University Of California, San Diego

NIH Research Projects · FY 2025 · 2016-05

While tremendous advances in pharmacotherapy for adults have been achieved in recent years, expanding the benefits of these new therapies to infants and children remains a significant challenge. Important developmental changes in pharmacokinetics, pharmacodynamics, disease presentation and progression all impede direct translation of adult therapeutics into pediatrics. The discipline of pediatric clinical pharmacology provides necessary training in developmental physiology to leverage existing knowledge and guide rational therapeutics for infants and children. However, the current pool of pharmacologists and pediatric sub-specialists with formal training in clinical pharmacology is both small and aging. Additional pediatric clinician-scientists with training in clinical pharmacology are urgently needed to ensure that therapies are optimized for infants and children. In 2015, under the leadership of Edmund Capparelli, PharmD (expert in pediatric pharmacokinetic (PK) modeling) and Adriana Tremoulet, MD, MAS (expert in pediatric clinical trials), training in pediatric clinical pharmacology at UC San Diego was formalized as a NICHD-supported T32 Training Program. We are proud of this first iteration of a formal program to provide cutting-edge research training in pediatric clinical pharmacology. We now propose a renewal of our T32 pediatric clinical pharmacology training program, entitled “Academic Training in Therapeutic Advancement for Child Health” (ATTACH). In this renewal, we proudly seek to (1) modernize our program with new program leadership, (2) recruit more world-renowned faculty to teach our fellows state-of-the-art technologies, (3) launch a systematic tool to assess performance of our program and assist in program improvement, (4) emphasize trainee and faculty mentor training, and (5) enhance recruitment methods to attract a more diverse pool of applicants and trainees to stimulate diversity in the workforce. Our key objectives of the first cycle of our training program will remain the same: (1) To increase the number Pediatric clinical pharmacologists and Clinician-Scientists engaged in clinical pharmacology research as applied to child health; (2) to attract outstanding young pediatric clinicians to UCSD and to expand their translational capacity through clinical pharmacology methods; (3) to facilitate career development of ATTACH trainees under the guidance of world class, established investigator-faculty mentors; and (4) to cultivate the early careers of women and minority investigators in pediatric therapeutics. With our impressive group of mentors with outstanding training track records, UCSD ATTACH Fellows will be poised to be tomorrow's leaders in pediatric clinical pharmacology.

NIH Research Projects · FY 2025 · 2016-04

This project leverages a cyanobacterial model system to answer the following questions: what are the molecular interactions that mark the passage of time in a cell, where do they occur in the cell, how do they mediate temporal regulation of events, and why does biological timing matter for fitness? The circadian biological clock is an oscillatory timer that drives 24-h rhythms of biological activities. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. However, the circadian clock is pervasive well beyond mammals, promoting fitness in diverse organisms throughout the phylogenetic tree. The circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. A new in vitro preparation comprising the oscillator proteins KaiA, KaiB, and KaiC, along with the kinases CikA and SasA and the transcription factor RpaA, reconstitutes the circadian rhythm of binding of RpaA to its target promoter with a real-time readout. This project will apply the in vitro clock and other technical and conceptual advances towards biochemical, cytological, genomic, and physiological objectives that will answer the target questions. The in vitro clock will reveal the molecular events that occur when the clock resets to an environmental timing cue, identify the sites of action of nucleotides that modulate the timing circuit, and determine how RpaA and a second transcription factor that is regulated by environmental signals, RpaB, work together to influence circadian phasing. The discovery that the kinases SasA and CikA impart tolerance to fluctuating oscillator component concentrations will overcome past hurdles for establishing a circadian circuit in Escherichia coli as a naïve model system for exploring clock connections to cellular physiology and for biotechnology applications. High-resolution cryo-electron tomography and focused ion-beam milling will be used to visualize clock-controlled daily changes in intracellular organization and the clock complex itself. The molecular basis and fitness advantage of circadian control of natural transformation will be determined. A bar-coded transposon library first used to identify all genes required for photoautotrophic growth will be used to identify new loci that contribute to fitness in a day-night cycle. Paired with physiological and metabolic assays, these experiments will answer the question: why does the timing of molecular events matter? Together, these approaches will elucidate clock mechanisms and the value of the clock to diurnal physiology, and will advance biotechnological opportunities for controlling metabolism in both photosynthetic and traditional bacterial production systems.

