University Of California, San Francisco

NIH Research Projects · FY 2026 · 2023-08

In the U.S., about 12% of women have impaired fecundity and 7% of couples have infertility, with 1/3 attributable to female factors. Underlying mechanisms remain largely unknown, however, even when more proximal pathologies are identified, thus precluding the development of accurate diagnostics and personalized therapies. In addition, pregnancies in subfertile women, conceived naturally or as a result of infertility treatments, have greater risk of complications such as pre-eclampsia, preterm birth, and fetal growth restriction that have life-long effects on the offspring. Thus, dissecting the mechanisms underlying reproductive success and compromise at the genomic, molecular, and cellular level is crucial to the health and well-being of this and future generations. Engaging investigators from multiple disciplines and building a sustainable pipeline of junior investigators, including those underrepresented in science and medicine, is also essential to this effort, as is the promotion of public literacy about reproductive health and science. These are core principles of our NIH National Center for Translational Research in Reproduction and Infertility (NCTRI) at the University of California San Francisco (UCSF), funded since 2007 and for which this new proposal is submitted with a new central theme focused on the inter-related roles of endometrial inflammation, epigenetics, and developmental processes of the peri- implantation uterus and early conceptus as central determinants of early pregnancy success or failure. This focus is motivated by the fact that the clinical association between pathological endometrial inflammation and female infertility, while well-established, lacks a deep mechanistic understanding. Our proposed Center is comprised of three inter-related Research Projects and a pilot project (to be determined), supported by an administrative core (A), and an education/outreach core (B). Project 1 (Roan/Huddleston, co-Leads) focuses on the phenotypes and functions of endometrial lymphocytes in the normal and inflamed human endometrium and decidua, including determinations of T and B cell antigen specificities. Project 2 (Erlebacher) focuses on how epigenetic processes active within endometrial and decidual stromal cells control, and are in turn controlled by, endometrial and decidual inflammation. Lastly, Project 3 (Blelloch/Fisher, co-Leads) addresses how primitive trophoblasts of the extra-embryonic tissues that comprise the fetal portion of maternal-fetal interface differentiate into the subtypes that determine the polarity of the implanted conceptus with respect to the decidua. We believe our Center will also advance reproduction and infertility research more generally by setting an example of successful transdisciplinary collaboration built upon the shared use of rare clinical specimens analyzed through complementary, multi-omics approaches combined with mechanistic investigations using model systems. Our Center also is designed to attract students, fellows, and junior scientists to careers in reproduction and infertility research, and to engage the community with regards to the importance of infertility research.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Nearly 20 million older adults undergo major elective surgical procedures, yet very few receive advance care planning (ACP). This is a critical missed opportunity to ensure optimal and patient-aligned medical decisions and communications. Despite ACP being incorporated into national quality metrics and society guidelines for surgical care for older adults, there are few examples of effective integration into the pre-surgical phase. Efforts to date have mostly focused on improving surgeons’ use of ACP but barriers remain significant, including varying levels of familiarity and comfort to conduct ACP conversations, lack of dedicated time during the pre-surgical care episode for these often-delicate conversations, and lack of appropriate patient-facing ACP tools to help patients and caregivers make complex decisions about their surgical treatment. Our team has designed and tested a theory-based, interactive ACP patient-facing technology solution (PREPARE) based on the new ACP paradigm of preparing people for communication and medical decision-making. Despite consistent evidence that PREPARE increases ACP engagement and patient and clinician empowerment to discuss ACP, a gap remains in extending PREPARE’s use to pre-surgical populations. We hypothesize that by including PREPARE into the electronic health record (HER)-centric pre-surgery workflow for older adults and including automated reminders, we can empower patients and surgical teams to engage in ACP discussions. Given the limited time and resources in the surgical setting to conduct ACP, we will be testing 3 delivery strategies in increasing resource intensity (PREPARE alone, PREPARE with text/phone reminders, or the additional of a healthcare navigator). To ensure generalizability, we will conduct our work in 3 healthcare systems (HCS): Univ. of CA, San Francisco (UCSF), Univ. of CA, Irvine (UCI) and M Health Fairview (UMN, a collaboration among the Univ. of MN Medical School, Univ of MN Physicians, and Fairview Health Services). We will first establish trial infrastructure (UG3) to conduct (UH3) an NIH Stage Model III (efficacy-effectiveness) three arm RCT in 3 HCS. Patients aged 65 or older, or with serious illness, who are referred for major elective surgery will be randomized to Arms: (1) Letter about ACP, PREPARE advanced directive (AD), PREPARE website; (2) Letter, AD, PREPARE plus reminder text/phone messages; (3) Letter, AD, PREPARE plus reminders plus a healthcare navigator on ACP documentation (discussions and care plans, primary outcome) and patient-reported ACP engagement. Using mixed methods, we will assess patients’ and surgical care teams' experience with surgery ACP. ACP note content will be evaluated using natural language processing (NLP) and data mining to begin to identify assess thematic completeness of ACP notes. This work is innovative because we are coalescing existing collaborations between HCS into a transdisciplinary group of surgeons, geriatricians, and informaticians across three health systems that will develop infrastructure and rigorously test a novel patient-centered system-level approach to integrating ACP into the surgical care episode, the first step towards goal-concordant surgical care.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Emergent behaviors arise when individuals follow rules about how to interact with one another and their environment to produce a group entity with new properties or abilities1,2. Common examples include a school of fish, a flock of birds, and a colony of ants. In an emergent behavior, individuals often simultaneously communicate with multiple other individuals while processing sensory cues from the environment2,3. All this information is integrated to produce a behavioral response that is remarkably cohesive across the group. Though complex information integration is a hallmark of how humans process the world, the mechanisms underlying communication in groups have been difficult to probe experimentally. I have discovered the first example of emergent group behavior in C. elegans in which individuals coordinate motion without being in physical contact: in response to a simple environmental cue, a population of several hundred individuals will synchronously perform an inward bidirectional spiral that eventually concentrates the initially dispersed population at a central point. The behavior occurs when a lid is placed on an open dish containing a sufficient density of worms in the absence of food and is dependent on intact sensory systems. Furthermore, I have demonstrated that a cue from a high density population can be sensed across a shared airspace to induce spiral-like behavior in individual worms that would not otherwise show spiral motion. I therefore propose that C. elegans uses a volatile cue to gauge population density, and spiraling behavior is triggered by concentration of this cue when the dish is closed. However, I also demonstrate that accumulation of a cue is not sufficient to induce spiraling if inter-worm distance is too great indicating that local cues between neighboring worms are also involved in coordinating spiraling. In Aim 1, I will elucidate the cues that individuals produce and perceive to facilitate emergent spiraling in C. elegans including both long-range density cues and short-range cues from nearby individuals. In Aim 2, I will take advantage of the tractability of C. elegans to ask how brain activity produces emergent spiraling behavior. C. elegans are transparent, and the brain activity of individuals can be directly visualized by following calcium transients in freely moving worms4. I will explore the hypothesis that neural networks reduce the noise of inputs to produce maximally coherent behavior across individuals in the collective. Together these findings will illuminate principles underlying emergent behavior at unprecedented resolution which may allow us to identify similar processes occurring in other systems or design artificial systems which exhibit spiral behavior.

