellowship training vehicle for the applicant at Stanford University, under an interdisciplinary mentorship team bridging Stanford’s Division of Nephrology and Department of Health Policy, with collaboration from the University of Southern California. This team brings expertise in bone mineral disease, USRDS data, and econometrics that will equip the trainee with the necessary skills to advance her career in health services research with a focus on the ESKD population.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Cancer immunotherapy has revolutionized oncology. Chimeric Antigen Receptor (CAR) T cells in particular have shown dramatic successes in some liquid tumors, with multiple FDA approved products in overlapping heme indications. Yet patient responses to engineered cell therapies are highly variable and unpredictable. Given both the extraordinary cost and extraordinary curative potential of engineered T cell therapies, matching the right cell to the right patient remains a large unmet clinical need. Yet how patient T cells variably respond to the wealth of different potential synthetic genetic alterations remains unexplored. Our long term goal is to develop personalized cellular immunotherapies and the diagnostic tests necessary to nominate the optimal engineered cell for a specific cancer patient. As a first step towards this goal, we will develop scalable pooled screening methodologies to rapidly assay approved and proposed cellular immunotherapy constructs at scale across large numbers of patients (Specific Aim 1). In cohorts of liquid and solid tumor patients, we will dissect the intrinsic variability of patient T cells synthetically engineered with CARs in repetitive stimulation assay models (Specific Aim 2). Finally, we will determine predictive correlates of patient specific engineered T cell function using high dimensional immune cell profiling and patient clinical metadata (Specific Aim 3). We hypothesize that engineered immune cell performance is highly variable and patient specific, and measurements of this variability across cancer patient’s T cells will nominate future predictive diagnostic strategies for personalized cellular immunotherapy choice. In this K08 Mentored Clinical Scientist Research Career Development Award application, the applicant, Dr. Theodore Roth, MD, PhD is a Clinical Pathologist in the Stanford University Department of Pathology. In addition to the enclosed Research Plan, this proposal encompasses a five-year Training Plan to complete his mentored career development with the primary goal of becoming and independent academic physician scientist running a research group devoted to developing personalized cellular immunotherapies. Dr. Roth will undergo training in diagnostic assay development (Training Aim 1), high dimensional immune profiling technologies (Training Aim 2), and successful assumption of research independence (Training Aim 3). Dr. Roth’s remaining mentored training will be overseen by a multidisciplinary group of distinguished physician scientists, including his primary mentor Dr. Ansuman Satpathy and co-mentor Dr. Crystal Mackall, along with a senior Advisory Committee composed of Drs. Ed Engleman, Howard Chang, and David Miklos. Stanford University provides an outstanding environment to complete Dr. Roth’s transition to research independence, with a highly active clinical cell therapy program overseen by his co-mentor, Dr. Crystal Mackall, and vibrant research communities in immunotherapy, genetics, diagnostic assay development, and high dimensional molecular technologies. Completion of the Research and Training Plans will propel Dr. Roth’s independent research career as an academic physician scientist.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Resistance to therapy is responsible for 90% of cancer patient mortality. Our understanding of how cancer cells resist therapy-induced death is limited. My goal is to better understand the molecular mechanisms of cancer cell therapy resistance, with an eye toward improving therapy. Identifying and characterizing novel resistance mechanisms should facilitate the design of more effective cancer therapies. Immunotherapy and ionizing radiation are two common cancer therapies. These therapies kill cancer cells, in part, via ferroptosis: a non-apoptotic cell death mechanism characterized by lipid peroxidation. Sensitizing cancer cells to ferroptosis may improve the efficacy of these and other cancer therapies. To effectively sensitize cancer cells to ferroptosis, we must identify how cancer cells normally resist ferroptosis. However, the mechanisms by which cancer cells resist ferroptosis are poorly understood. I recently showed that the transcription factor nuclear factor erythroid 2 like 1 (NFE2L1, or NRF1) is required to protect cancer cells from ferroptosis. My overarching hypothesis is that NFE2L1 is central to an important pathway promoting ferroptosis resistance in cancer cells. The goal of this research, as part of my overall training plan, is to test this hypothesis and determine how NFE2L1 protects cancer cells from ferroptosis. Ultimately, this research has the potential to inform advances in cancer therapy. I propose two Specific Aims. In Aim 1, I will test whether certain post-translational modifications of NFE2L1 are required for ferroptosis resistance. These studies will be conducted using genetic and pharmacologic approaches to perturb NFE2L1 N-glycosylation and cleavage and assess the effects on ferroptosis sensitivity. To support the successful execution of this aim, I will be trained in glycobiology by my co-sponsor, Dr. Carolyn Bertozzi, who pioneered this field. In Aim 2, I will elucidate the role of nicotinamide N-methyltransferase (NNMT) in ferroptosis resistance. Preliminary data suggest that NNMT, which encodes an enzyme involved in nicotinamide adenine dinucleotide metabolism, is a direct target of NFE2L1. I hypothesize that NNMT regulates cancer cell ferroptosis sensitivity by altering redox homeostasis. Through my proposed experiments, I will determine whether NNMT is a direct target of NFE2L1. I will also assess ferroptosis sensitivity in cells in which NNMT is knocked out or overexpressed. Mass spectrometry will be used to measure the abundance of redox molecules in these cell lines to analyze the role of NNMT in ferroptosis-related redox regulation. I will be trained in mass spectrometry by my collaborator, Dr. Monther Abu-Remaileh, who is an expert in metabolomics. With the guidance of my expert team of mentors at Stanford University, my proposed experiments will elucidate this novel pathway promoting cancer cell ferroptosis evasion and identify possible strategies to minimize cancer therapy resistance.