NIH Research Projects · FY 2026 · 2016-04

PROJECT SUMMARY/ABSTRACT The ability to non-invasively predict or monitor early patient response to cancer therapy will have significant implications for patient care with the potential to spare non-responding patients the high morbidity and cost associated with failed treatments. Due to its wide accessibility, lack of radiation, relatively low cost, and excellent visualization of the liver, ultrasound is a demonstrated and potentially ideal modality to repeatedly image and monitor patient responses over the course of therapy. In the first phase of this work, we demonstrated that current 2D dynamic contrast-enhanced ultrasound (DCE-US) is fundamentally limited in quantification due to tumor heterogeneities, resulting in consecutive sampling errors with substantial over- or underestimation of treatment response in patients. We then showed that these limitations could be overcome by a novel 3D DCE-US imaging approach using innovative matrix array transducer technology to provide motion-compensated, accurate quantitative and volumetric assessment of tissue perfusion of liver metastases. In this competitive renewal proposal, framed as an industry-academic partnership with Philips who is committed to our proposal, our aim to clinically advance 3D multi-parametric tissue and perfusion characterization with ultrasound (3D MPUS) to predict or monitor early response in patients with metastatic colorectal cancers to the liver (mCRC). For this, we have proposed a set of specific aims where we will: i) work with Philips to enhance 3D DCE-US data acquisition to overcome previously reported limitations, enable simultaneous acquisition of raw RF/Bmode data for tissue characterization, and develop an analysis software suite for MPUS quantification, ii) carry-out a multi-site clinical feasibility, acquisition repeatability and quantification reproducibility assessment of 3D MPUS in 300 patients, and iii) determine whether 3D MPUS quantification can predict or monitor early therapy response. For this, baseline and early changes of tumor perfusion and tissue parameters (obtained before and at 2-3 weeks after initiation of therapy) will be correlated to treatment response. Treatment response as defined on abdominal radiological scans acquired at 2 months following treatment initiation will be determined using standard RECIST 1.1 reporting. Our proposal builds on the recent FDA approval of ultrasound contrast agents for liver tumor imaging, increased clinical availability of commercial 3D US abdominal probes, and our initial clinical validation of 3D DCE-US and extensive experiences in QUS.

NIH Research Projects · FY 2024 · 2016-04

Following a decade of significant strides forward, the global malaria eradication agenda has stalled, due in part to the accelerating emergence of insecticide-resistant mosquitoes and drug-resistant malarial parasites. The World Health Organization and others have called for the development of new strategies to help defeat this devastating disease that infects over 2 million people and killing over 400,000 annually, predominantly young children in impoverished regions. Gene-drives, which can bias inheritance of desired traits, offer a novel and promising strategy either to eliminate disease causing insect vectors, or to immunize them against pathogens. Such super-Mendelian CRIPSR-based gene-drive systems encode bipartite transgenic cassettes consisting of the Cas9 endonuclease and a guide RNA (gRNA), which directs DNA cleavage at the genomic site of insertion. In reproductive cells, such targeted cutting of the homologous chromosome results in copying the drive element at the cleavage site through homology directed repair, resulting in nearly all progeny inheriting the drive element and its cargo. My group has contributed to developing the first CRIPSR-based gene drive (or active genetic) systems in flies, mosquitoes, mammals, and bacteria. We also pioneered allelic-drive systems designed to bias inheritance of a favored allelic variant at a separate genetic locus. In addition, we have developed, and extensively tested, two types of self-copying drive neutralizing systems, both of which carry gRNAs, but no source of Cas9. ERACRs delete and replace gene-drives, while e-CHACRs copy themselves while mutating and inactivating the Cas9 transgene carried on a gene-drive. Small population cage experiments in flies and mosquitoes have shown that highly efficient gene-drives rapidly spread through target populations, and that ERACRs and e-CHACRs can reliably replace (ERACRs) or halt (e-CHACRs) a gene-drive element. In this grant, we propose first to develop a flexible two-component (split-drive or CHACR) system that can be genetically converted (or hacked) into a single full-drive system. The split and full drive elements are inserted into genes essential for viability or reproduction, and also carry recoded cDNAs of the targeted genes to restore function of those loci. These recoded systems benefit greatly from a phenomenon we discovered and refer to as lethal/sterile mosaicism, which dominantly eliminates loss-of-function alleles (mistakes) in the target gene generated by imprecise DNA repair events rather than the intended copying event. Next, we will develop and test next-generation ERACR and e-CHACRs able to eliminate or halt our recoded-drives, and also test a self-limiting drive system that slowly targets Cas9 for mutagenesis. In parallel to these drive experiments, we will delve into the mechanisms and timing of the drive process using a unique set of image-based genetic elements we have developed. We anticipate that the intellectual advances and implementable game-changing technologies from these studies will contribute importantly to solving critical global challenges in human health.