NIH Research Projects · FY 2025 · 2023-08

Project Summary As a member of the E. Alejandro Sweet-Cordero laboratory my efforts are directed at executing the computational aspects of the grants, manuscripts, and the general goals of the lab as well as mentoring other lab members. The goals of our projects are to study the mechanism of cancer progression and obtain clinically relevant insights for pediatric and adult solid tumors. The R50 NCI Research Specialist Award would provide me with the necessary funds to continue to further develop my computational tools and ongoing support for the following NCI-funded projects: (1) Development of Advanced Preclinical Models for Pediatric Solid Tumors (R01CA243555). There are three main goals to this project: a) verify that matched patient tissue and patient- derived xenografts (PDX) or PDX-derived cell lines are transcriptionally and molecularly similar; b) utilize high- throughput analyses to nominate drug targets for testing, and c) study drug resistance using single-cell RNAseq technologies. (2) Development of Novel Protein-based Therapeutics for Lung Cancer (R01CA225103). The main goals of this grant are to understand the biology of CLCF1-CNTFR as it relates to adult lung cancer and evaluate the synergistic outcomes of the engineered “decoy” CNTFR receptor (eCNTFR) with other compounds (e.g., eCNTFR+ PDL1 checkpoint inhibitor). (3) Investigate the Role of Long Non-coding RNAs (lncRNA) in Sarcoma Pathogenesis (R01CA211657). The goals of this grant are to elucidate the role of lncRNA EWSAT1/2 in chromatin remodeling at EWS-FLI repressed enhancer sites, utilizing ChIRP-Seq and ATACseq. Other lncRNAs revealed from a prior lncRNA CRISPR-mediated interference screen will also be investigated for their role in Ewing Sarcoma using both in vitro and in vivo models. My ongoing efforts to develop software and computational pipelines will not only go toward the goals of these NCI grants but also align with my long-term career goals and benefit the broader cancer research community.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Lung function measurements are routinely compared to racial/ethnic norms, biasing interpretation and perpetuating asthma disparities. The race/ethnicity-based lung function reference equations used to calculate these norms do not account for genetic ancestry—the genetic origin of one’s population, which can explain over 15% of lung function variation within a racial/ethnic group. Consequently, race/ethnicity-based equations misestimate lung function, often resulting in delayed disease detection and inadequate treatment, especially among populations disproportionately affected by asthma. Dr. Witonsky (candidate) derived equations that use genetic ancestry instead of race/ethnicity to more accurately predict lung function. While genetic ancestry- informed equations appear to remove racial/ethnic bias from lung function measurement, establishing their clinical utility and equity requires evidence that they better predict asthma-related outcomes. In addition, further research is needed to disentangle the social and genetic determinants of genetic ancestry differences in lung function. The proposed mentored research will address these knowledge gaps using data from existing and new cohorts of Black and Hispanic/Latino individuals with and without asthma via three specific aims: (1) to evaluate genetic ancestry-informed, race/ethnicity-based, and “one size fits all” lung function equations for predicting asthma-related outcomes, (2) to quantify the proportion of genetic ancestry differences in lung function that is explained by social exposures, and (3) to quantify the proportion of genetic ancestry differences in lung function that is explained by known lung function-associated genetic loci. In support of this research and Dr. Witonsky’s goal of becoming an independent clinical investigator, this K23 proposal includes formal training with experts in the areas of asthma translational and clinical research (Dr. Prescott Woodruff, primary mentor); advanced statistical and predictive analytic methods (Dr. Stephen Shiboski, co-mentor); social epidemiology and health disparities research (Dr. Luísa Borrell, co-mentor); genetic epidemiology (Dr. Elad Ziv, co-mentor); and statistical genetics (Dr. Noah Zaitlen, advisor). In addition, professional development planning will involve structured meetings with Dr. Woodruff and a leader within Dr. Witonsky’s Division of Pediatric Allergy, Immunology, and Bone Marrow Transplant (Dr. Morna Dorsey, advisor). As a faculty member in the Department of Pediatrics at the University of California, San Francisco, Dr. Witonsky will have access to world-class biomedical and research facilities, workshops and seminars, and an NCATS-funded K Scholars Program. Completion of the proposed research and career development activities in this application will inform the development of an R01 proposal and enable Dr. Witonsky to develop an innovative research program applying computational precision health methods that integrate clinical, social, and genomic data to reduce asthma disparities.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Osteoarthritis (OA) is the leading cause of pain and disability worldwide, and there are currently no disease modifying treatments available. While obesity-induced OA involves both metabolic and biomechanical factors, a key link is excess fat, or adipose tissue – a source of inflammatory mediators implicated in the pathogenesis of OA. The mechanistic influence of adiposity, biomechanical alterations, and metabolic syndrome have been difficult to determine and disentangle. To separate these factors, we used a mouse model of lipodystrophy (LD), in which the animals completely lack fat but maintain normal body mass. The LD mouse demonstrates many clinical signs observed in individuals with obesity-induced OA (sclerotic subchondral bone, systemic inflammation, insulin resistance, metabolic disturbance, and muscle weakness). Unexpectedly, we observed that LD knee joints are protected from OA. When fat was transplanted into LD mice protection from OA was reversed, implicating that adipose tissue, and factors secreted by adipose tissue called adipokines – but not body weight – are critical mediators of joint degeneration. These results suggest that adipose tissue and the mediators (adipokines) secreted by adipose tissue adversely affect cartilage health. In the mentored K99 portion of this grant, we will generate bioengineered designer adipose implants using murine induced pluripotent stem cells (iPSCs) to provide a platform to deconstruct adipokine signaling and investigate the mechanisms linking adipose tissue and joint health. This approach, which was not possible previously without creating complex and expensive transgenic mice, addresses a gap fundamental in our understanding of obesity and OA. In the independent R00 portion of this grant, we will leverage recent advances in regenerative medicine to develop and test a self-regulating cell-based implant that can provide biologic drugs to combat OA, laying the platform for Dr. Collins’ independent research career, and the groundwork for a first R01. The value of this platform is the flexibility to interchangeably deliver a wide range of potential therapeutics. Using this novel and flexible platform, we will hijack adipokine signaling to deliver anti-inflammatory mediators in a tunable and well- controlled manner as a novel regenerative therapy for OA. Since this iPSC platform could readily accommodate edits and alterations of targets of interest in a variety of cell types, the potential for this therapy is far-reaching, as many chronic diseases (cancer, cardiovascular disease, diabetes, etc.) have links to pathologic inflammatory signaling.

