University Of California, San Diego

NIH Research Projects · FY 2026 · 2023-08

Abstract Multiple organ dysfunction is the most common cause of death in injured patients who do not die from brain injury or uncontrolled hemorrhage. Older patients are at highest risk for morbidity with reduced probability to return to productive life after trauma and shock. Current therapy is symptomatic without addressing the underlying mechanisms for the increased morbidity seen in aged individuals. In the past, we advanced the revolutionary idea that multiorgan dysfunction after trauma/shock is due to leak of the powerful digestive enzymes across the intestinal mucosal barrier systemically. Specifically, enteral blockade of digestive proteases in multiple forms of acute shock and sepsis significantly reduces diverse cell dysfunctions, morbidity and mortality; this intervention is currently being tested in FDA-approved Phase III clinical trial after completion of multiple successful Phase II studies in cardiac and gastrointestinal surgery patients. In this application we hypothesize and provide preliminary results that demonstrate chronic escape of digestive enzymes in old animals from the bowel into systemically, with subsequent infiltration into peripheral organs. Digestive enzymes cleave extracellular matrix proteins and membrane receptors, causing cell and organ dysfunctions in the old. When exposed to trauma/shock, the old are subjected to acute leak of digestive enzymes (just like the young) but in addition, must contend with a chronically accumulated digestive enzyme load in their tissues, leading to significantly increased multiorgan dysfunction after shock. Our Overall Objective is to use pretreatment of old rats with inhibitors of digestive enzymes prior to acute trauma/shock to reduce their high level of multiple organ dysfunction and mortality. Our Specific Aims are to (1) determine the accumulation and activities of digestive enzymes in tissues outside the gastrointestinal tract of old versus young rats of both genders. Preliminary results indicate that aged animals have significantly increased levels of digestive enzymes in peripheral tissues and consequently reduced organ function. (2) measure the protease activity and organ dysfunction in the old after treatment with digestive enzyme inhibitors. Our rationale is that one-week blockade of digestive enzymes with competitive inhibitors at low level (µM) reduces their activity in tissues outside the intestine and improves cell and organ function without significantly affecting digestive enzyme activity and digestion inside the small intestine (at mM concentrations). (3) determine multiorgan dysfunction and mortality after trauma/shock in pretreated old animals with attenuated digestive enzyme activity in their tissues outside the intestine. Our studies will bring to light a new mechanism for development of organ and cell dysfunction in the old that is translatable to humans. We will determine the first time the accumulation and temporal activity of digestive enzymes (including, but not limited to serine protease, lipase, amylase) in tissues outside the intestine, develop new approaches to attenuate their activity and reduce multiorgan dysfunction upon exposure to trauma/shock.

NIH Research Projects · FY 2026 · 2023-08

Project Summary During chromosome segregation, each daughter cell receives a complete complement of the genome, and this is repeated for every cell division. Therefore, chromosome segregation must be extraordinarily accurate and robust to ensure the health of an adult human; otherwise, disasters like cancers can occur. Cancer cells exploit and rewire their chromosome segregation machinery to meet their insatiable need of uncontrolled cell division. Successful chemotherapeutic drugs kill cancer cells through disrupting this obligate need. Thus, understanding mechanisms of chromosome segregation has far-reaching implications to human health. Kinetochores execute chromosome segregation by connecting chromosomal centromeres to spindle microtubules. This connection must be flexible to accommodate the fleeting passage of the DNA polymerases that replicate centromeres during the S phase; it must also be strong to withstand the pulling force of spindle microtubules during mitosis. Cells coordinate these opposing attributes of kinetochores temporally and regulate the transition between them. Because kinetochores and their associated regulators are highly conserved among the eukaryotic kingdoms of life, we will use the yeast Saccharomyces cerevisiae as a primary research organism to study how kinetochores are assembled. The central hypothesis is that kinetochore assembly is a highly cooperative process that involves multiple protein-protein and protein-DNA contacts, which are controlled by cell cycle signals. To understand how kinetochores are assembled, Specific Aim 1 will apply a quantitative proteomics platform to define the steps of kinetochore assembly; making use of stable isotope based mass spectrometry (MS) to analyze native kinetochores as well as reconstituted kinetochores assembled from concentrated cell extracts. Two key interfaces govern kinetochore assembly: the first one is between centromeres and inner kinetochores, while the second one is between inner and outer kinetochores. Specific Aim 2 will probe the centromere-inner kinetochore interface and focus on how phosphorylation of specific inner kinetochore components may regulate it. Specific Aim 3 will dissect the inner-outer kinetochore interface and study its cell cycle control with an ultimate goal of reconstituting the kinetochore that retains its physiological properties. All together, these studies are aimed at understanding how kinetochores are assembled. Understanding kinetochore assembly has broad relevance, because the rules and methods of study apply to all systems in which signals are integrated to control macromolecular assemblies. Our collaborative team, equipped with interdisciplinary expertise and shared interest in kinetochore biology, is uniquely qualified to carry out the proposed projects and to make impactful advance in this area of considerable biomedical significance.

NIH Research Projects · FY 2025 · 2023-08

PROJECT ABSTRACT At a time when youth suicide and school shooting incidents are increasing, a growing number of U.S. schools are implementing commercially available social media surveillance (SMS) technology that can allegedly identify mental health and safety threats by monitoring students' social media posts. Although SMS technology is increasingly being purchased and used by schools, such use is often not disclosed to students or parents. There is also a lack of transparency regarding the methods used by companies to collect information about students, and there are no guidelines to inform how schools use and respond to the information they receive. Moreover, there is typically no evaluation of practice by schools, and there has been no systematic assessment of the ethical, legal, and social implications (ELSI), including how it affects the students whom it purportedly aims to help. We recently conducted a brief survey of 529 students, parents, teachers, and school administrators on perceptions of SMS. Compared to administrators, students were less supportive of using SMS, more skeptical of whether it could address violence, bullying, and mental health issues, and parents felt it could lead to data misuse and discrimination. These preliminary data point to differing perspectives among stakeholder groups and suggest more work is needed to assess the ways in which SMS is currently implemented and the range and complexity of possible effects on students. The long-term goal of this research is to promote the health, safety, and privacy interests of students and the institutional obligations and interests of schools by identifying and mitigating harms from school-based SMS. The overall objective of this application is to document the ways in which SMS is currently being used by school administrators, assess the perspectives of students and parents, and analyze the ELSI issues. Our hypothesis, based on our preliminary work, is that administrators engage in a wide range of implementation practices and that some youth are vulnerable to adverse effects of school-based SMS. The work proposed in this application is needed to inform student-centered policy recommendations in this area where little transparency and regulation currently exist. We propose the following specific aims: 1) Document school administrators' reasons for using SMS and current implementation practices; 2) Assess the perceived effects of school-based SMS on students and identify student characteristics associated with vulnerability to adverse effects; and 3) Conduct a detailed analysis of the ELSI issues with school-based SMS and develop policy recommendations. This project will generate new insights about perceptions and practices related to SMS in schools. Results could also inform a research agenda in this area, including whether systematic intervention studies of some school-based SMS systems are warranted, and if so, offer study design considerations that center the mental health needs of students and youth. More generally, this work will inform current and future practices and policies related to the use of SMS and other forms of digital surveillance in broader areas of public health.