NIH Research Projects · FY 2026 · 2016-04

Today, 20% of adolescents and 26% of young adults have a diagnosable internalizing (e.g. anxiety, depression) or externalizing problem (e.g. impulsivity, aggression, conduct disorders). Neurodevelopmental disabilities and mental health disorders in adolescents and young adults have increased over the last two decades worldwide, and environmental contaminants may explain a part of this increase. The most used insecticides worldwide include organophosphates (OPs: inhibitors of acetylcholinesterase (AChE)), pyrethroids and neonicotinoids. Toxicological and epidemiological evidence links OP exposure with altered neurocognitive performance. Additionally, a small number of studies have described associations with internalizing symptoms and some externalizing behaviors (i.e. low inhibitory control). It is plausible neonicotinoids may also affect brain processes as they share some toxicological mechanisms like OPs and emerging findings indicate pyrethroids may affect brain processes and neurobehavior. Further, emerging studies suggest that pesticides may transiently affect cognitive performance and mood. Assessing transient health outcomes is complex, which has led to limited knowledge on this field. We propose a renewal of our 2016 study of adolescents and young adults (ages 18-23 years in 2022, n≈554) and builds on the planned 2022 follow-up exam of the NIH-funded Study of Secondary Pesticide Exposures among Children and Adolescents (ESPINA). The proposed study incorporates a novel mobile health assessment tool to evaluate the real-world neurotoxic effects of pesticides. ESPINA is a study established in 2008 of participants residing in Pedro Moncayo, Ecuador, a county with one of the highest concentrations of flower plantations in the Americas. Leveraging our smartphone-based ecological momentary cognitive testing (EMCT) platform, we will assess whether recurring and seasonal pesticide exposures in adolescence and young adulthood affect cognition and mental health. Using a burst sampling design 5 times/year for 2 years during high- and low-pesticide spray seasons, we will test the hypotheses: 1) pesticide spray seasons are associated with cyclical alterations in internalizing symptoms, externalizing behaviors, and cognitive performance assessed using EMCT; 2) pesticide exposures (AChE inhibition and OP and pyrethroid urinary pesticide metabolites) assessed during 5 agricultural periods are related to elevated depressive and externalizing symptoms, and decreased cognitive performance assessed using EMCT; 3) Urinary pesticide metabolites (OP, neonicotinoids, pyrethroids) are associated with increased mental health symptoms from 2016 to 2022 when all participants were adolescents, and whether age, sex and COVID-19-related factors (e.g., exposure; psychosocial stressors) modify the associations between environmental exposures and mental health. Identifying cognitive and mental health sequelae related to agricultural pesticide spray seasons on people living in agricultural settings will advance this understudied field of research and has significant implications for public health.