NIH Research Projects · FY 2026 · 2023-08

Summary Organismal and cell rejuvenation are exciting new approaches to counteract aging, and recent breakthroughs have brought them to the forefront of aging research. For examples, systemic factors in young blood was found to rejuvenate various mouse tissues and brain function, and partial reprogramming with four stem cell transcription factors (TFs) (Yamanaka factors) rejuvenate tissues and cells and extend the lifespan of mice. These discoveries demonstrate that “young” and “old” can be described as different states, and the “old” state can be reversed back into a “young” state through transcriptional reprogramming. We hypothesized that there might exist many solutions to human cell rejuvenation through transcriptional reprogramming, and some of the solutions may be safer and more potent than Yamanaka factors. In a recently completed project supported by an NIH/NIA R21 grant, the Li lab developed a systematic approach to test this hypothesis and to find the solutions. Using a human cell culture model of replicative aging employed by Hayflick (continuously passaged human fibroblast cells), we developed a high throughput screen using Perturb-seq to identify the potential rejuvenating TFs -- those that when over-expressed or repressed in old cells, are capable of reprogramming the global gene expression program from the old state back to a younger state. We identified four TFs/chromatin modifier (E2F3, EZH2, STAT3, ZFX) that when over-expressed or repressed individually, are able to rejuvenate human fibroblast cells aged in vitro. Here we propose to further test the rejuvenating effect of these four factors in human fibroblast cells aged in vivo in their natural tissue environment, and in mouse liver. We will also develop new technologies to screen for more potent rejuvenating TF combinations and test them in aged human fibroblast cells and in mouse liver. If successful, this proposed study will identify TFs/TF combinations that can rejuvenate in vitro and in vivo aged human fibroblast cells and mouse liver. This will set the stage for transgenic mouse study and translation to human therapies. The methodologies developed in this proposal can be generalized to identify combinatorial transcriptional programs that produce any desired cellular phenotypes, e.g., the reversion of the cellular state from disease to normal in cell culture models of diseases.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Embryos cultured external to the body are exposed to non-physiologic conditions that can impair the health of the future adult. Moreover, culture stress reduces immediate embryo viability and implantation success, motivating multiple embryo transfers and leading to high rates of multiple pregnancies. These pregnancies are risky for fetus and mother and exhibit a high healthcare cost burden. To address this, embryos are screened based on early-stage morphology to identify the healthiest for transfer, but this approach achieves limited success and is highly dependent on the individual conducting the test. An accurate and objective screening method would increase implantation rates and reduce the need for multiple transfers, in addition to yielding healthier offspring. The objective of this project is to develop a new technology for noninvasive IVF embryo selection. Dr. Paolo Rinaudo, Co-I on this grant, is a Reproductive Endocrinologist working at the UCSF Center for Reproductive Health which performs >2000 IVF procedures per year and has commitment to research. Through experience derived from two decades of work in IVF, he identified that current methods for screening embryos are inadequate, with the gold standard, morphological selection, achieving a positive predictive value of just 30%. While an embryo may appear morphologically healthy, it may not be molecularly healthy, which may affect the long-term development of the fetus. Among his two decades of research on cultured embryos and IVF is the finding that ex vivo culture can stress embryos and drive Warburg-like metabolism. In addition, culture stress has now been correlated to long-term negative health outcomes, including abnormal placental development, metabolic dysfunction, hypertension, and diabetes. Dr. Rinaudo hypothesized that metabolically active embryos lower their culture droplet pH, similar to how Warburg metabolism acidifies the environment around cancer cells, thereby affording a simple way to select the best embryos for transfer. His research has confirmed this, but the technique cannot be translated to practice because there is no effective and reliable pH measuring method for the culture droplet due to its tiny volume. Armed with this concept, he reached out to Dr. Adam Abate, a physicist and engineer at UCSF. Dr. Abate is a leader in microfluidics and nanotechnology with a record of developing and translating health care technologies to practice, having founded multiple companies commercializing his inventions, including Fluent Biosciences (genomics), Mission Bio (oncology diagnostics) and Scribe (cell & gene therapy). Together, Drs. Rinaudo and Abate have designed a novel hydrogel biosensor that non-invasively measures culture droplet pH. Being an IVF clinician, Dr. Rinaudo is aware of the regulatory and practical constraints of IVF operations and thus ensured the design is feasible for this setting by making it simple, non-invasive, and cost-effective. This grant will develop the technology and validate its efficacy in a mouse model of IVF, providing critical data for a follow-on R01. The project is thus based on a rigorous scientific foundation, decades of clinical experience, and comprises a team capable of translating science discoveries to the clinic.

NIH Research Projects · FY 2025 · 2023-08

1 Clonal hematopoiesis (CH), a highly prevalent condition in the elderly, arises from somatic mutations that endow 2 a proliferative advantage to a subset of hematopoietic cells. CH increases the risk of myocardial infarction and stroke 3 independent of traditional risk factors. Relative to other common genetic variants giving rise to CH, the JAK2V617F (JAK2VF) 4 mutation that increases JAK/STAT signaling occurs at a younger age and imparts a substantial risk of premature coronary 5 heart disease (CHD). In CH mutated cells make up only a small fraction of blood cells, however, patients maintain an 6 elevated risk of CHD, suggesting mutant cells may have an outsized impact on their environment. Using murine models of 7 Jak2VF CH we found that IL-1 specifically promotes cell intrinsic proliferation of Jak2VF macrophages, but not wild type 8 (WT) macrophages even within the same atherosclerotic lesions. Administration of antibodies to IL-1 reduced Jak2VF 9 macrophage proliferation and improved features of plaque stability. scRNA-Seq analysis of lesion from mice with Jak2VF 10 expression in all bone marrow cells identified a prominent inflammatory myeloid population that was enriched for genes 11 associated with IL-1 signaling. We found that inflammatory myeloid cell abundance was decreased in the absence of the 12 pyroptosis executioner Gasdermin D (Gsdmd), potentially due to suppressed IL-1 secretion. Therefore, Specific Aim 1 13 will investigate the ability of Jak2VF cells to promote inflammatory phenotypes in WT cells within the same lesion through 14 IL-1 signaling. Mechanistically we have shown that the AIM2 inflammasome is activated in lesions from Jak2VF mice. 15 Deletion of Gsdmd from Jak2VF cells leads to increased DNA double strand breaks in lesions. Bone marrow derived 16 macrophages expressing Jak2VF, but lacking Caspase 1/11 have increased reactive oxygen species, suggesting DNA damage 17 occur upstream of inflammasome activation. Thus, Specific Aim 2 will determine if increased oxidized DNA in Jak2VF 18 macrophages promotes inflammasome activation and inflammation in lesions. Specific Aim 3 will then investigate if 19 truncation mutations in the CH variant protein phosphatase Mg2+/Mn2+ dependent 1D (PPM1D) that leads to inactivation of 20 DNA repair proteins, promotes atherosclerosis. The research proposed here will be accomplished by expanding my 21 conceptual knowledge of DNA damage in macrophages and atherosclerosis and will be complemented by training in single 22 cell sequencing technology to understand how immune cell populations change in lesions based on CH variant status. This 23 training will be supported by rigorous mentoring and collaboration with my scientific advisors during the K99 phase. 24 Together the proposed research and training will elucidate mechanism by which DNA damage and inflammasome activation 25 drive accelerated atherosclerosis in CH which will provide me the knowledge and skills to successfully transition into an 26 independent academic researcher. 27 28 29 30