NIH Research Projects · FY 2025 · 2023-08

Project Summary To be a productive member of a collective, a cell must precisely and dynamically partition its own contents. Therefore, pathways that generate asymmetry in the cell, commonly referred to as polarity, are essential for development and homeostasis. How cells create de novo polarity and harness asymmetry to diversify cellular populations during morphogenesis remain important and open areas of study. The molecular logic that generates and sustains highly conserved polarity domains has been thoroughly studied in animals and yeast and has led to the creation of synthetic circuits capable of generating polarity in vitro. However, there are still significant challenges to interrogating these pathways in situ, where technical limitations make it difficult to track individual cells over days in developing animals as they transit through multiple identities. To overcome this hurdle, the proposed work will interrogate polarity pathways within developing plant tissues, where observation of subcellular dynamics can be paired to long-term tracking of full developmental decisions at single-cell resolution. Plants harness polarity for many of the same functions as animal cells, including regulation of asymmetric cell division and organelle positioning. Importantly, our recent progress investigating the formation and functions for cell polarity in developing Arabidopsis leaves highlights that these polarity circuits have both commonalities with and differences from canonical polarity pathways in animals. Therefore, our investigations will advance understanding of polarity mechanisms broadly and introduce an experimentally tractable and independently evolved system to test the generality of polarity models. Specifically, our aims are to 1) determine the molecular interactions that create polarity within leaf progenitors, 2) delineate the pathways that couple polarity to organelle topography for tissue formation, and 3) identify the control points where extrinsic signals regulate polarity pathways. We have developed imaging platforms, new genetic tools, and analysis pipelines that will allow us to make rapid progress on our aims. Taken together, we expect that we will identify novel means of harnessing polarity in cells, with potential future applications as tools to exert spatial control for bioengineering purposes and human health.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Despite the scaling up of evidence-based interventions to End the HIV Epidemic (EHE) in the U.S., several communities most affected by HIV lag behind the EHE goal of reducing HIV infections by 90% by 2030. Black/African American (AA) people with HIV (PWH) account for a large proportion of HIV diagnoses in the U.S. Specifically, AAs represented 42% of all new HIV diagnoses reported in 2019 despite comprising only 13% of the total United States population. Furthermore, AA PWH have poorer outcomes along the entire HIV care continuum, including a greater number with undiagnosed HIV, poorer linkage to and retention in HIV care, lower rates of receiving antiretroviral therapy (ART), lower ART adherence rates, and lower likelihood of achieving viral suppression. While AA PWH with strong community cohesion report positive HIV outcomes (e.g., high ART adherence, viral suppression), in regions where there is low community cohesion among AA PWH, these individuals may be overlooked, despite the unique experience of having weaker community cohesion. As such, it is imperative to focus research efforts on investigating psychosocial determinants of health among AA PWH with low community cohesion. We have shown that high psychosocial resources (e.g., resilience, personal mastery, social support) positively influences health outcomes such as medication adherence, and physical and mental health-related quality of life. Nevertheless, these quantitative-based studies may not account for the unique, individual experiences of AA PWH who lack strong community cohesion. Assessing the relationships between psychosocial determinants of health, medication adherence, and healthcare utilization is critical toward identifying AA PWH most at-risk for poor health outcomes. Accordingly, the proposed F31 project will follow-up on an initial study of AA PWH in order to: 1) Characterize ART adherence trajectories and their associations with HIV characteristics among AA PWH in an EHE jurisdiction with low AA community cohesion; 2) Identify modifiable psychosocial determinants of health associated with ART adherence trajectories among AA PWH in an EHE jurisdiction with low AA community cohesion; and 3) Explore qualitative relationships between positive modifiable psychosocial determinants of health (e.g., resilience), ART adherence, and healthcare utilization among AA PWH in an EHE jurisdiction with low AA community cohesion. The proposed research will use advanced qualitative and quantitative research methods to examine these relationships. The opportunities afforded via this F31 mechanism will facilitate the applicant’s professional development toward becoming an independent investigator dedicated to researching behavioral medicine topics such as psychosocial determinants of health outcomes among diverse populations.