NSF Awards · FY 2024 · 2024-09

This project explores how the collective behavior of ant colonies depends on their physiology, and how this can shape adaptation to changing climates. The work extends a long-term study since 1988 of a population of colonies of the red harvester ant in New Mexico. A crucial constraint for these seed-eating ants in the desert is water stress. A colony must spend water to get water and food: a forager loses water to evaporation while out searching for seeds, and ants get their water from the fats in the seeds that they eat. Like many insects, ants are coated with a waxy, greasy substance, cuticular hydrocarbons (CHCs), spread by grooming, which function to prevent water loss. An ant colony operates without central control, using simple interactions among ants to adjust its activity to changing conditions. Colonies differ in how they regulate foraging activity: some reduce foraging activity in dry conditions, thus conserving water but sacrificing food intake. This project investigates how the collective regulation of foraging depends on differences among colonies in their CHCs. Foragers with less effective CHCs return to the nest more desiccated and less likely to leave again on the next foraging trip, thus reducing the colony's overall foraging activity. The project will investigate how colonies differ in CHCs, how much the CHC profile can change in hot, dry conditions, and how this shapes foraging behavior. Adaptation of resistance to desiccation may be crucial in determining survival in the intensifying drought in the southwest US. The project examines how the cuticular hydrocarbon (CHC) profile is associated with water loss, whether colonies more susceptible to water loss are likely to forage less in dry conditions, and whether, as drought continues to lower the food supply, this previously adaptive response now increases the risk of starvation. Aim 1 uses analytical chemistry to examine variation among colonies in CHC profile, particularly in the waterproofing compounds of n-alkanes and alkenes and its effects on water loss. Functional assays and chemical analysis will test for plasticity in CHC profile in response to current water pressure deficit, during a colony's lifetime. The long-term demographic study will be continued, monitoring about 300 colonies per year so that the locations and ages of all colonies are known, and the data will be used to examine heritability of CHC profile from parent to offspring colony. Aim 2 examines how colony differences in CHC profile are associated with the regulation of foraging behavior in dry conditions(CHC). Demographic data on colony survival will be used to determine how the effects of colony differences in CHC profile and in foraging behavior determine colony fitness. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-09

How new species are formed is a grand challenge across biology. Particular combinations of genes from different populations may not interact favorably during hybridization, creating unhealthy offspring due to genetic incompatibilities. The specific genes involved in this process are seldom identified, but genes involved in mitochondrial function are prime candidates and the focus of recent research. This project will investigate how different combinations of mitochondrial and nuclear genes create reproductive barriers in hybridizing swordtail fish (Xiphophorus), a model system for genetic incompatibilities. Genomic tools will be used to identify which specific combinations of nuclear and mitochondrial genes influence overall hybrid health. Specific aspects of mitochondrial function such as respiratory efficiency will also be investigated as a mechanistic basis for hybrid incompatibilities. Genetic incompatibilities will also be investigated in different environments and developmental stages because incompatibilities may only manifest in certain conditions. This work includes generating hybrids in the laboratory by targeted crossing experiments and examining natural populations with ongoing hybridization. These activities will be used to recruit students from diverse backgrounds to STEM research, especially in the opportunity-rich field of bioinformatics. Freshmen will be explicitly targeted though the development of a new program called “Power in the Powerhouses” as part of the University of Texas at Austin’s highly successful Freshman Research Initiative to recruit and train the next generation of STEM researchers. Coevolution between nuclear and cytoplasmic genomes can create coadapted genomes within a population that may be disrupted during hybridization, creating reproductive isolation and acting as a common mechanism of speciation. Under this hypothesis, selection during introgressive hybridization should act to maintain coadapted cytonuclear genotypes. To test this hypothesis, genome-wide patterns of ancestry will be generated from three naturally hybridizing pairs and three lab-generated hybrid pairs of swordtail fish species (genus Xiphophorus). Selection should especially favor matched ancestry between mitochondrial genomes and the subset of nuclear-encoded genes that interact with mitochondrial-encoded gene products. Mitonuclear incompatibilities will be identified through statistical associations between nuclear alleles and mitotypes in natural and lab-bred hybrids. Lethal