NIH Research Projects · FY 2025 · 2016-04

PROJECT SUMMARY / ABSTRACT The overall objective of the T32 “Translational Vision Research Training at UCSD” program is to train postdoctoral fellows to be become outstanding translational vision research scientists. The program builds on the exceptional training record of faculty from the University of California San Diego School of Medicine (ranked among the top 10 institutions for NIH Training grants) in general, and the Shiley Eye Institute in particular who have mentored scientists to translate basic science results into improved clinical care. Moreover, UCSD is highly collaborative with strong programmatic support for the translation of vision research from bench to bedside in areas such as genetics/proteomics, clinical research, bioengineering, ophthalmic imaging and image analysis, drug delivery, and data science, biomedical informatics and artificial intelligence. The short-term objectives of the T32 Training Grant program are to provide 2 postdoctoral fellows each year with training and instruction by mentors who direct NIH-funded laboratories. The program will provide a coordinated framework for the recruitment and training of postdoctoral fellows in three trainings tracks that focus on the Shiley Eye Institute and UCSD faculty's core strengths, (1) clinical/human subjects research and (2) genomics/proteomics and 3) data science/informatics/artificial intelligence. Each Training Track has an integrated mentor group, with an outstanding track record in training full-time postdoctoral scientists, and National Eye Institute support for their research. Twenty-two faculty mentors representing 11 UCSD departments, the Institute of Genomic Medicine and the new Halıcıoğlu Data Science Institute will participate in the program. The program benefits from resources provided by the Clinical Translational Research Institute, Vision Research Center core grant, the UCSD Supercomputer Center. The multi-disciplinary vision research training experience includes didactic instruction appropriate to the area being studied, research experience with a lead mentor as well as professional development seminars, lectures and journal clubs in various disciplines. Instruction in research ethics and leadership, laboratory management, and grant writing will be required. To oversee the training program, solicit and review applications, and monitor/facilitate progress through each postdoctoral fellow's Individual Development Plan (IDP), the administrative structure of the training program includes a Program Director, Linda Zangwill, PhD, co-Director, Radha Ayyagari, PhD and an Executive Committee. In addition, an External Advisory Committee provides independent evaluation of the training program. Every postdoctoral fellow in the T32 Translational Vision Research Training Program at UCSD will be expected to exit well prepared for a vision research career in academia or industry.

NIH Research Projects · FY 2026 · 2016-04

This application proposes to continue to develop, implement, and evaluate the highly successful Sustained Training on Aging and HIV Research (STAHR) research training program, which has the goal of expanding the pool of NIMH-funded new investigators performing research of HIV, aging, and mental health. Since the initiation of the program in 2015, the lifespan of people with HIV (PWH) has continued to improve but the healthspan has not improved to the same extent, raising concerns about premature aging. Many key research questions regarding the complex relationship between HIV, aging, and the brain remain unanswered. This emerging field encompasses issues of physical and psychiatric disease, polypharmacy, cognitive impairment, and psychosocial stressors on the one hand, and positive aspects such as resilience and neuroplasticity on the other. Much remains to be learned about mechanisms and interventions for preventing and treating mental illnesses, including cognitive and mood disorders, in older PWH. Fostering the development of researchers with knowledge and expertise in both aging and HIV is critical for the promotion of urgently needed research in this field. The STAHR program builds on the infrastructure and expertise available at two major research programs at the University of California, San Diego (UCSD, the Stein Institute for Research on Aging and the HIV Neurobehavioral Research Program) and addresses several priorities of the NIMH Division of AIDS Research and the NIH Office of AIDS Research. The program targets clinical and translational postdoctoral fellows and junior faculty from across the country who have experience in aging or HIV research, but are interested in gaining expertise in the intersection of these two factors. Four new scholars are selected each year and are matched with a primary mentor at their home institution and a co-mentor at UCSD. Training goals are to increase knowledge and skills in HIV and aging mental health research and support career development, with mentoring being a key focus. Each scholar works with their mentors to develop an Individual Development Plan. Although many of the training activities are performed remotely throughout the 3-year program, we have all scholars and mentors convene annually for a 2-day Workshop at UCSD when public health allows. First year scholars also attend a 4-week Summer Institute that will be part in-person at UCSD and part remote. In its first funding cycle, the STAHR program trained a diverse group of 20 early-career scientists (50% underrepresented minorities). The program’s success thus far is evidenced by the impressive track record of publications, awarded grants, career milestones and the positive program evaluations. Thus far, the program has grown its network of mentors across the U.S., which has supported trainee development and fostered collaborations. With this renewal, the network will continue to grow and serve as a resource for trainees during and after the program. Overall, STAHR strives to develop high quality independently funded investigators, who will make lasting contributions to mental health research in HIV and aging.