NIH Research Projects · FY 2026 · 2023-08

Work over the last decade has provided tantalizing clues that the genetic mechanisms that underlie evolutionary trajectories may be more constrained and deterministic than previously appreciated. Whether the genetic basis of evolution is predictable or stochastic is a fundamental question in evolutionary biology, and the ability to predict evolutionary responses has critical implications for the fields of agriculture, medicine and conservation. Unfortunately, we are far from understanding the factors that underlie the predictability of evolutionary responses or the fitness consequences associated with alternative evolutionary trajectories in nature. Surveys of replicate populations adapting to the same environmental conditions provide an opportunity to quantify the repeatability of evolutionary trajectories and determine the factors affecting observed patterns of variation. Several genetic factors are hypothesized to affect the probability of a gene or mutation’s use during the process of adaptation. However, to date there have been few empirical tests determining the contribution of individual factors to patterns of gene re-use (parallelism) in natural populations. In the proposed work, we built upon our prior findings and combine innovative population genomics, genetic engineering, and experiments to answer three core biological questions: 1) How does parallelism translate across biological levels? 2) Which genetic factors affect parallelism? 3) What are the fitness consequences associated with genetically distinct, yet phenotypically similar, evolutionary trajectories? Our laboratory is well positioned to answer these profoundly important questions about the repeatability and predictability of evolutionary trajectories. Our model system for addressing these questions is the threespine stickleback (Gasterosteus aculeatus), a species with bountiful genetic and genomic resources. Stickleback are considered a textbook example of parallel phenotypic evolution; thousands of independently derived stickleback populations have repeatedly adapted to a variety of freshwater environments. We will survey several focal freshwater populations to examine the scaling of papalism across biological levels and to test whether the source and type of genetic variation or epistatic interactions predict the frequency of gene use during adaptation. The creation of genetically modified lines that differ only in their allele frequencies at candidate loci will allow us to test whether fitness coefficients can predict the probability of gene use (magnitude of genetic parallelism) in nature. Together this work will test several fundamental evolutionary questions and take a first step toward determining whether the development of a predictive evolutionary framework is possible.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Hematopoietic Stem Cells (HSCs) produce all cells of the blood lineage throughout life. Defects in HSC self- renewal can lead to immunological defects, anemia, and bone marrow failure. Enhanced HSC self-renewal can result in hematopoietic malignancies. Thus, precise regulation of HSC self-renewal is essential for maintaining hematopoietic and human health. Our previous work has shown that adult HSCs tightly control protein synthesis and that modest changes in protein synthesis impair HSC self-renewal and function. However, the mechanisms that regulate mRNA translation in HSCs remain largely unknown. Transfer RNAs (tRNAs) are non-coding adaptor RNAs critical for mRNA translation that are encoded by hundreds of genes in the mammalian genome, with multiple functional genes capable of decoding virtually every codon. We previously showed that the tRNA repertoire influences neuronal function but the effect of changes in tRNA expression on hematopoietic cells is unknown. In preliminary studies, we found that loss of n-Tr22, a member of the five gene arginine UCU tRNA family significantly impairs HSC maintenance and self-renewal, and this is exacerbated in a sensitized genetic background lacking the ribosome rescue factor Gtpbp2, resulting in a complete loss of adult, but not fetal HSCs. We hypothesize that the sensitivity of adult HSCs to the n-Tr22 mutation may be due to differences in the tRNA repertoire between adult HSCs and restricted progenitors, and between HSCs at different developmental stages. The impact of tRNA mutations may be further influenced by differential codon usage in the transcriptome of these cell populations. Finally, there may be cell-type-specific differences in the signaling pathways activated by loss of a tRNA. We propose to test this hypothesis by using chromatin immunoprecipitation and sequencing to determine the tRNA repertoire in the hematopoietic system. We will also analyze how this tRNA mutation influences the maintenance and function of HSCs in the presence and absence of the Gtpbp2 mutation using flow cytometry and long-term multilineage reconstitution assays. Finally, we will determine the effects of the loss of n-Tr22 and Gtpbp2 on protein synthesis and gene expression by incorporation of a puromycin analog, ribosome profiling, and RNA-sequencing. The results from this grant will not only shed light on the role of tRNAs in regulating mRNA translation in the hematopoietic system, but also provide a means to understand the role of these genes in the phenotypic heterogeneity common to many human hematopoietic disorders.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Approximately 35% of children in the U.S. have overweight or obesity (OW/OB). To date, the most successful weight loss program for children is Family-Based Treatment (FBT), which is delivered to both children and parents and includes nutrition and physical activity education, behavior therapy techniques, and parenting skills. Data suggest that 2/3 of children who participate in FBT do not respond. This lack of success suggests that there are unaddressed mechanisms that could be impacting treatment response. The Behavioral Susceptibility Theory purports that food cue responsiveness (FR) is the primary driver of eating onset, while satiety responsiveness (SR) is the primary driver of eating offset. High FR is a risk factor that influences weight loss maintenance among children who participate in FBT. We developed a new model for the treatment of children with OW/OB, called Regulation of Cues (ROC), which is based on the Behavioral Susceptibility Theory. The ROC program targets these mechanisms for overeating, by focusing on reducing FR and increasing SR. We pilot tested the ROC model with children who overeat with OW/OB, and showed that the ROC program is feasible, acceptable and is a promising treatment for children. The objective of this proposed study is to further evaluate the ROC program among children with OW/OB who are high on FR and their parents. We propose a multisite 4-arm randomized controlled trial at UC San Diego and University of Minnesota that will compare ROC, FBT, ROC+ nutrition education and caloric restriction (ROC+) and a health education comparator (HE) over 18 months. We will recruit and randomize 280 children with OW/OB who are high on FR and their parents and will conduct assessments at baseline, throughout treatment, post-treatment, and at 6- and 12-month follow-up. The primary aim of this study is to evaluate changes in child weight (BMIz/%BMIp95) as well as overeating episodes among children enrolled in ROC, FBT, ROC+ and HE. Secondary aim 1 will compare ROC, FBT, ROC+ and HE on child SR, FR, inhibition, and energy intake over the course of the treatment and follow-up. Secondary aim 2 will compare ROC, FBT, ROC+ and HE on parent outcomes, including weight loss (BMI), overeating episodes, satiety responsiveness, food cue responsiveness inhibition, and energy intake over the course of treatment and follow-up. Finally, exploratory aim 1 will evaluate effects of mediators (food cue responsiveness, satiety responsiveness, inhibition) and moderators (food cue responsiveness, satiety responsiveness, demographics, baseline BMIz/%BMIp95/BMI) of treatment effects on weight loss over time among children and parents. This study could provide a more durable weight loss program for children with OW/OB and high FR and has the potential to change the paradigm of weight-loss treatment for children. This study will also contribute to the study of basic appetitive mechanisms and food intake and inform clinical decision making for children with OW/OB.