mitonuclear incompatibilities have already been identified using this approach in one pair of hybridizing Xiphophorus. Mitochondrial- and nuclear-interacting genes should also show concordant clines in ancestry with geography. Compromised energetic phenotypes as well as reduced organismal fitness should result from incompatible combinations of mitochondrial and nuclear genes, likely in an environmentally-dependent context. Therefore, in addition to standard metrics of organismal health, whole-organism metabolic rates and mitochondrial DNA copy number will be assessed in parental Xiphophorus species and their hybrids in response to thermal and hypoxic stressors. Multiple respiratory phenotypes in isolated mitochondria will also be investigated, including those dependent on mitonuclear interactions. Phenotypes will be assessed in adults and embryos, as lethal mitonuclear incompatibilities can prevent embryos from developing. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-09

This project focuses on the heart of one of the most imperiled ecosystems, coral reefs, where climate change and heat waves have killed many corals. Corals live in a fine-tuned symbiosis with single celled algae, and heatwaves severely disrupt this symbiosis: even a few degrees can cause the partnership to breakdown. The team's preliminary data suggest that this sensitivity arises from how well individual coral and algal partners interact with one another at the cellular level such that corals with better genetic matches to their symbiont seem to resist heat better. This project is identifying genes and regions of the genome that interact to result in a functional partnership and testing this relationship across a range of environmental conditions. The results of these experiments, in combination with mobile sequencing platforms that generate data in real time, are bolstering conservation efforts by finding optimal populations for restoration projects driven by local communities in Palau. This allows quick translation and scaling the results of the experiments into conservation strategies by quickly identifying colonies with optimal host-symbiont combinations, improving the efficiency and efficacy of local coral restoration projects in Palau. The project also provides research training for postdoctoral scientists, graduate students and a research associate, and public outreach through the production of video and multimedia products. For ecosystems dependent on symbioses, climate resilience depends on successful interactions between partners (i.e. GxGxE interactions). In corals, this variation has been investigated at the level of coral genetic variants (within or between species) or genus-level symbiont variants. Yet, despite a great deal of careful work, these data have not yet revealed strong impact of specific coral genes on bleaching variation. This project is 1) characterizing population-level variation in both the host and symbiont populations to identify pairs of genetic loci in the host and symbiont genome that are highly correlated (i.e. exhibit strong linkage disequilibrium), termed ‘matched’ loci; 2) testing for these matched loci in three species of Acropora from three locations in Palau that historically have exhibited dramatically different thermal profiles and identifying how matching relates to bleaching resistance; 3) reciprocally transplanting colonies from all three locations to test for local adaptation at the level of the holobiont, specifically focusing on whether the ‘matching’ profile changes in different environmental contexts; 4) identifying and characterizing colonies whose dominant symbiont partner switched after transplantation to elucidate the selective forces shaping symbiont genotypes in light of both their environmental and host-specific context; and 5) partnering with local governments and communities to implement restoration plots using rapid and mobile sequencing platforms to test if identifying and using genetically optimal host and symbiont partners in restoration efforts helps to improve conservation outcomes. This project is jointly funded by Biological Oceanography (GEO/OCE) and Organismal Response to Climate Change (BIO/IOS). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT The detection and characterization of cell-free DNA (cfDNA) is increasingly being used to detect cancer – this modality is frequently referred to as a liquid biopsy. Epigenetic characterization of cfDNA is an emerging approach for sensitive detection and quantification of cancer burden. Trends in cancer growth are evident from cfDNA among patients with advandced and metastatic cancer; however, there is a significant translational need for the development of early-stage cancer or even pre-cancer detection assays via liquid biopsy. Malignant tumor cells shed DNA into the bloodstream of cancer patients as cfDNA, commonly in the form of nucleosome-sized fragments. There is broad interest in cfDNA methylation as a cancer biomarker modality, ranging from targeted biomarker panels to whole-genome characterization of cfDNA methylomes. For detecting 5mC methylation, cfDNA is currently processed with bisulfite or enzymatic conversion of unmodified cytosines into uracils, which is detected by short-read sequencers. This approach introduces biases such as GC skews, oxidative DNA damage, PCR amplification