NIH Research Projects · FY 2025 · 2016-04

Summary Aortic aneurysms and dissections are the most serious and deadly manifestations of Marfan Syndrome (MFS), and current therapies to prevent aortic dilation are only moderately effective. MFS is caused by mutations or deletions in fibrillin-1 (Fbn1), a component of extracellular microfibrils, which surround and connect elastic fibers to smooth muscle cells (SMCs) in the aortic media. Reduced fibrillin function alters signaling between extracellular matrix and SMCs, resulting in SMC apopotosis and extracellular matrix degradation. In addition, the altered signaling leads to increased production of reactive oxygen species (ROS) and nitric oxide (NO), increasing oxidative and nitrosative stress and activating protein kinase G (PKG) via the NO/cGMP pathway. Although the increased ROS, reactive nitrogen species (RNS), and PKG activation contribute to aneurysm formation in MFS, neither the sources of ROS/RNS nor the effects of oxidative/nitrosative stress on SMC functions are fully understood. The vitamin B12 analog cobinamide, on which we hold several patents, is a strong and versatile antioxidant that can neutralize ROS and RNS, including NO. During the last grant period, we showed that mice with an activating PKG1 mutation (Prkg1R177Q) that causes thoracic aneurysms and dissections in humans, develop aortic dilation associated with increased oxidative stress and media degeneration⸺elastic fiber fragmentation, increased matrix metalloproteinase activity, media fibrosis, and SMC apoptosis; cobinamide treatment completely prevented these changes. Preliminary data show that cobinamide also reduces aortic dilation and prevents elastic fiber fragmentation and SMC apoptosis in a mouse model of MFS (Fbn1C1041G/+), while reducing markers of oxidative stress and excess PKG signaling. We hypothesize that increased ROS/RNS combined with PKG activation from increased NO synthase (NOS2) drive abnormal SMC functions and aortic pathology in MFS, and that an optimized dose schedule of cobinamide will prevent aortic dilation and improve survival in mice with MFS, especially when combined with a -blocker. In Aim 1, we will determine the mecha- nisms and consequences of excess ROS/RNS generation in human fibrillin1-deficient or mutant (iPSC-derived) SMCs, using shRNA knockdown and pharmacological approaches to inhibit ROS/RNS-generating enzymes and PKG in vitro. We will assess contributions of excess NO synthase and PKG activity to the progression of aortic disease in vivo, by inducing SMC-specific knockout of NOS2 or PKG1 in Fbn1C1041G/+ mice. We will also test whether ROS detoxification by SMC-specific catalase overexpression ameliorates aortic pathology. In Aim2, we will determine the optimal cobinamide dose and starting time to prevent aortic dilation and death from aortic dissections in mice with moderate (Fbn1C1041G/+) and severe (Fbn1mgR/mgR) MFS, respectively. In addition, we will combine cobinamide with the β-blocker propranolol, because cobinamide prevents aortic media degeneration, while propranolol reduces mechanical stress without affecting degenerative changes in the media. These studies could lead to considerably improved treatment of the aortic disease in patients with MFS.

NIH Research Projects · FY 2025 · 2016-04

There is abundant evidence that obesity confers increased risk for at least 13 forms of cancer. The incidence of breast, colon, and liver cancer are all increased in obese populations, and the epidemiologic evidence for the obesity-breast cancer connection is particularly strong. One in eight women will be diagnosed with breast cancer during their lifetime. Breast cancer incidence increases approximately 10-fold for women over the of age 60, compared to age 50 or younger. This increase in breast cancer risk is associated with an increase in obesity. Indeed, obesity increases the risk of triple-negative breast cancer in premenopausal women and estrogen receptor positive breast cancer in postmenopausal women. A rarer form of inflammatory breast cancer is dramatically increased (up to 5-fold) in both groups. More importantly, obesity shortens disease-free survival in both pre- and postmenopausal women. Patient mortality in breast cancer is primarily caused by distant metastases. Obesity at the time of diagnosis is associated with increased risk of distant metastasis and mortality. Studies in rodents have confirmed these relationships, showing that dietary-induced obesity and high-fat diets lead to increased incidence and growth of tumors in oncogene and carcinogen-induced breast cancers. Despite this body of correlative evidence, the mechanisms of obesity-induced breast cancer risk remain poorly understood. One possibility is that the obesity causes insulin resistance in the liver and compensatory elevation in circulating insulin to control glucose levels. At the same time, other tissues, including tumors, may not be insulin resistant and so are exposed to increased insulin signaling. Indeed, we have shown that reducing insulin resistance by treating with omega-3 fatty acids reduces breast cancer growth in mice. We have also shown that time-restricted feeding (TRF) versus unrestricted feeding of a high-fat diet improves insulin resistance despite sustained obesity and equal caloric intake. Furthermore, we showed that TRF inhibited obesity-driven breast tumor growth and corrected tumor circadian rhythms, and that the TRF impact on tumor growth was mediated by reducing insulin levels. A number of important questions remain unanswered. Firstly, how does insulin drive tumor growth? Is it a direct effect on the tumor cell, or on the microenvironment? Secondly, does correction of the circadian rhythms in the tumor cell by TRF contribute to the reduced tumor growth? Thirdly, how do nutrients and insulin entrain the circadian clock in tumors? Due to the link between obesity, insulin resistance and breast cancer in pre- and postmenopausal women, and the translational potential of time-restricted feeding, we will investigate the effect of deleting the insulin receptor, mTORC1 signaling, or components of the circadian clock in tumor cells to test whether loss of these signals alters tumor growth in vivo and the response to TRF. We will also test whether TRF enhances chemotherapy to inhibit tumor growth. Accumulating evidence from TRF-related clinical studies support the translational relevance of our proposal. Translational, mechanistic findings from these studies will impact on breast cancer prevention and therapy.