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Although individually rare, Mendelian diseases are collectively common. Nearly 1% of people have a medical condition that can be traced back to a single gene. This is particularly true for patients with cardiovascular disease. Genetic testing is now commonly employed in clinical practice. As a result, it has become clear that we have an incomplete understanding of how genetic mutations cause Mendelian disease. Some patients with deleterious mutations display very severe symptoms while others are almost entirely unaffected. This is true even for childhood-onset disorders with severe cardiovascular symptoms, like Marfan Syndrome. Understanding the range of disease severity has important implications for diagnosis, prognosis, and management. In the past, cohort studies and case series have been used to gather this type of information, but these yield incomplete and biased views of disease heterogeneity. Therefore, new methods for studying Mendelian disease genetic and phenotypic diversity are urgently needed. Population-scale biobanks linked to electronic health records (EHRs) can provide a less biased view of genotype-to-phenotype relationships. Subjects are included in these datasets regardless of their medical history. EHR data also provides detailed phenotypic information on each subject. Finally, these biobanks now include hundreds of thousands of individuals, capturing rare genetic variation on an unprecedented scale. As a result, we hypothesize that population-scale biobanks can provide new insight into the genotype-to-phenotype relationships that underlie congenital cardiovascular syndromes (CCSs). This hypothesis will be tested in two specific aims. In Aim 1, we will use biobanks to develop quantitative scores that reproducibly summarize CCS- related phenotypic severity. These traits have multiple applications. In Aim 2, we will use them to build computational models that predict the phenotypic effects of CCS-related rare variants directly from sequence context. Once validated, these models should reduce diagnostic uncertainty in clinical practice. I am a clinical geneticist and physician-scientist devoted to improving the quality of healthcare provided to Mendelian disease patients. Long term, I plan to develop an independent research program that uses complex clinical and genetic datasets to improve our understanding of Mendelian disease risk, variability, and progression. My K38 research proposal is entirely consistent with these goals. In addition, it will provide valuable career development. New technical skills in EHR data analysis and statistical genetics will be acquired, as will academic skills like grant writing. During the award, I will be mentored by leaders in the fields of biomedical data science and human genetics, including Dr. Atul Butte and Dr. Neil Risch. Finally, the K38 award will serve as springboard for future funding opportunities and research independence. Therefore, the K38 StARRTS award will serve as a critical milestone in my development as a physician scientist.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Traumatic brain injury (TBI) is a leading cause of death in the US, and treatment options are limited. Therapeutic clinical trials in TBI have yielded disappointing results owing in part to the difficulty in accounting for clinically important heterogeneity within TBI. Early delivery of therapy is essential after TBI to reduce secondary brain injury, but unrestricted treatment of all brain injuries could be harmful. TBI stimulates a complex cascade of immunologic responses, both centrally and peripherally. These peripheral immune responses to TBI could serve as an early sensor of risk phenotype given the rapid, readily measurable response in the blood. An improved ability to risk-stratify patients on admission will streamline patient selection for aggressive interventions—such as invasive neuromonitoring—versus selection of those patients who can safely be observed reducing potential harms. Holly E Hinson, MD MCR is a Neurologist and Neurointensivist at Oregon Health and Science University where she cares for patients with severe acute brain injury. The objective of this application is to develop supervised learning models of actionable short- and long-term outcomes post-TBI and to interrogate if pre-specified immunoregulatory proteins add predictive power to the models over clinical features alone. Her central hypothesis is that immunoregulatory proteomic signatures improve our ability to classify a low-risk clinical phenotype after TBI. Dr. Hinson’s preliminary data suggest peripheral cytokine levels are associated with actionable clinical events acutely after TBI. The project employs a highly-sensitive, single molecule immunoarray (SIMOA) to detect immunoregulatory proteins complemented with an unbiased proteomic approach utilizing global discovery mass spectrometry. She will develop and assess a series of models incorporating proteomic signatures to classify: acute progressive intracranial hemorrhage (Aim 1A), acute neurologic deterioration (Aim 1B), and long-term outcomes measured by the 6-month Glasgow Outcome Scale (Aim 2). She will develop these models in a well-defined, clinical trial population (development set), and test their ability to correctly classify outcome in an independent, prospectively enrolled cohort at OHSU (test set). Under a multidisciplinary team of expert mentors, the project will generate new insights into low-risk phenotype recognition and outcome classification after acute TBI. The proposed patient-oriented research project will be enhanced by a structured didactic program in the principles of predictive modeling and patient phenotyping (including proteomics), which will provide Dr. Hinson with the critical skills she will need to conduct independent, innovative translational clinical research in the field of neurotrauma.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Cells alter their gene expression landscape to change their cellular state. The ability of cells to quickly transit between cell states is essential for many processes in biology—from development to wound healing and regeneration, processes which often go awry in pathological conditions like cancer or fibrosis. Thus, there is a biomedical need to understand the basic mechanisms driving these rapid cellular transitions. The epithelial- mesenchymal transition (EMT) is one such cellular transition that has reiterative roles in human health and disease. While much is known about the transcriptional programs that promote EMT, there are additional levels of gene expression control that impact cell state transitions and by extension EMT. The role of post- transcriptional regulation, and how it drives and contributes to a spectrum of EMT states, is not well understood. Here, we examine the role of post-transcriptional regulation, with emphasis on transcript turnover, during EMT. Neural crest cells undergo a tightly regulated EMT and offer a tractable model system in which to investigate the basic mechanisms of RNA turnover during EMT. We hypothesize that, in addition to pro-EMT transcriptional activation, transcripts that inhibit EMT (anti- EMT) or serve to maintain a previous cellular state must be degraded to drive EMT and cell state transitions. Using neural crest EMT as a model system, we seek to test this hypothesis by answering the following questions: 1) How are anti-EMT and residual transcripts targeted for turnover; and 2) What are the targets of RNA turnover during EMT and how does specific RNA turnover contribute to hybrid EMT states? To answer these questions, we will: 1) Apply a combination of unbiased multi-omic and candidate gene approaches to identify the RNA-binding proteins that promote RNA turnover during EMT; and 2) Apply RNA-sequencing approaches to broadly identify the targets of RNA turnover, the mechanism of how they are turned over, and how this contributes to hybrid EMT states. This Proposal seeks to understand the mechanisms of RNA turnover during EMT. The results of these studies will greatly advance current understanding of the basic cellular mechanisms driving EMT, providing novel targets for modulating EMT in human health and disease.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Gliomas are the most common malignant primary brain tumors in adults. Among gliomas driven by mutant isocitrate dehydrogenase, tumors harboring a 1p/19q codeletion are classified as oligodendrogliomas. Current therapies such as radiation and chemotherapy are highly toxic and cause long-lasting and life-altering deficits in cognitive and physical abilities. Importantly, although oligodendroglioma patients live for years with standard treatment, tumors inevitably recur and cause patient death. Since the 1p/19q codeletion is a hallmark of oligodendrogliomas, identifying metabolic vulnerabilities associated with the 1p/19 codeletion can lead to precision medicines for oligodendroglioma patients. Glycolytic metabolism, in particular, fuels biosynthesis and bioenergetics and is central to tumor proliferation. The glycolytic gene enolase 1, which is located on chromosome 1p36.23, is lost in oligodendrogliomas due to the 1p/19q codeletion, leaving these tumors dependent on enolase 2 (ENO2) for continued glycolysis. Our studies indicate that inhibiting ENO2 using a safe, potent ENO2 inhibitor (POMHEX) downregulates glycolysis in patient-derived oligodendrogliomas. However, ENO2 inhibition leads to a compensatory activation of pyruvate dehydrogenase (PDH), a key tricarboxylic acid (TCA) cycle enzyme. Importantly, combining POMHEX with the novel safe PDH inhibitor CPI- 613 completely abrogates glycolysis, the TCA cycle and oligodendroglioma growth. We will, therefore, test the hypothesis that targeting ENO2 and PDH is a precision therapy strategy for oligodendrogliomas (Aim 1). Successful translation of novel therapies is hindered by the lack of companion biomarkers that report on response to therapy. Magnetic resonance imaging, which is the mainstay of glioma imaging, fails to accurately report on response to therapy. Deuterium Magnetic Resonance Spectroscopy (DMRS) following administration of 2H-labeled substrates such as glucose is a safe clinically translatable method of imaging glycolytic flux in vivo. In Aim 2, we will examine the ability of 2H-glucose to report on response to ENO2 and PDH inhibition in oligodendrogliomas in vivo at clinically relevant field strength (3T). Our proposal is innovative and impactful because it will validate ENO2 and PDH as precision targets for oligodendrogliomas in this era of genomic medicine. Since the safety of POMHEX and CPI-613 has been established in primates and humans, and since DMRS can be readily deployed on clinical MR scanners, our therapies and companion biomarkers have the potential to be rapidly translated to the clinic. In essence, by simultaneously targeting metabolism for therapy and for imaging treatment response, our studies will enable precision medicine that improves outcomes and quality of life for oligodendroglioma patients.