bias, and alignment artifacts. Characterizing cfDNA methylomes from patients remains challenging, particularly with conventional sequencing approaches. We recently demonstrated a novel approach for single-molecule methylation analysis of cfDNA that overcomes these issues. We developed a nanopore-based sequencing approach for efficiently characterizing methylation profiles from the cfDNA of cancer patients. The passage of methylated DNA through the nanopore generates a unique electrical signal compared to unmodified DNA without cytosine conversion, which can then be classified with machine learning algorithms. We generated up to hundreds of millions of reads per cfDNA sample from colorectal cancer patients, with single nanograms or less of analyte per patient. In this project, we seek to extend our work to (1) characterize the early-stage colorectal cancer cfDNA methylome landscape, and (2) to develop a classification model for early detection of colorectal cancer. Using cfDNA samples from patients at the Stanford Cancer Institute (SCI) and the PLCO clinical trial, we will generate cfDNA methylomes that will be the basis of biomarker signatures for colorectal cancer detection. In Aim 1, we will derive cfDNA methylome profiles from confirmed and pre-diagnostic CRC, and characterize how cfDNA methylomes are affected by tumor subtype and stage. We will also deconvolute cfDNA using matched primary tumor and immune cell references. In Aim 2, using sequenced cfDNA we will build a machine learning model that will determine statistically significant changes in cfDNA methylation between cancer patients and healthy controls. The machine learning model will also quantify tumor burden and whether it is likely that a sample is indicative of cancer. We will use the SCI and PLCO patients as independent cohorts to perform training and validation.

NSF Awards · FY 2024 · 2024-09

Modern Earth is covered by lush tropical forests, extensive grasslands, and soaring redwoods—in striking contrast to landscapes through much of Earth’s early history that consisted largely of bare rock and microbial mats. Plants have dramatically altered Earth’s landscape and climate (like the shapes of rivers and patterns of rainfall). However, there is currently little consensus on how the development of plants, starting with the first ground-hugging mosses and liverworts around 470 million years ago, followed by the eventual rise of trees around 380 million years ago, affected nutrient and oxygen levels both on land and in the oceans. This research combines field, laboratory, and modeling approaches to examine the effects of early land plants on the Earth system. This study focuses on the Canadian Arctic Archipelago which contains some of the best-preserved sedimentary rocks chronicling this key time period of early plant evolution. The team of researchers are studying fossil plants, pollen, and spores and geochemical elements to understand how weathering changed on land, how plant material was delivered to the ocean, how the availability of critical nutrients like phosphorus changed on the land and in the oceans, and how oxygen and sulfur levels changed in the ocean. The broader impacts activities stemming from the research include educational and mentorship opportunities for students in middle-school through graduate school. Graduate students will be co-mentored by the Principal Investigators, and undergraduates will also be recruited to analyze collected samples. The Yale Peabody Museum and the Yale Pathways to Science program will provide platforms for community-oriented outreach efforts, including educational events fostering scientific literacy and engagement in local middle-school students. The team will also take advantage of the unique opportunity provided by recent Peabody renovations to develop a new public-facing exhibit on “Ecosystem Engineering” focused on land plants and their impacts on Earth’s landscapes and ecosystems. The University of California, Riverside’s Camp Highlander program is fostering local high-school student engagement with Earth sciences. Finally, field-conducted telepresence outreach through the new “Annals of the Arctic” program, integrated with existing summer programs at Stanford, Yale, and UCR, will provide public-facing exposure to day-by-day realities of geologic fieldwork in remote terrains. This will increase the accessibility of geologic research and provide a venue for direct illustration of geologic concepts, human experiences of the dynamic nature of polar ecosystems, and their vulnerability to ongoing environmental change. Reconstructing the biotic, biogeochemical and climatic impacts of the evolution of land plants has been hampered by the commonly fragmentary and disassociated records of geochemical and paleontological change across the lower-middle Paleozoic transition, and by the limited integration of empirical observations with the mechanistic framework that can be provided by biogeochemical and Earth-system models. To address these fundamental questions, we are generating new, high-resolution field-based geochemical data (biomarker, programmed pyrolysis, carbon isotope, lithium isotope, osmium isotope, phosphorus speciation and phosphate-oxygen isotope, iron speciation, and trace metal abundances) and sedimentological and paleontological (plant body