NIH Research Projects · FY 2026 · 2016-03

PROJECT SUMMARY Colonic epithelial wound healing is the primary therapeutic endpoint for inflammatory bowel disease (IBD), but a fundamental and unified understanding of wound healing mechanisms remains elusive. In the initial cycle of funding for this project, we developed and leveraged a three-dimensional tissue imaging technique (called Deep Mucosal Imaging) to reconstruct, in an unbiased manner, the quantitative evolution of colonic epithelial lineages through different stages of wound healing in murine acute colitis. These studies demonstrated that the injury-associated relaxation of homeostatic crypt boundaries facilitated the specification and clonal expansion of an injury-induced stem cell population that we refer to as founder progenitor cells (FPCs). These colonic FPCs represent primitive proliferative cells and are not marked by known markers of reserve or revival intestinal stem cells. However, each FPC has the clonogenic potential to regenerate 10-100 crypts. Thus, understanding the primary mechanisms through which FPCs are specified will reveal important new knowledge about the basic parameters of colonic epithelial wound healing, which could ultimately hold translational potential for patients with IBD. Our goal is to define the molecular and cellular pathways through which FPCs are specified. In preliminary studies, we found that both mucosal basal and superficial cells contributed to the FPC pool. Furthermore, profiling of FPCs shows enrichment of spliceosomal transcripts, suggesting a role for alternative splicing in specifying this highly regenerative cell population. Based on these findings, our hypothesis is that regenerative function may arise from multiple cell populations but converges onto the specification of the unitary but transient FPC population. We will test the validity of this model across multiple forms of colitis and define key signals that mediate FPC specification. To test this hypothesis, we propose 2 Specific Aims. We will: 1) define lineage dynamics, temporal windows, and spatial contexts for repair- associated colonic epithelial cell plasticity, and 2) define alternative RNA splicing as a marker of proliferative cell identity in repair. If successful, these studies will link specification of a highly regenerative progenitor population to lineage plasticity and splicing-mediated cellular adaptations, unifying key mechanisms and identifying potential targets for critical programs of repair.

NIH Research Projects · FY 2025 · 2016-01

Project Summary Rearrangements of genomic segments or structural variation (SVs), include changes (increase/decrease) of copy number, inversions, translocations, and other mechanisms that change or rearrange the DNA content of a cell. Complex SVs can mediate many constitutional diseases; highly pathological germline rearrangements can damage the viability of the embryo; and somatic rearrangements can increase the pathology of many diseases, including cancer. The genomic footprint of complex SVs is a changed karyotype, defined by a collection of sequences of oriented genomic intervals whose coordinates are drawn from a reference genome, so that every sequence corresponds to a haploid or marker chromosome. The goal of this renewal proposal is to develop tools to elucidate the karyotype of a donor. In Aim 1, we will develop methods to reconstruct focal amplifications using long-read technology. In Aim 2, we will develop methods to elucidate genome scale karyotypes for constitutional disorders using optical genome maps. Highly rearranged regions will be clarified using a mix of experiments and computation in Aim 3. Finally, in Aim 4, we will extend the notion of a karyotype to predict the 3-dimensional conformation of ecDNA using Hi-C. The development of our aims will require novel graph theoretic and combinatorial optimization methods applied to new sequencing technologies. Their successful implementation will provide novel algorithms and software tools for improved karyotyping, deeper insights into gene regulation and DNA repair for focal amplifications, and better understanding of disease pathology mediated by complex variations.