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The coordination of genetic programs and the physical organization of cells sculpt developing tissues, drive regeneration and, when dysregulated, facilitate disease. How these two processes are coordinated in space and time is poorly understood. Cell-cell interfaces are important organizing centers for morphogenic instruction. Here signals influencing gene expression that ultimately dictate cell fate decisions are integrated with changes in cell mechanics and, together, are necessary to build and maintain multicellular architectures. With an appreciation that gene regulatory networks and mechanics conspire at cell-cell interfaces to instruct morphogenic processes, an emerging challenge is to experimentally define the details of their intersectional operations in diverse morphogenic contexts. One intriguing example of cell-cell communication is the ubiquitously important Notch receptor pathway which has the intrinsic capacity to regulate both cell mechanics and gene expression, yet mechanisms of these distinct activities are unclear. As a postdoctoral fellow, the PI developed biomimetic microfluidic models of human tissues that were employed to identify several new mechanisms controlling 3D multicellular behavior operating at cell-cell and cell-extracellular matrix interfaces. The PI discovered that the highly conserved Notch family of receptors possess a previously undescribed cortical signaling function that permits Notch to connect changes in cell mechanics to transcriptional output. As an independent laboratory, the Kutys Lab has completed the first investigation into cortical Notch signaling in epithelial tissues and has identified morphogenic consequences and previously unappreciated signaling mechanisms. Over the next five years, the goal of the Kutys Lab is to continue its multidisciplinary approach of developing next generation biomimetic human tissue systems, molecular technologies, and microscopy-based methods to define the coordination of morphogenic behavior and signaling at cell adhesive interfaces. We will focus these efforts in three Areas: 1) engineering new tools to deeply understand how the cortical Notch pathway regulates signaling and adhesion mechanics in epithelia and the contexts in which it is invoked to regulate important biological processes in vitro and in vivo, 2) comprehensively defining how Notch receptor localization and activation are influenced by biophysical interactions with cell-cell adhesions and cortical actin during epithelial crowding and in endothelial cells exposed to shear stress, and 3) integrating proteomic approaches with 3D biomimetic systems to broadly profile and dissect how tissue architecture influences molecular control systems operating at cell-cell interfaces. The results of this research and the integration of the enabling technologies will contribute to the overall objectives of my research program. Together, these studies will offer fundamental insight into an important new arm of Notch signaling, how cell-cell adhesions might be dynamically regulated, and the molecular basis for how transcriptional and adhesive programs might be coordinated within complex 3D tissues.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT There is increasing recognition that post-tuberculosis (TB) lung disease (PTLD) is common and causes significant morbidity and mortality. However, changes in lung impairment are heterogenous with some patients improving and others worsening after completion of TB treatment. Moreover, spirometry – the standard method of assessing lung function – is not routinely available in high TB burden countries. Thus, to prioritize post-TB patients who may benefit from early interventions, there is an urgent need to better facilitate early identification of patients at risk for developing PTLD. The overall objective of this application is to evaluate novel approaches to facilitate early identification of patients most at risk for PTLD. The central hypothesis is that patient on-treatment (adherence behavior) and novel post- treatment (cough frequency and acoustic features) factors will improve risk stratification of PTLD. The central hypothesis will be tested by pursuing three specific aims: 1) characterize the evolution of lung function post-TB and its impact on health-related quality of life, 2) evaluate cough frequency and acoustic features measured by a novel mobile app as a non-invasive, inexpensive proxy for spirometry, and 3) evaluate adherence and cough feature trajectories as novel predictors of PTLD. The results of this work will provide preliminary data for an NIH R01 application evaluating app-based cough measurement as a monitoring tool for rarer but serious post-TB outcomes including COPD, TB recurrence and mortality. Dr. Huddart’s career goal is to become an independent investigator focused on understanding drivers of poor outcomes among TB patients in order to inform interventions to avert TB-related morbidity and mortality. To support her path to independence, the proposed work will be paired with a dedicated, multidisciplinary mentorship team and training in patient-centered outcomes assessment (Aim 1), machine learning (Aim 2), and dynamic outcome modelling (Aim 3). UCSF is an outstanding environment that is committed to junior investigators with extensive resources for research and career development. Thus, the K01 award will provide Dr. Huddart with the critical mentorship, training, resources and experience to become an international leader in TB outcomes research.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Most women treated for breast cancer will experience some form of drug-related toxicity and subsequent impairments in Health-related Quality of Life (HRQOL), yet toxicity is assessed inconsistently in oncology trials. Although the potential for side effects of treatments is of great importance to patients in making informed choices about their treatment, the toxicities are often under-reported. When assessing symptoms of trial participants, patients and providers do not always attribute symptoms to the study drug, which can result in misclassification of the maximum tolerated dose. Furthermore, many drug toxicities such as neuropathy, fatigue and diarrhea are often underreported by providers in trials, and thus a patient-centered assessment may lead to earlier recognition of reversable side effects. A major gap in knowledge is how to analyze and utilize patient level toxicity data in real time, and how to present the data to providers in a format that can result in early toxicity mitigation. While the number of lower- grade toxicities may increase given the reporting of patient outcomes, acting on these lower grade toxicities can mitigate serious adverse events (SAEs). We have recently instantiated an electronic patient reported outcomes (ePRO) platform across 26 sites in I- SPY2 where we collect adverse events and quality of lie information. I-SPY2 is an adaptive platform trial for high risk, early-stage breast cancer that continuously evaluates the efficacy of new neoadjuvant breast cancer therapies. The overall objective of this proposal is to refine and implement new methodology using interpretable machine learning that can be used to underpin a framework to redirect treatment and avoid more serious illnesses. Such methodology does not exist in clinical trials today and can hugely benefit patients, their providers and the clinical care team by tracking the inflection points of patient distress that could otherwise be missed but may require more immediate intervention. The methods will be developed through a computational framework in discussion with providers, at different stages of treatment, such as when the severity of a single symptom really impacts physical functioning (primary outcome), or when constellation of symptoms herald a significant deterioration in overall health. The central hypothesis of this proposal is that the methodology that we are developing on who will develop chronic conditions and symptoms that may affect quality of life will mitigate the event of a serious adverse reaction and improve overall quality of life, particularly physical functioning. We will test our methodology in a group of I-SPY patients and Breast Care Center early-stage participants at UCSF.