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Chronic pain (CP), a highly prevalent, costly, and disabling biopsychosocial condition, is being increasingly addressed with cognitive behavioral interventions such as Acceptance and Commitment Therapy (ACT) and Cognitive Behavioral Therapy (CBT). Such interventions seek to maximize physical, social, and occupational functioning by changing how one manages pain via addressing pain-related thoughts, beliefs, and behaviors. The current CP literature is limited by the use of between-subject designs and self-report measures that lack ecological validity and may be subject to bias. One component of function, physical activity, is able to be objectively measured in an ecologically valid way using accelerometry. In fact, prior research has combined accelerometry and pain ratings collected using ecological momentary assessment (EMA) to better reflect the real-time, real-world relationship between pain and physical activity. Such research has indicated cross- sectionally a dynamic within-person pain-physical activity relationship. We hypothesize that this dynamic relationship is alterable by cognitive behavioral intervention and seek to examine this research question in Veterans with CP, as they represent an underserved and apt population for CP research given disproportionately high rates of this condition. In Aim 1, we model the relationship between EMA-assessed pain and objective physical activity over a 7-day baseline period, within-person, using parallel latent growth curve modelling (LGCM). We hypothesize that at baseline, the growth trajectories of pain and physical activity will be related such that increases in pain will be associated with decreases in physical activity. In Aim 2, we use multigroup analysis to examine if participation in a cognitive behavioral intervention alters the relationship between the growth trajectories of EMA pain and objective physical activity. We hypothesize that, post- intervention, the relationship between the growth trajectories of pain and physical activity will be significantly altered compared to baseline. In our Exploratory Aim, we conduct follow-up interviews to qualitatively explore how treatment impacts how individuals relate to their pain. Therefore, the proposed research will examine the relationship between pain and physical activity and if and how it changes after cognitive behavioral intervention. The use of structural equation modelling (i.e., LGCM) and qualitative methods will be combined to portray a more complete picture of the experience of CP and treatment. Thus, findings from this study will address existing limitations in the understanding and conceptualization of function in CP, provide information on how to improve function in individuals with CP, and promote the improvement and refinement of cognitive behavioral interventions for the treatment of CP. Importantly, this project will advance the applicant’s long-term goal of becoming an independent clinical researcher committed to applying advanced technological and statistical tools to the improvement of treatments for CP.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Stress and chronic pain disorders are comorbid conditions wherein stress exacerbates pain. Mindfulness meditation, a non-opioidergic and self-regulatory technique that trains non-reactive awareness to arising sensory and affective events, reduces clinical and experimentally induced pain and stress. Our recently completed psychophysical and functional magnetic resonance imaging (fMRI) study found that four, 20-minute sessions of mindfulness training in healthy participants reduced stress ( 39%, p = .023) and pain intensity ( 32%, p < .001) in response to noxious heat (49°C). Stress reductions also predicted lower pain intensity (p = .013). The perigenual anterior cingulate cortex (pgACC), a cognitive-affective brain area involved in reappraisal, and the amygdala, a key brain area involved in sensory and emotional processing, are associated with modulating pain and stress and share extensive anatomical connections. Our laboratory has demonstrated that increased pgACC activity underlies mindfulness-induced reductions in pain and anxiety in healthy individuals, while mindfulness-induced deactivation of the amygdala has been shown to correlate with pain and stress relief across separate studies. Despite these findings, no study has determined whether mindfulness meditation promotes stress relief and analgesia by modulating the pgACC and corresponding neural activity in and connectivity between nociceptive and negative affect-specific brain regions. To this end, we will analyze the blood-oxygen-level-dependent fMRI data corresponding to our behavioral findings to test the following hypotheses. We will first determine if higher pgACC (HYP1a) and lower amygdala (HYP1b) activity during mindfulness is associated with higher pain/stress relief. We will next determine if increased pgACC-amygdalar functional connectivity correlates with mindfulness-induced pain (HYP1c) and stress relief (HYP1d). Our preliminary analyses found that mindfulness-induced analgesia correlates with reductions in machine-learned, multivariate fMRI signatures sensitive and specific to nociception (neurologic pain signature; NPS, p = .03) and stimulus-induced negative affect (p = .01). We will similarly determine if reductions in negative affect brain responses correspond to stress relief (HYP2a). Finally, we will confirm if increased NPS- (HYP2b) and negative affect signature- (HYP2c) pgACC connectivity is associated with pain and/or stress relief. The proposed research and training will be accomplished under the mentorship of my Sponsor, Co- sponsors, and Co-Mentor, who are experts in fMRI clinical trials on pain, meditation, and/or stress. Activities include a) training in pain-evoking procedures and statistical and computational techniques, programming, and neuroanatomy for analysis of behavioral and fMRI data b) ethical interactions with individuals with chronic pain and stress, c) scientifically validated mindfulness training, and d) fostering career development strategies. The knowledge and skills gained will prepare me to successfully navigate a career as an independent cognitive neuroscientist identifying the brain mechanisms of novel treatments for pain and comorbid health ailments.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Unexplained infertility accounts for 25% of infertility cases. One of the challenges in identifying factors related to infertility, particularly unexplained infertility is that there are no live births. A hallmark of genes essential in oogenesis and during preimplantation development is a failure to progress to the point a viable pregnancy can be recognized. As such, identifying genes essential to the these crucial stages is an important step in understating unexplained infertility. Prior research suggests that Bmal1-/- mice produce embryos which are largely lost before the blastocyst stage, even when implanted into pseudopregnant wildtype mice. Dr. Cassin’s preliminary data suggests that initial deficits occur much earlier as Bmal1-/- oocytes are largely observed to be abnormal, likely leading to the phenotype previously described. Further, preliminary work in this grant reveals that BMAL1 is expressed in developing follicles and throughout preimplantation development. BMAL1 has been shown to regulate many of the most essential regulatory functions in early development such as cap-dependent translation, and mRNA regulation, but the role of Bmal1 has not yet been investigated. This grant proposes the examination of Bmal1 during oogenesis and the maternal to zygotic transition (MZT). The overarching hypothesis of this grant is Bmal1 is an essential maternal factor during oogenesis, aids in the regulation of many essential early genes transcribed by the embryo, and regulates a cell-autonomous embryonic clock. Aim1 will address the expression and phenotype of Bmal1 activity during oogenesis. The guiding hypothesis for Aim1 is a primary deficit in oogenesis as a result of Bmal1 m-KO contributes to poor oocyte quality and defects in MZT progression. This Aim will be addressed through the use of embryo culture and immunohistochemical (IHC) techniques. The guiding hypothesis for Aim2 is BMAL1 is an essential factor for preimplantation through the regulation of both maternal mRNAs as well as early embryonic genome activation. As BMAL1 has many regulatory functions Aim2 combine rescue experiments with several -omics techniques to investigate the specific function that BMAL1 has in the MZT. Aim3 examines the regulation of the MZT by Bmal1. The guiding hypothesis of Aim3 is the cell- autonomous clock of the developing embryo is regulated through Bmal1. To address Aim3 live cell imaging of reporter mice will be used to observe movement of BMAL1 throughout the cell. This work specifically focuses on one gene, Bmal1. However, because Bmal1 is a gene involved in many regulatory pathways, this grant opens up a new area of research, the circadian genes in early development. Further, it identifies a novel potential factor in unexplained infertility, and identifies a new direction in the field of infertility. Dr. Cassin will be trained by her mentors in the fields of embryology and circadian biology. Together with training in presenting, scientific writing, and bioinformatics, Dr. Cassin will possess all the skills necessary to establish a successful, independent lab following the mentored phase of the grant.