fossils, palynomorphs, graptolite and conodont biostratigraphy) records from key sections in the Canadian Arctic to reconstruct first-order ecological and environmental changes—in both continental and marine settings—concurrent with the radiation of early land plants. The Silurian–Devonian transition is an under-characterized but key interval for both land plant evolution and marine redox state, and these data will be integrated with long-term records to distinguish perturbations from more permanent state shifts. These new empirical records will be coupled to biogeochemical modeling over a range of scales—from local critical zone and seafloor diagenetic processes to continental climate and ocean and atmospheric carbon-oxygen cycle modeling—to develop a more robust process-based understanding of plant-biogeochemical feedbacks and reconstruct the long-term consequences of early land plant evolution for the broader Earth system. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Alcohol Use Disorder (AUD) contributes to 88,000 deaths per year in the United States. Although several pharmacological treatments are available, adherence to these treatments is low and approximately 60% of individuals relapse within 6 months. Further, these treatments modulate the brain in a relatively global fashion. Evidence from neuroimaging studies of AUD patients has shown that elevated cortico-striatal activity in response to alcohol cues predicts relapse. Thus, there is an emerging interest in developing novel, neural-circuit specific therapeutic tools to enhance AUD treatment outcomes. Transcranial magnetic stimulation (TMS) is one such non-invasive, neural-circuit specific tool. Through electromagnetic induction, repetitive pulses of TMS can be applied to the cortex to change neural activity within a cortical circuit. Subsequently, TMS for AUD has been developed as a strategy to reduce brain reactivity to alcohol cues within the prefrontal cortex and its downstream targets in the striatum. I recently led an analysis of a randomized, double-blind clinical trial applying 10 days of TMS to the medial prefrontal cortex among AUD patients. Relative to sham, individuals who received real TMS were 3 times more likely to remain sober and experienced a reduction in average brain reactivity to alcohol cues. Despite these positive results, there was considerable variability in that approximately 1/3 of individuals who received real TMS relapsed to alcohol and reductions to striatal reactivity to alcohol cues were not uniform. My NIAAA-sponsored F31 demonstrated that individual patterns of brain response to alcohol cues are highly variable among AUD patients and often occur outside of the prefrontal cortex. Given this spatial variability, delivering TMS to a fixed point on the scalp leads to a situation in which TMS electrical fields do not always overlap with an indvidual’s brain response to alcohol cues. In a proof-of-concept retrospective analysis, I found that patients who had overlap between alcohol cue-reactivity patterns and TMS electrical fields had the best clinical outcomes. This K99/R00 proposal seeks to build upon this analysis by developing prospective functional connectivitiy - guided TMS (fcg-TMS) clinical trials for AUD. First, using an existing dataset, we aim to characterize the spatial topography of TMS cortical targets as defined by peak functional connectivity with the striatum during alcohol cue presentation (Aim 1, K99 phase). Then, we aim to evaluate the efficacy of one session of functional- connectivity guided TMS (fcg-TMS) on reducing striatal response to alcohol cues, a key biomarker of treatment efficacy (Aim 2, K99 Phase). These K99 aims will be complemented by a training plan that includes 4 domains: clinical trial design, fcg-TMS, laboratory leadership, and complex modeling of relapse and brain networks in AUD. Following these foundational trainings and experiments, Aim 3 (R00 phase) of this proposal will evaluate the efficacy of 15 sessions of fcg-TMS clinical trial in reducing drinking, relapse rates, and striatal response to alcohol cues. Cutting edge tools will be used to analyze the data presented in this proposal, including prospective, computational electrical field modeling, advanced functional connectivity analysis, and multivariate statistics.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Night or rotating schedules under shiftwork are unavoidable, especially in healthcare work. Shiftwork can disturb circadian rhythms due to unusual light exposure and has been linked to many long-term negative health outcomes such as cardiovascular and cardiometabolic disorders. Studying disturbances in circadian rhythms in shift workers has proven challenging due to limitations in measuring circadian rhythms in humans under real- world conditions. However, with recent developments in wearable sensors, we can now capture continuous, remote, and longitudinal measurements of physiological signals (e.g., heart rate, breathing rate, biomechanics), behavioral patterns (e.g., activity tracking), and other external stimuli (e.g., light exposure), enabling the characterization of circadian disturbances and supporting identifying actionable strategies for shift workers to regain and maintain internal synchrony. This proposal seeks to examine the impact of the coordination of central and peripheral