NIH Research Projects · FY 2024 · 2023-07

PROJECT ABSTRACT Reducing the global burden of low birthweight (LBW) remains a high priority for the World Health Organization (WHO). In Africa, malaria in pregnancy contributes to approximately 20% of LBW cases and affects nearly 12 million pregnancies every year. To curb the risk of malaria and LBW in Africa, the WHO recommends intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp with SP), a malaria chemoprevention strategy for pregnant women living in malaria-endemic settings. However, over the past two decades, widespread parasite resistance to SP has called for an urgent need to identify alternative antimalarials that could replace SP. While several antimalarials have been studied to date, the most promising candidate appears to be dihydroartemisinin-piperaquine (DP). Randomized controlled trials from our group and others have shown DP to be safe in pregnant women and far superior to SP in preventing malaria. Yet, these studies yield conflicting results on whether DP is superior to SP in preventing LBW. Mediation analyses conducted by our group confirm that the reason for this paradoxical finding is that SP, an antimalarial with known antibiotic and anti-inflammatory properties, improves LBW through mechanisms independent of its antimalarial activity (e.g., potentially through preventing sexually transmitted and reproductive tract infections, changing the gut or vaginal microbiome, and reducing maternal inflammation). Moreover, upon further investigation, the benefits of IPTp with either DP or SP appear to be context-specific, largely driven by the heterogeneity of the ‘non-malarial’ effects of SP between sites. Thus, in order to inform WHO on the optimal IPTp regimen, which may require a tailored approach for each setting, further evidence is needed to define the mechanisms driving the non-malarial effects of SP and for whom and where prevention of the malarial and ‘non-malarial’ mechanisms are most relevant. The objectives of this K99/R00 are to: characterize the mechanisms that mediate the effect of SP and DP on birthweight (Aim 1), assess the extent to which these mechanisms and other factors are causing heterogeneity between sites (Aim 2), and develop a model to estimate which antimalarial combination (either DP, SP, or a combination of DP+SP) would be the most optimal regimen for each unique epidemiological setting (Aim 3). Our research will leverage existing data from eight clinical trials conducted across ten study sites. The proposal will build on the applicant’s background in malaria, clinical trials, and epidemiology and include new training in: (1) the potential ‘non-malarial’ targets of SP affecting maternal and child health, (2) advanced computational statistics, (3) causal inference methods to target and tailor interventions. The training plan will be guided by an exemplary mentorship team who are experts in the field of causal inference, statistics, malaria, and maternal and child health. The combined research and training plan will competitively position the applicant for a successfully independent research career as an infectious disease epidemiologist focused on improving global maternal and child health policies.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY For patients with cirrhosis, liver transplantation is a well-established therapy, restoring liver function and reversing portal hypertension within days to weeks of the surgery itself. But cirrhosis also leads to insidious extra-hepatic effects such as muscle wasting, malnutrition, and functional impairment that may take months to reverse, if at all, which can compromise health and wellbeing (“global functional health”) after transplantation. In the ambulatory setting, these chronic cirrhosis manifestations can be captured by measures of “frailty”, the chronic biological state of decreased physiological reserve and increased vulnerability to health stressors, and operationalized using the Liver Frailty Index (LFI)—which our team developed from grip strength, chair stands, and balance. When assessed in the ambulatory setting, LFI predicts adverse health outcomes including hospitalizations and mortality. Frailty is now a well-accepted construct in hepatology/transplantation: in 2019, the American Society of Transplantation (AST) endorsed the use of standardized frailty metrics, including the LFI, for ambulatory liver transplant evaluation. However, approximately 1/3 of cirrhosis patients are hospitalized with acute illness immediately prior to transplant, in whom pre-morbid, ambulatory, frailty metrics may not be available. In this setting, transplant clinicians have, in many instances, been applying this construct in the acute care setting to inform transplant decisions—often informally through an “eyeball test”—despite a lack of studies evaluating the construct of frailty or tools to measure frailty in acutely ill patients. Unlike in the ambulatory setting where frailty represents factors that would not reverse with liver transplantation, a single frailty assessment in the acute setting may simply reflect the severity of acute liver-related decompensation, which, in theory, could reverse with a new liver. On the other hand, frailty trajectories may be informative of a patient’s ability to recovery a major stressor such as transplant surgery, so application of frailty as a trajectory may be more clinically appropriate. But testing of these hypotheses has not yet been done. In this proposal, we will leverage our 9-center research network to develop and validate a novel inpatient frailty index optimized for this acutely ill population, investigate models incorporating single and longitudinal assessments of frailty for the prediction of 1-year post-transplant global functional health, and associate inpatient frailty assessments with 1- year post-transplant healthcare utilization. Impact: Our proposal will result in a pragmatic, objective tool to standardize assessment of frailty in acutely ill patients with cirrhosis undergoing liver transplantation and clinical prediction models to guide use of single and longitudinal assessments of frailty for transplant decision- making in this clinically dynamic population. Understanding the precise relationship between acute care frailty and outcomes is essential to facilitate appropriate and systematic implementation of frailty in transplantation.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ ABSTRACT Establishing precise synaptic connections is critical for normal brain function. Synaptic dysfunction can lead to neuronal hyperexcitability, contributing to disorders including epilepsy. Microglia are the dominant immune cells in the brain and play multiple roles in synaptic development, modulating neuronal excitability, and engulfing excess excitatory synapses. However, the mechanisms by which microglia impact synapses have largely been investigated with fixed tissue histology or in limited regions of the adult brain using rodent models. In fact, microglial engulfment of whole synapses has not been directly observed in the developing brain. In this proposal, I will use a zebrafish model system to study microglial-synapse interactions in the intact developing brain. My recently published work identified a population of synapse-associated microglia (SAMs) enriched in the zebrafish hindbrain and defined its transcriptional profile by single-cell and regional bulk sequencing. In this proposal, I will examine this microglial subset using a combination of live imaging and candidate gene deletion in both physiology and in the context of hyperexcitability. Aim 1 will determine if microglia engulf synapses during development and the impact of immune activation or after deletion of a core lysosomal protease known as cathepsin b (ctsba) - a top candidate from my transcriptomic work. Aim 2 will further assess these phenotypes in the context of chemically induced hyperexcitability and use startle behavior recordings to assess the impact on neural circuit function. Finally, in Aim 3 (R00 phase), I will define the molecular mechanisms regulating lysosome activity during microglia phagocytosis and transcriptionally profile microglia following neuronal hyperexcitability. Together these studies will open a distinct direction using a new model to identify molecular pathways that regulate microglia-synaptic interactions with the potential to investigate non-neuronal therapeutic interventions that impact development and disease states such as epilepsy.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Pulmonary fibrosis is a chronic and intractable disease with a 5-year survival rate comparable to pancreatic or lung cancers. Deterioration of respiratory function in pulmonary fibrosis is caused by progressive replacement of normal tissue for gas exchange to dense fibrotic scar with fibrillar collagens. Pathologic fibroblasts accumulate at the sites of fibrogenesis and work as effector cells for excessive collagen deposition. Development of therapeutic strategies for targeting pathologic fibroblasts is hindered by the lack of understanding to cellular lineage and molecular detail of pathologic fibroblasts. In our previous study, we performed single-cell RNA-sequencing of normal and fibrotic lungs of mouse and human with a specialized protocol to identify all collagen-producing cells. We identified several fibroblast subsets that localize in different compartments of the lung. One of the fibroblast subsets emerge in fibrotic lungs of both mouse and human and show the highest levels of collagen gene expression and enhanced migratory capacity. These fibroblasts are characterized by specific expression of Cthrc1 (collagen triple helix repeat containing 1) and localized within fibroblastic foci of idiopathic pulmonary fibrosis, suggesting their pathologic role in pulmonary fibrosis. We recently generated and validated a novel mouse strain, Cthrc1-CreER, which allows us to specifically manipulate the pathologic fibroblast population in pulmonary fibrosis. The goal of this K99/R00 proposal is to elucidate the role and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis by using our innovative murine tools. Aim 1 (K99 phase) will reveal the role and fate of pathologic fibroblasts by ablating Cthrc1+ cells or lineage-tracing Cthrc1-CreER-labeled cells over the course of bleomycin-induced pulmonary fibrosis. Aim 2 (K99 phase) will reveal the transcriptional and epigenetic landscape of pathologic fibroblasts by performing RNA-seq, ChIP-seq, and ATAC-seq of purified Cthrc1-CreER-labeled cells at multiple time points of pulmonary fibrosis to seek master regulators for activation and deactivation. We will also seek the transcriptional regulations of CTHRC1+ cells in human pulmonary fibrosis. Aim 3 (R00 phase) will demonstrate the role of genes regulating pathologic fibroblasts by using intratracheal adoptive transfer of fibroblasts with lentiviral gene modifications and by conditionally knocking out candidate genes in fibroblasts in pulmonary fibrosis. These studies using the murine genetic tool highly specific for pathologic fibroblasts will shed light on cellular function and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis. This proposal is also designed to provide the candidate with training opportunity to obtain skill sets for murine genetic approach in search of therapeutic targets and functional genomics approach integrating RNA-seq, ChIP-seq, and ATAC- seq. The success of this project will enable the candidate to establish his expertise in the field of pulmonary fibrosis and lead to the candidate’s transition to scientific independence over the course of award period.