NIH Research Projects · FY 2026 · 2023-08

Project Summary / Abstract Natural product leads from marine life continue to inspire new drugs, with nine approved for clinical use since 2010. A historical challenge with developing marine organism-derived bioactive chemicals has been one of limited supply. Often, natural drug leads are isolated from rare or environmentally sensitive marine invertebrates and algae, which challenges the pre-clinical evaluation of promising candidates when demands outweigh natural supplies. Some terrestrial plant-derived drugs can now be produced in genetically engineered microbial cell factories, an approach that would be attractive for marine-derived molecules. However, the biosynthetic machinery responsible for the biosynthesis of bioactive natural products in marine animals and algae is largely unknown. Until now. We have discovered and validated the first steps of biosynthetic pathways in corals and algae that lead to over 6,000 terpenoids, including the anti-inflammatory pseudopterosin and anticancer halomon. We propose a multidisciplinary project to investigate the molecular basis of terpenoid diversification in marine eukaryotes that harbor terpenoids with promising biological properties. Significant outcomes of this research project will include a new paradigm for terpenoid biosynthetic logic in marine eukaryotes, and the application of this basic knowledge toward the microbial production of marine animal and algal molecules. We propose three specific aims, namely: 1) To develop a marine eukaryotic genome mining platform for algae and corals; 2) To functionally characterize marine eukaryotic terpene synthases for high yield terpene production; and 3) To discover and characterize terpene tailoring enzymes (halogenation/oxygenation) associated with bioactive coral and algal natural products.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to unravel mechanisms driving abnormal immune activation and cognitive impairment in people living with HIV (PLWH) through the lens of epigenetic programming. Despite overall advances in lifespan and health for PLWH who are on suppressive ART, a subset of individuals on ART continue to demonstrate neuroimmune abnormalities and associated clinical neurological syndromes including cognitive complications. Cerebrospinal fluid (CSF) studies are a window into the CNS of PLWH, revealing a role for abnormal myeloid cell activation and persistence viral replication in the CNS, despite apparent systemic viral suppression with ART. Our own single cell genomic studies of fresh CSF cells from PLWH have shown that a rare microglia-like myeloid cell population resides in the CSF in PLWH; that these cells are linked to HIV disease status; and that these myeloid cells can harbor HIV DNA. Additionally, our research has shown that epigenetic features of myeloid cells are rapidly altered in HIV infection and this aberrant myeloid epigenetic state associated with HIV infection persists despite the immediate initiation of ART during acute HIV infection. However, despite myeloid cells being recognized as crucial cellular mediators of CNS abnormalities in PLWH, the epigenetic landscapes of CNS myeloid and other immune cells in PLWH remain uncharted. Our central hypothesis is that HIV leaves epigenetic “scars” at regulatory regions of proinflammatory gene networks in distinct CSF myeloid cell subsets, contributing to HIV-related cognitive impairment despite ART. This hypothesis will be tested in our established HIV Associated Reservoirs and Comorbidities Study (HARC) cohort at Yale that includes large volume lumbar puncture from study participants with and without HIV and will be further explored utilizing postmortem brain specimens from the National NeuroAIDS Tissue Consortium (NNTC). In PLWH, we will longitudinally assess fresh CSF myeloid and T cell single cell epigenetic and transcriptional cell states over the course of ART treatment and ask whether there is damage to the epigenomes of CSF myeloid and T cells sustained during HIV infection that persists over time as epigenetic “scars”. Using machine learning, we will then assess for an association between epigenetic perturbations in PLWH and CNS outcomes, including cognitive impairment and abnormal CSF soluble biomarkers of inflammation and neuronal injury. We will also explore single cell epigenetic cell states of myeloid and glia in postmortem brain tissues from choroid plexus and periventricular zones of HIV-infected individuals who died on suppressive ART and matched controls. Lastly, we will apply an innovative new single cell assay for multifactorial chromatin profiling to assay histone modifications. These proposed, in-depth, multiomic single cell analyses of distinct myeloid cell subsets in the CNS and blood, combined with cognitive assessments, will reveal the effects of HIV infection on immune cell epigenomes, and will uncover transcriptional and epigenetic states most strongly related to cognitive complications in PLWH. The results will advance efforts to biotype CNS complications in PLWH based on immune and viral pathogenesis.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Childhood cancer is the leading cause of death by disease past infancy in the U.S. While survival rates are improving, stark racial/ethnic disparities remain. Hispanic children have a significantly higher incidence of several cancers, and inferior overall survival than their non-Hispanic White (NHW) counterparts. Although clinical trial enrollment is linked to higher survival and NIH mandates appropriate inclusion of minorities in clinical trials, Hispanic children with cancer are significantly underrepresented in cancer clinical trials compared to NHWs. Given that Hispanics will comprise 33% of the U.S. childhood population by 2030, there is an urgent need to identify effective interventions to overcome low trial accrual to equitably benefit Hispanics. Novel data from our group suggest that inadequate research literacy (capacity to understand and act on information to make decisions about research) is a barrier to participation in clinical trials. There are major gaps in knowledge about interventions to improve research literacy and clinical trial participation for Hispanic children. This R01 is informed by the PI’s peer navigation intervention developed during her K08 award, “COMPRENDO” (ChildhOod Malignancy Peer REsearch NavigatiOn). COMPRENDO (adapted from the evidence-based patient navigation model) involves trained peers (parents) who deliver culture, language, and health literacy- concordant education to parents/guardians of children with cancer during informed consent for therapeutic clinical trials. In our pilot feasibility study, COMPRENDO was feasible, highly acceptable by stakeholders and increased parental informed consent comprehension and voluntariness (willingness to participate in research without feeling pressured). The logical next step is to test COMPRENDO effectiveness on a larger scale. We will study COMPRENDO in a multisite effectiveness-implementation randomized clinical trial (RCT) in 4 sites with diverse populations of Hispanics. Our long-term goal is to implement generalizable, targeted, effective, and reproducible peer navigation interventions to enhance minority accrual to clinical trials. The overarching objective of this R01 is to increase clinical trial accrual in Hispanic children with cancer by improving research literacy in their parents in a Hybrid Type 1 design to both test COMPRENDO effectiveness and explore multisite implementation determinants. Hispanic parents will be randomized to COMPRENDO or usual care [informed consent with the oncologist only]. We will measure child-centered (accrual) and parent-centered (comprehension, voluntariness, decisional regret, decision-making self-efficacy, and satisfaction) outcomes. Impact: This research is significant as it addresses an understudied area by yielding actionable knowledge on how peer navigation can improve research literacy. Developed with stakeholder input, COMPRENDO is innovative for its targeted peer navigation and its implementation evaluation in a multisite RCT to inform scale up. COMPRENDO has the potential to increase clinical trial accrual in Hispanics across the age continuum, enabling the equitable translation of discoveries and therapies, which is a top priority for NCI.

NIH Research Projects · FY 2025 · 2023-07

Project Summary While >90% of TB cases are curable, treatment is dependent on diagnostics based exclusively on the detection of Mycobacterium tuberculosis (Mtb) in patient sputum, despite the limitations of sputum as a diagnostic sample. Sputum is difficult to process, almost impossible to obtain from patients with paucibacillary TB, such as people living with HIV (PLWH) and children and is not diagnostic for patients with extrapulmonary TB. A low- cost, rapid test using blood instead of sputum could transform TB diagnosis for ALL patients and reduce the current diagnostic gap between patients with and without HIV co-infection. Most active TB disease (ATB) is still diagnosed with sputum smear microscopy, despite its low sensitivity and specificity. This lack of diagnostic rigor contributes to the ongoing underdiagnosis of ~3 million TB cases/year, resulting in continued transmission and poor patient outcomes, especially among PLWH in whom mortality is highest. Our long-term goal is to transform TB diagnosis with a blood test that accurately distinguishes patients with ATB from latent TB infection (LTBI), and other pulmonary disorders regardless of co-infection with HIV. The objective of this study is to demonstrate the analytical and clinical performance of a novel assay for rapid diagnosis and treatment monitoring of ATB that exploits the precise molecular affinity of CRISPR and promiscuous nuclease activity of Cas12a to detect ultra-low concentrations of Mtb cell free DNA (cfDNA) circulating in blood. Preliminary analytical studies indicate assay detection limits of <1fg/µL of Mtb cfDNA in blood and no cross-reactivity with non-tuberculosis mycobacteria (NTM) DNA. Preliminary clinical studies including adults and children with presumptive TB, as well as samples from a diagnostically challenging cohort of symptomatic immunocompromised children living with HIV, demonstrated high diagnostic sensitivity and specificity in a pooled adult and pediatric group (>90%), and high diagnostic sensitivity (85%) in children. We will achieve our proposed objective through the following specific aims: AIM 1: Validate a lateral flow version of the CRISPR-TB assay for low resource settings. Our current working assay requires a PCR step and fluorescence reader. Preliminary studies indicate we can significantly simplify this workflow using isothermal amplification and a novel lateral flow (LF) strip for detection of Mtb cfDNA. We will expand on our pilot studies to validate the existing LF assay using contrived and clinical samples. AIM 2: Define the dynamics of the Mtb cfDNA signal for treatment monitoring. Preliminary studies indicate the Mtb cfDNA signal decreases to extinction after treatment initiation but can have non-linear dynamics. We will define the variability and trajectory of the Mtb cfDNA signal in biobanked serial samples from 34 patients treated for drug resistant TB over a year, and 50 prospectively collected patients with drug susceptible TB treated over six months. AIM 3: Determine the clinical sensitivity and specificity of the CRISPR-TB assay in field settings. We will prospectively collect and test blood samples from 450 patients at risk for TB in TB-endemic field settings with high (South Africa), medium (Pakistan) and low (Mexico) HIV burden.