body clocks internally and with the external environment in the real-world. We hypothesize that internal and external synchrony of the body’s clocks is critical for regaining stability after perturbations such as shiftwork. In this proposal, we seek to develop a quantitative framework for assessing body clock synchrony in the real-world using wearable sensors. In the long term, we seek to leverage this work to interrogate the impact of the coordination of body clocks on health state and develop timely interventions to regain stability. To do this, in Aim 1, I will improve circadian phase prediction algorithms using real-world light diets to enable the study of circadian rhythms outside of the laboratory and in real-world settings. In Aim 2, I will assess internal synchrony leveraging continuous, ambulatory monitoring of physiological signals and estimation of central clock phase to develop a quantitative framework for measuring the coordination of central and peripheral body clocks. This will enable deeper understanding of their coordination and contribution to recovery from disturbances. In Aim 3, I will develop predictive algorithms using machine learning to enable the evaluation of light diets to improve synchrony in shift workers to translate tools to empower them to maintain synchrony and reduce risk of long- term negative health outcomes. Completing these aims and working in collaboration with my sponsor, Dr. Jamie Zeitzer, and co-sponsor, Dr. Todd Coleman, will enable me to develop the skillset necessary to study disease dynamics in the real-world using wearable sensors and pursue a future career as a robust, independent, and interdisciplinary researcher. My sponsor and co-sponsor are well equipped to provide training in measuring complex human physiological signals, developing engineering skills in statistical signal processing and estimation theory, and communication and leadership skills to facilitate the translation of my work to a wider audience. The support of my mentors as well as the plentiful resources and collaborative environment of the Bioengineering PhD program and Stanford University are opportune for the successful completion of this work and complementary training goals.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The idea of evolutionary constraint is central to understanding of how populations have and will adapt(ed) in response to heterogeneous selective pressures. Constraints therefore determine how organisms with relevance to human health, like pathogens and cancers, adapt to different drugs, hosts, or environments. Constraints can be formed by strict trade-offs, when mutations adaptive in one environment have antagonistically pleiotropic costs in alternate environments, or by more dynamic processes, when the ability to select for hypothetically possible costless generalism is limited by mutational accessibility, speed of selection, or ecological opportunity for selection. These overlapping processes can make evolutionary constraint difficult to measure and these different processes difficult to disentangle. Despite the centrality of constraint to evolutionary theory, these difficulties mean that the processes forming constraints at different levels remain poorly understood— leaving open questions about how they affect evolution. In this proposal, I leverage high throughput microbial experimental evolution lineage tracking methods that provide unprecedented power to measure the distributions of fitness effects for mutations in multiple environments. These methods allow us to generate quantitative insights into the dynamics of evolutionary constraint. Aim 1 will ask whether constraints in laboratory experimental evolution resemble natural variation by measuring the joint distribution of fitness effects (JDFE) for natural isolates of S. cerevisiae in two nutrient environments mimicking natural conditions (synthetic wine must and synthetic beer wort) and then experimentally evolving a barcoded laboratory strain in these and an alternating media environment to determine whether the laboratory evolved JDFE resembles the naturally evolved JDFE. Aim 2 will explore how evolutionary constraints change over time by leveraging a rebarcoding system to experimentally evolve S. cerevisiae in the same conditions as Aim 1 for 5 evolutionary `steps' (~400 generations) and measure the JDFE at each `step'. Most laboratory experimental evolution examines shorter time scales, but we know from long-term experiments and natural observations that evolutionary dynamics change over time as populations near fitness peaks and/or change their environment. Thus, Aim 2 will allow us to quantitatively measure how constraints change over time. Through this work, I will build a powerful system for conducting high-throughput, long term evolution in multiple environments. The proposed research will significantly expand my training in the areas of quantitative genetics, population genetics, and molecular biology while encouraging increasing independence. It will therefore compliment my PhD training in evolutionary ecology to allow me to build an integrative research program that links genetic, evolutionary, and ecological processes to understand how constraints determine eco- evolutionary dynamics. Additionally, I will participate in professional development and outreach activities.