NIH Research Projects · FY 2024 · 2023-07

Project Summary People living with HIV (PLWH) have an estimated 1.6-6.0 times increased risk of developing oropharyngeal squamous cell carcinomas (OPSCC) compared with individuals in the general population. Approximately 70% of OPSCC are caused by HPV, and PLWH also have an increased prevalence of oral HPV infection compared to the general population. While a smaller proportion of oral squamous cell carcinomas (OSCC) are attributed to HPV, PLWH also have an increased risk of developing HPV-associated OSCC. Disparities exist among racial/ethnic groups for OSCC/OPSCC outcomes and Black individuals with OPSCC and OSCC have worse survival, a later stage of diagnosis, and less frequent cancer directed treatment than other racial/ethnic groups. HPV-associated OSCC/OPSCC are potentially preventable through HPV vaccination. Although oral cancer screening is not recommended for the general population, early detection is associated with improved survival and screening may be important in racial/ethnic minority groups. Oral health care providers may be the first point of contact for OSCC/OPSCC prevention strategies. PLWH have dental insurance provided through the Ryan White Care Act. Even so, more than half of PLWH have unmet oral care needs and 58-64% do not receive regular dental care. PLWH also face stigma and/or discrimination from living with HIV, including in the dental office, and may also have other intersectionality with sexual and gender minority (SGM) groups, for which there is additional stigma. Understanding the experiences of PLWH and the barriers/facilitators of oral health, through a lens of health equity, is needed before interventions can be designed to engage PLWH with intersecting identities in OSCC/OPSCC prevention activities. As a first step, we propose to conduct a qualitative study among PLWH of diverse racial/ethnic and SGM identities to: (1) explore individual, interpersonal, and structural oral health equity factors that serve as barriers or facilitators of accessing oral health care, (2) to explore knowledge and perceptions of HPV vaccination and OSCC/OPSCC, and (3) to collect recommendations on how to increase access to oral health care and engage PLWH in OSCC/OPSCC prevention. We will conduct approximately 12-18 focus groups with 5-8 individuals grouped by common identity group. These groupings may be by race/ethnicity, gender identity, sexual preference, or a combination of identities. The results of this study will be used to: 1) inform the design of a multilevel, culturally appropriate intervention to increase engagement with oral health care and HPV- associated OSCC/OPSCC prevention activities. The PI will design a randomized controlled trial to evaluate the intervention’s impact on HPV vaccination, OSCC/OPSCC knowledge, and retention in oral health care; and 2) assess the risks and benefits of OSCC and novel OPSCC screening among PLWH from diverse racial/ethnic identity groups. Screening PLWH at high risk for HPV-associated OSCC/OPSCC and from of groups with poor 5-year survival from OSCC/OPSCC may prove beneficial in reducing cancer-related mortality.