NIH Research Projects · FY 2026 · 2023-07

Over 34 million Americans (~10% of population) have diabetes, 90-95% of which is type 2 diabetes (T2D). T2D is a leading cause of health complications in the US, and minority populations with diabetes are more likely to experience microvascular complications, macrovascular disease, and subsequent death than their White counterparts even when access to care is comparable. The pathophysiology of hyperglycemic organ damage, and why some patients are relatively spared, remains largely unknown. Aggressive glycemic control is known to decrease the frequency of diabetic complications, particularly microvascular, however, few patients are able reach recommended glycemic targets. Inherited variation is known to contribute to the risk of T2D complications. However, genetic associations studies of diabetic complications have only recently begun to reveal the specific genes and pathways responsible for increased susceptibility. While these findings show the promise of this approach, there is an urgent need to better understand the mechanisms by which hyperglycemia leads to organ damage and increase genetic discoveries in diabetic complications. To achieve this goal, we hypothesize that genetics can further enhance the biological insights into diabetic complications by using large-scale sample size, consideration of pleiotropy, environmental modulation and genetic subtyping. The following Specific Aims are proposed to test this hypothesis 1) Genomic and pleiotropy analyses of diabetic complications in 185K subjects with T2D across five racial- ethnic groups; 2) Gene x environment (GxE) interaction analyses of diabetic complications to consider the role of environmental modulation on genetic risk T2D complications in up to 1.3M subjects with and without T2D; and 3) genetic risk profiles and causal inference in diabetic complications to identify causal risk factors and disentangle the relationship between the factors and T2D and its complications. This work has the potential to elucidate the mechanisms of diabetic complications and provide insights into biology and knowledge critical to guide the development of potential clinical predictors, strategies for prevention and guide development of new therapies.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT The human immunodeficiency virus 1 (HIV-1) is the RNA retrovirus that causes acquired immunodeficiency syndrome (AIDS), a disease that has killed over 40 million people worldwide and infected more than twice that. Continued high infection rates has made understanding of HIV biology and vaccines a high priority. A key molecule involved in both infection and vaccine efforts is the HIV-1 Envelope protein (Env). Experimental structural biology techniques have characterized the basic structure of Env, but they are unable to provide details about the extensive N-linked glycan shield nor inform on the flexibility and dynamics of Env. In this proposal, we envision using all-atom molecular dynamics simulations as a `computational microscope,' to provide insights into the dynamics of Env that are unattainable with current experimental techniques. Together with top flight experimentalists, we will develop and simulate a series of models to explore the dynamics of Env as well as its interactions with co-receptors and the cell glycocalyx, in unprecedented detail. In addition, we will use advanced simulation techniques to optimize immunogen design as well as provide critical information about hitherto unseen druggable sites in Env.

NIH Research Projects · FY 2026 · 2023-07

Delineating a parietal-anterior cingulate-claustrum circuit underlying cognitive control and attention Treatments are urgently needed for cognitive dysfunction in psychiatric patients. Given the link between such dysfunction and outcome in patients, large numbers of clinical trials were conducted with companies attempting to be ‘first-to-market’. In the rush however, preclinical studies used had limited validity to the cognitive domains reportedly targeted. Thus, circuit-engagement of the cognitive domain tested was rarely if-at-all verified and all clinical trials to-date have failed. New paradigms have emerged with reported relevance to domains affected in psychiatry, but little opportunity to validate circuits underlying these behaviors, let-alone drugs that modulate such circuits and behavior, have arisen. This application will utilize a circuit-targeted approach to confirm the utility of the 5-choice continuous performance test (5C-CPT) to measure cognitive control and attention across multiple psychiatric disorders. Specific Aim 1 will optimize the touchscreen 5C-CPT for parametric manipulation. The 5C-CPT exists for mice, rats, and humans, with EEG & fMRI versions. The task has always been standard however, primarily in 5-hole operant chambers, but a touchscreen version with parametric manipulations within the task would improve translatability to human testing and enable task performance-based consistency. Backward masking of stimuli have been used in cognitive control tasks previously, but only recently used in human 5C-CPT studies. Here, we will demonstrate that such masked trials enable parametric assessment of 5C-CPT performance in mice. Specific Aim 2 will determine the pharmacological sensitivity of the touchscreen 5C-CPT. After developing the task, it is important to confirm that it is sensitive to manipulations, including those available for use in humans for pharmacological predictive validation. We demonstrated that modafinil improves healthy human participant performance of the standard 5C-CPT, while scopolamine impairs mouse performance. Here, we will confirm that modafinil and scopolamine similarly affect this masked touchscreen 5C-CPT, while predicting that a dopamine D4 receptor agonist would improve cognitive control. We will confirm that modafinil rescues scopolamine-induced deficits, avoiding receptor tautological complications. Specific Aim 3 will confirm the role of the anterior cingulate cortex (ACC)claustrum and claustrumparietal cortex (PC) circuit underlying this masked touchscreen 5C-CPT performance. Consistent with human CPTs, we confirmed the necessity of the PC for mouse 5-choice (5C-)CPT performance. We hypothesize that a ACC to claustrum projection is important during more cognitively demanding trials (from parametric manipulations), while a claustrum to PC projection occurs is important for selecting whether to respond or not during trials. Using fiber photometry and optogenetic techniques, we will confirm both the activation and necessity of this circuit respectively, including changes in activity as a direct result of pharmacological manipulation. Thus, circuitry underlying cognitive control will be identified, as will pharmacological treatments affecting this circuit that are readily testable in healthy human participants.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Molecular mechanisms of bacterial immune signaling through DNA damage The availability of tens of thousands of bacterial genome sequences, plus new bioinformatics tools and new understanding of bacterial genome organization, has enabled the discovery and experimental characterization of dozens of anti-bacteriophage and anti-plasmid defense systems in bacteria. Since a typical bacterial genome encodes 3-6 distinct defense systems, a key question is whether and how these systems can coordinate their activities to synergistically fight an infection. In prior work on the widespread and diverse CBASS (Cyclic oligonucleotide-Based Anti-phage Signaling System) defense systems, we identified two transcriptional regulators – CapW and the two-protein CapH+CapP system – that boost CBASS gene expression in response to DNA damage. Together, CapW and CapH+CapP are associated with ~10% of CBASS systems, and are also found adjacent to a broad range of known and predicted bacterial defense systems including Pycsar, DISARM, and BREX. These findings suggest that CapW and CapH+CapP may mediate activation of antiviral defense in response to a universal signal of cell stress, DNA damage. Here, I will first identify the small-molecule or nucleic acid ligand that binds and activates CapW upon DNA damage. I will combine biochemical assays for CapW binding to both its target DNA and its ligand with x-ray crystallography to characterize the conformational changes imposed by the ligand to control CapW-DNA binding. This work will establish a mechanism for CapW, a widespread bacterial transcription factor. Next, I will test the idea that CapW and CapH+CapP mediate cooperation between antiviral defense systems by sensing DNA damage. Specifically, we hypothesize that DNA-targeting immune systems like restriction-modification and CRISPR-Cas create DNA damage that is sensed by CapW or CapH+CapP to activate a secondary defense system (CBASS or others) to reinforce the defensive response. I will systematically test this model by infecting cells encoding both a restriction-modification system and a CapW- or CapH+CapP-associated CBASS system to determine if the combination of these systems yields synergistic antiviral immunity. Additionally, I will test whether DNA damage sensing plays a role in defense-system synergy, using structure-based mutations to either CapW or CapP that eliminate DNA damage sensing. Together, these experiments will reveal the molecular mechanism of CapW, and the role of DNA damage sensors in mediating synergy in bacterial defense systems. The findings have the potential to establish a new paradigm in which DNA targeting defense systems constitute a first line of antiviral defense, and DNA damage-activated systems constitute a second line of defense with orthogonal mechanisms. Thus, instead of viewing bacterial defense systems in isolation, this work will establish how they cooperate to compose a comprehensive bacterial “immune system”.