NIH Research Projects · FY 2025 · 2023-07

A novel cartridge-based sequencing solution for decentralized M. tuberculosis resistance detection Rifampin-resistant tuberculosis (RR-TB) is one of the principal causes of death associated with antimicrobial resistance. Newer all oral bedaquiline-containing RR-TB regimens, now recommended worldwide, will shorten treatment and improve outcomes. However, the lack of timely drug susceptibility data due to the slow growth rate of M. tuberculosis and the need for high containment biological laboratories are major barriers to scale-up of bedaquiline and other new and repurposed drugs. Xpert MTB/RIF Ultra and other commercial molecular TB tests can identify rifampin resistance, but cannot inform complete treatment regimens because they are limited to analyzing only a small number of genetic loci (i.e., “hot spots”). Targeted next-generation sequencing (NGS) is able to sequence entire genes rapidly without need for culture, and therefore could transform RR-TB clinical management. Achieving near-patient clinical application, however, has two major bottlenecks: (1) a complex, unstandardized workflow for preparing clinical samples for NGS, and (2) the cost and infrastructure requirements of industry-standard Illumina platforms. Partnering with global TB diagnostics leader Cepheid, we address these barriers by utilizing the ultrasonication, microfluidics, and thermocycler capabilities of the existing Xpert Ultra cartridge, already in use in over 180 countries, to unite a vetted direct-sample-to-answer system with a nanopore gene sequencing platform for the first time. Xpert CartSeq, a pioneering cartridge- based sequencing solution suitable for lower levels of healthcare, achieves robust DNA extraction and sophisticated library preparation while minimizing user variability and mitigating technical skill requirements. Through its development as detailed in this proposal, we aim to catalyze the clinical application of NGS in high burden settings.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Current treatments for many musculoskeletal disorders are often suboptimal. Understanding the signals controlling skeletal homeostasis and repair is of high relevance, since this knowledge is critical for developing effective therapies for common conditions such as osteoporosis and fractures. In this proposal, we study fibrous dysplasia (FD) as a way to understand the regulators of human bone formation, and test if these pathways could then be used to enhance bone repair. FD accounts for 2.5% of all bone lesions and can occur as part of McCune-Albright Syndrome (MAS). FD is caused by genetic mutations in the GNAS locus, leading to a constitutively active Gs-GPCR protein, hence increasing cAMP levels and causing aberrant cellular signaling. Medical treatments for this disfiguring disorder are sorely lacking. This proposal uses new tools, including mouse models, human induced pluripotent stem cells (iPSCs), skeletal stem/progenitor cells, and advanced genetic strategies, to address the critical knowledge gaps and to find novel therapies for these medically significant conditions. We propose three specific aims: Aim 1: Identify novel compounds that directly target Gsα-regulated cAMP and Wnt production. We previously showed that stopping excess Gs-GPCR pathway activity in mice could dramatically reverse FD-like bone lesions. Using a new artificial intelligence computational approach, we found 71 candidate compounds predicted to selectively bind the GsαR201H protein. Preliminary studies demonstrate that some of these show the desired inhibition of GsαR201H-induced basal cAMP production. This Aim takes our top candidates and further characterizes their ability to block cAMP and Wnt activity, as potential molecular tools for manipulating GNAS. We also test if the lead compounds can reverse existing FD lesions in mouse calvarial cultures. Aim 2: Test if Wnt inhibition can prevent FD bone lesions in mice. How the GNAS mutations in FD cause dramatic bone formation is still poorly defined. We recently found that three proteins, Wnt4, Wnt5a, and Wnt9a, are upregulated in both mouse and human FD bone lesions. This Aim tests if blocking these proteins can reverse FD bone lesions in mice, and examines how each Wnt protein impacts FD lesion pathology. Aim 3: Determine the pathways that are dysregulated in human FD bone lesions and assess the roles of WNT signaling in human skeletal stem/progenitor cells during fracture repair. This aim uses advanced genetics on human FD bone samples to identify the malfunctioning cell types that cause FD. We also test how overactivating WNT4, WNT5a, or WNT9a in human skeletal stem/progenitor cells affect bone formation in a fracture healing model. These results will identify new targets for treating FD and for promoting fracture healing. This application address key knowledge gaps about which Wnt signaling molecules drive FD and how they impact human osteogenesis. The proposal comes from an established, strong, collaborative, multi-institutional team with extensive experience in GPCRs, FD, bone biology, and bone analytical methods. 1

NIH Research Projects · FY 2024 · 2023-07

SUMMARY The dorsal endopiriform nucleus (DEn) is a highly conserved structure found in mammalian brains that is developmentally related to the claustrum and has been linked to the claustrum's role as the “seat of consciousness”. The DEn is connected to more limbic regions than the claustrum, has a very dense projection to infralimbic cortex, and receives strong inputs from sensory cortices especially olfactory and gustatory regions. While the DEn's role in behavior remains largely unknown beyond contributing to the generation of seizure activity, it has long been known to have extremely high levels of mu, delta, and kappa opioid receptor (MOR, DOR, and KOR) protein and mRNA. In preliminary studies we discovered a mostly non-dopaminergic ventral tegmental area (VTA) projection to the DEn; these neurons show a significantly stronger hyperpolarization in response to the MOR agonist DAMGO than any other VTA projection we have investigated. This is an exciting find because opioid reward, including intra-VTA opioid reward, is dopamine independent in opioid naïve animals. Thus, this non-dopaminergic, MOR sensitive projection may contribute to this opioid reward. Since the DEn receives inputs from a variety of sensory cortices, this is also a potential site of direct interaction between reinforcement encoding from the VTA and sensory cue information. Here we propose to anatomically map the VTA innervation of the DEn, characterize the VTA  DEn synaptic connectivity including its MOR sensitivity, and test whether selective modulation of this circuit connection produces reward or promotes learning or salience signaling. Together these studies will lay the groundwork for understanding a previously unstudied but highly opioid sensitive output of the VTA to an opioid receptor enriched brain region situated to integrate information from across cerebral cortex.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Regulatory T cells (Tregs) are a small subset of T cells that are vital to immune self-tolerance. They function by dominantly controlling the activities of other immune cells. In mouse models of type 1 diabetes (T1D), a single infusion of islet-specific Tregs prevents and stably reverses autoimmune diabetes. In these mouse models, infused Tregs accumulate in pancreatic islets and arrest autoimmune aggression against islet beta cells by expressing immune suppressive functions locally. Early-phase clinical trials of Treg cell therapy in patients with T1D have shown that it is feasible to produce billions of patients’ own Tregs for infusion and the therapy is well tolerated and safe. These pioneering efforts have paved the way for the development of next-generation Treg therapy aiming at establishing efficacy. The research program described in this proposal focuses on a critical need for a strategy to increase effectiveness by targeting human Tregs to the pancreatic islets and to monitor the targeting efficacy in patients. The proposed strategy is guided by the overarching hypothesis that shared, dominant immunopeptides on the surface of beta cells are highly specific anchors for islet targeting. Tregs can be engineered to target these anchors to deliver their immune regulatory function locally in the islets. Moreover, successful engagement of Tregs with beta cells can be monitored using an engineered biomarker released into the peripheral blood. Aim 1 will define the immunopeptidome of pancreatic beta cells. Aim 2 will develop a cellular engineering strategy to target Tregs efficiently and safely to pancreatic islets. Aim 3 will develop a barcode biomarker that is released by activated Tregs into the peripheral blood. These projects together aim to develop islet- specific TCR activation relay (iSTAR) Tregs. We have built a team of three investigators with complementary expertise in immunology, beta cell biology, and technology development. The proposed program synergizes our expertise to tackle the challenges in precision targeting of pancreatic islets for the preservation of beta cell mass in T1D.