NIH Research Projects · FY 2024 · 2023-07

Project Summary. While recent efforts have identified the need for methodologies capable of inactivating oncoproteins, current intervention strategies have left much of the proteome “undruggable.” In response the NCI has recognized this challenge as outlined in PAR-22-216 by incentivizing the development of new molecular targeting agents based on specific signaling pathways activated during the process of tumorigenesis or tumor progression. This program proposes an important next step in reaching these goals by providing a platform for the rapid development and characterization of heterobifunctional molecules capable of inducing degradation of cancer related metalloenzymes with new molecular targeting agents. In this program, our team will develop proteolysis targeting chimeras (PROTACs) containing state-of-the-art metal-binding pharmacophores (MBPs) with the ultimate goal of achieving isoform-selective degradation of jumonji C-domain containing lysine demethylases. Current strategies to inhibit conserved catalytic domain Jumonjis (JMJCs) involve targeting the α-ketoglutaric acid (2OG) substrate-accepting active site of JMJCs with inhibitors that can coordinate to the iron ion in the active site. Targeting the histone-binding helper domain of JMJCs generates additional isoform selectivity. However, no inhibitor has been shown to be selective for only one isoform of JMJCs. We have chosen to take a targeted degradation approach in order to increase the surface area of the peripheral interaction by recruiting an E3 ligase. This program will demonstrate how the potential protein- protein interaction induced by these chimeras can be leveraged to induce selective degradation of metalloproteins (`MetalloPROTACs') through rational MBP and linker design and will serve as a platform for the study of cancer biology, as well as laying the foundation for future development of therapeutic agents.

NIH Research Projects · FY 2024 · 2023-07

Abstract Treatment resistant depression (TRD) is present in about 2.5 percent of the population and is associated with considerable personal suffering and societal costs. Repetitive transcranial magnetic stimulation (rTMS) is a safe and effective treatment for TRD that is increasingly being used in clinical practice across the world. However, while there is convincing evidence for the effectiveness of rTMS treatment, the overall remission rate achieved in patients is typically considerably less than 50%. Coupled with the fact that a course of rTMS takes considerable time to administer (up to 6 weeks), this limited efficacy substantially impacts clinical utility. Intermittent theta burst stimulation (iTBS) is a new form of TMS that involves the patterned application of stimulation pulses in both the theta and gamma frequencies. iTBS has recently been shown to be non-inferior to standard rTMS but can be applied in a dramatically shorter time, typically three minutes per day compared to 20-40 minutes. However, the use of iTBS has not been shown to enhance overall treatment efficacy compared to rTMS. Several methods are under investigation to try to improve the efficacy of iTBS treatment, in part through the individualization of treatment parameters. In this study, individualization of iTBS will include frequency and location optimization. Frequency individualization involves stimulating at each patient’s endogenous brain rhythms derived from theta- gamma coupling during a working memory task. Our findings suggest that such frequency individualization of iTBS produces greater effects on neuroplasticity, cognitive function, and mood enhancement in healthy subjects. Next, we have developed methods to individualize coil placement using electric field (E-field) modelling to limit anatomic variability and maximize dosing for each patient. We will aim to individually target the TMS e-field to a specific cortical region corresponding to Talairach coordinate (–45, 45, 35) in the left dorsolateral prefrontal cortex (DLPFC). We also previously reported that targeting this region with rTMS produces superior efficacy compared to conventional targeting. We now propose to undertake a 2-phase program to determine if individualized iTBS based on frequency and E-field modelling will enhance its efficacy in patients with TRD. In the R61 phase of this application, we will test if this novel approach produces target engagement by randomizing 75 patients with TRD to a course of frequency and location individualized iTBS, location individualized iTBS only, or standard iTBS stimulation. We aim to establish whether individualized frequency/location iTBS produces a greater change in theta connectivity as a demonstration of its capacity to alter activity in relevant frontal–parietal circuitry. If this is established, we will have achieved our go-criterion and will request to proceed with the three- year R33 phase. During this phase, we will conduct a larger scale randomized controlled trial comparing individualized frequency/location iTBS to standard iTBS in 144 patients with TRD and compare the effects on both target engagement (fronto-parietal theta connectivity) and clinical outcomes (depression severity). The overall goal of this research is to develop a more effective form of iTBS treatment for patients with TRD.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY In malignancies, CD8+ T cells can recognize and eliminate tumor cells, but often fail to cure disease due to their progressive loss of antitumor function resulting from chronic activation in the immunosuppressive tumor microenvironment. In response to infection in healthy tissues, T cells differentiate into tissue-resident memory cells (TRM), and after clearance of antigen can remain lodged in tissues to survey and provide protection from reinfection. When TRM-like T cells are found in cancer patient tumors, improved responses to immunotherapy and better patient outcomes are observed. However, whether TRM-like TIL represent ‘progenitors’ of exhausted TIL, or are a separate cell state outside the exhaustion spectrum is still unclear. To better understand the relationship among TRM-like TIL, exhaustion states, and TRM memory cells, we directly compared TRM from acute viral infection and exhausted TIL from tumors to find transcriptional differences between these distinct T cell states. Focusing on genes highly expressed by TRM that may mediate their enhanced functions in tissues, we asked which of those were downregulated as T cells became terminally exhausted, coincident with loss of function. This approach identified numerous genes related to protein regulation, including multiple under-characterized E3 ubiquitin ligases. Protein regulation by the ubiquitin proteosome system is an essential biological process for homeostasis, crucial for cell differentiation and function, and has been previously shown to be important for memory T cell identity. Correlated with our preliminary data, we found that exhausted TIL have an excess of unfolded proteins in their cytosol, and when we enforced expression of the identified E3 ubiquitin ligases in tumor-specific T cells, it allowed for better tumor control and improved mouse survival. Therefore, we propose to explore the relationship between protein homeostasis and TRM, exhaustion, and TRM-like TIL cell fates. Aim 1 seeks to understand if the identified E3 ubiquitin ligases influence exhaustion or TRM cell fate by using mouse models of cancer to enforce expression or knock out these ligases and study exhaustion T cell fate, or to enforce expression or knock out the ligases in acute viral infection and study TRM cell fate. Aim 2 seeks to identify the protein-interaction targets of these E3 ubiquitin ligases by in vitro BioID proximity labeling assay and mass spectrometry, then verify the targets in vivo TIL and determine if expression of these ligases in TIL can decrease unfolded protein abundance. Understanding the relationship between T cell exhaustion and TRM can provide vital new insights into the biology of these two differentiated T cell populations and inform efforts to manipulate T cell fates towards TRM-like TIL to benefit cancer immunotherapy.

NIH Research Projects · FY 2024 · 2023-07

Project Summary The proposed research aims to investigate the role of endolysosomal damage in ischemic brain injury. Recently, significant progress has been made in understanding the endolysosomal system, previously known as the lysosomal system. This endolysosomal system now includes three basic structures: (i) late endosome (LE), (ii) lysosome (L), and (iii) endolysosome/autolysosome (EL/AL). The LE receives incoming endolysosomal proteins (e.g., intraluminal cathepsins and structural proteins) from the Golgi apparatus and waste cargos from the endocytic and autophagic pathways. Although the LE contains both cathepsins and waste cargos, the LE’s cathepsins cannot efficiently degrade these waste cargos due to LE’s less acidic pH (6.0). The LE must fuse with the more acidic lysosome (L) (pH 4.0-4.5) to become a hybrid endolysosome (EL) to efficiently degrade these waste cargos. This rate-limiting LE-to-L fusion step is mediated by the N-ethylmaleimide sensitive factor ATPase (NSF)-dependent machinery. Our recent studies show that brain ischemia leads to NSF deficiency in neurons destined to die. We generated a neuron-specific NSF deficient mouse line to understand the role of NSF deficiency in the endolysosomal damage observed after both global and focal brain ischemia. In NSF-deficient mice (absence of ischemia), there is a prominent buildup of abnormal “multi-aggregated” endolysosomal structures, followed by autonomous neuronal death. This phenotype replicates the same neuropathological features observed in the wildtype (wt) littermates after both global and focal brain ischemia. Neuronal cathepsin B (CTSB) release is another key neuropathological feature observed in both our NSF- deficient mice without ischemia and the wt littermates after both global and focal brain ischemia. Most cathepsins have low or no activity at neutral pH, but CTSB uniquely exhibits endopeptidase activity at neutral pH. CTSB is the most dominant cathepsin in neurons. Our recent studies further show that conventional (all tissue) CTSB knockout (KO) in mice significantly protects the brain in a mouse focal brain ischemia model. Based on these new results, we hypothesize that post-ischemic NSF deficiency leads to endolysosomal damage and causes a large quantity release of its contents, e.g., CTSB, into the cytoplasm and extracellular space. This large quantity release of CTSB leads to ischemic brain injury. We will test this novel hypothesis by investigating: (i) how post- ischemic NSF deficiency leads to endolysosomal damage using neuron-specific NSF deficient mice without brain ischemia and their littermates subjected to brain ischemia (Aim 1); (ii) if NSF overexpression in transgenic (tg) mice can reduce both endolysosomal damage and brain ischemic injury (Aim 2); (iii) whether and how neuronal CTSB release leads to ischemic brain injury using neuron-specific CTSB KO mice (Aim 3); and (iv) the role of microglial and macrophage CTSB in prolonged post-ischemic inflammation using microglia and macrophage- specific CTSB KO mice (Aim 4). These studies will facilitate the development of novel therapeutics that target endolysosomal damage and potentially restores NSF activity after brain ischemia.

NIH Research Projects · FY 2024 · 2023-07

Rigorous biocontainment prevents laboratory mice from being infected with natural pathogens and allows researchers to perform animal experiments in a reliable and reproducible manner. However, such specific pathogen-free (SPF) mice do not necessily model immune responses that occur in nature, and this may contribute to the often-reported disconnect between pre-clinical research and human clinical trials. Athough many key advances in biomedical research have been made possible by the study of SPF mice, there is now compelling evidence that microbial or viral experience can greatly impact immune pathophysiology. In past two decades, regulatory T (Treg) cells have emerged as a dedicated immune population crucial for the negative regulation of immune responses. Considering that Treg cell homeostasis and function can be greatly influenced by unique signals present in different tissue environments, it is conceiable that the microbial exposure outside the current laboratory setting could heavily impact Treg cells and their ability not only to establish immunological tolerance against ‘‘self’’ or innocuous foreign antigens, but also to keep in check effector immune responses to pathogens. To gain a better understanding of Treg cell-mediated immune regulation in a more physiological setting, we have worked closely with the Animal Care Program at UC San Diego to establish a new protocol to generate, maintain, and extensively characterize a stable repository of physiological microbe-exposed environmentally conditioned (PC) animals. By taking this approach, two well-establsihed mouse models that affords Treg cell isolation and Treg cell-specific ablation will be first adapted to the “dirty” condition. Next, through performing extensive comparative immune phenotype analysis and single-cell sequencing studies of Treg cells isolated from both lymphoid and non-lymphoid tissues in SPF mice and PC mice, the impact of physiological microbial exposure on Treg cell biology will be determined. Finally, we will examine the suppressor function of Treg cells from PC mice to maintain immune homeostasis and to control ongoing inflammation in comparison with their SPF counterparts. Together, our study will not only further extend our fundamental knowledge of Treg cell-dependent immune regulation but also provide critical insights into the possibilities and limitations of targeting Treg cells to treat a wide array of human immunological diseases. .

NIH Research Projects · FY 2024 · 2023-07

Project Summary Presentations of respiratory failure can vary from mild requiring supplemental oxygen to more severe requiring invasive mechanical ventilation (i.e., acute respiratory distress syndrome or ARDS). Early in respiratory failure patients often have abnormalities in gas exchange, control of breathing, and/or pulmonary mechanics. During the ongoing COVID-19 pandemic, some studies have demonstrated that increased respiratory drive is associated with worse clinical outcomes while others have demonstrated contrary findings. We have also demonstrated that a noninvasive assessment of gas exchange using the alveolar gas meter (AGM) is predictive of who will require supplemental oxygen from COVID-19 infection. Additional studies predating the COVID-19 pandemic showed that increased inspiratory effort, large transpulmonary pressure changes, and elevated dead space were all associated with worse outcomes in ARDS. However, many of these studies focused on patients already intubated and mechanically ventilated. Less is known about the relevance of these parameters in spontaneously breathing individuals. For a few years now the notion of patient self-inflicted lung injury (P-SILI) has been gaining traction in the medical literature. The basic concept is that patients with impending respiratory failure from existing lung injury can cause worsening damage to their lungs. If patients are spontaneously breathing with excessively high respiratory drive, then they may generate excessively negative inspiratory efforts leading to major transpulmonary pressure changes. The results of these abnormalities are large tidal volumes like mechanically ventilating someone with injurious tidal volumes prior to the advent of low tidal volume ventilation (LTVV). Also, retrospective review of large cohort studies and randomized controlled trials of ARDS patients have demonstrated that spontaneously breathing patients prior to intubation often generated excessively large tidal volumes. Whether their subsequent lung injury was entirely due to their underlying ARDS pathology or partially due to superimposed P-SILI is not known. If a physiological signature of impending respiratory failure in spontaneously breathing individuals exists and it is associated with elevated respiratory drive, steps can subsequently be taken to mitigate the increased drive and study the longitudinal effects of these variables in patients who develop respiratory failure. Moreover, if such a signature exists and intervention abrogates some of the subsequent lung injury, this is a strong proof of concept for P-SILI in vivo. We aim to test three independent but related hypotheses. First, the AGM is a novel and simple technique which can be used to identify patients at increased risk for respiratory failure better than traditional metrics. Second, the breath hold maneuver is a validated technique to assess control of breathing that can identify patients with poor respiratory prognosis. Third, the airway occlusion pressure will provide additional predictive value in identifying high risk patients. This proposal will lay the groundwork for the PI to gain experience and training for a long and productive academic medical career.