NIH Research Projects · FY 2026 · 2015-09

Abstract Adolescent Brain Cognitive Development (ABCD) is the largest long-term study of brain development and child health in the United States. The ABCD Research Consortium consists of 21 research sites across the country, a Coordinating Center, and a Data Analysis and Informatics Resource Center. In its first five years, under RFA-DA-15-015, ABCD enrolled a diverse sample of 11,878 9-10 year olds from across the consortium, and will track their biological and behavioral development through adolescence into young adulthood. All participants received a comprehensive baseline assessment, including state-of-the-art brain imaging, neuropsychological testing, bioassays, careful assessment of substance use, mental health, physical health, and culture and environment. A similar detailed assessment recurs every 2 years. Interim in-person annual interviews and mid-year telephone or mobile app assessments provide refined temporal resolution of developmental changes and life events that occur over time with minimal burden to participating youth and parents. Intensive efforts are made to keep the vast majority of participants involved with the study through adolescence and beyond, and retention rates thus far are very high. Neuroimaging has expanded our understanding of brain development from childhood into adulthood. Using this and other cutting-edge technologies, ABCD can determine how different kinds of youth experiences (such as sports, school involvement, extracurricular activities, videogames, social media, unhealthy sleep patterns, and vaping) interact with each other and with a child's changing biology to affect brain development and social, behavioral, academic, health, and other outcomes. Data, securely and privately shared with the scientific community, will enable investigators to: (1) describe individual developmental pathways in terms of neural, cognitive, emotional, and academic functioning, and influencing factors; (2) develop national standards of healthy brain development; (3) investigate the roles and interaction of genes and the environment on development; (4) examine how physical activity, sleep, screen time, sports injuries (including traumatic brain injuries), and other experiences influence brain development; (5) determine and replicate factors that influence mental health from childhood to young adulthood; (6) characterize relationships between mental health and substance use; and (7) specify how use of substances such as cannabis, alcohol, tobacco, and caffeine affects developmental outcomes, and how neural, cognitive, emotional, and environmental factors influence the risk for adolescent substance use.

NIH Research Projects · FY 2026 · 2015-09

PROJECT SUMMARY In its initial funding period, the ABCD consortium used a rigorous epidemiological approach to recruit a diverse sample of 11,878 9- to 10-year-olds through our 21 research sites, of which 2,136 are twins or triplets. Expert workgroups (WGs) designed protocols using proven methods to assess, scan, and maintain this cohort with very high retention rates. The study complements well validated standardized assessments with novel real-time objective data collected wirelessly in the natural environment to securely capture indices of youth behaviors. These technologies add measurement precision, ecological validity, and an opportunity to reveal previously unknown effects of behavioral, physiological, and environmental factors in the development and fluctuation of substance use, screen media use, mental and physical health concerns, brain development, and their dynamic relationships. Bioassays (e.g., from hair, shed teeth, blood, and oral fluid) provide genome-wide genotyping, pubertal hormones, more precise estimates of exposures to substances of abuse and environmental toxins, and eventually more comprehensive estimates of relevant epigenetic effects. State-of-the-art harmonization methods for MRI and other data enable seamless data integration across sites into an invaluable open-science database that becomes available to the larger scientific community within a year of data collection. The Coordinating Center (CC) met all of its previous aims, and here describes how it will achieve an adjusted set of aims for the next 7 years. Specifically, we describe a flexible organizational framework created for management of the study that relies heavily on cross-disciplinary knowledge and extensive research experience of expert assessment WGs and strategic planning groups, and a dedicated team within the CC, Data Analysis, Informatics and Resource Center (DAIRC), and NIH to coordinate internal and external communications; support and integrate the development and revision of protocols and data quality reviews performed by WGs; establish, monitor, and maintain the standardization of study protocols across the consortium sites; measure and review study progress toward goals for retention, data quality, and scientific impact; based on these metrics, take supportive and corrective action to ensure that the goals are met; manage bioethics policies and consortium interactions with the single IRB (sIRB); promote the professional development of diverse junior investigators; establish a framework to resolve conflicts within the CC or within the wider consortium; and responsibly address any unanticipated scientific, methodological, logistical, or ethical challenges to ensure the study achieves its overarching objectives. We also describe how the CC and DAIRC will together implement quality control procedures to improve data quality across all data types; coordinate data sharing functions with the staff of the NIMH Data Archive; manage a pipeline for enhancing the study with novel assessment technologies; and facilitate rigorous analyses of ABCD data that set new standards for use of data from this powerful, but complex and challenging, high-dimensional study.

NIH Research Projects · FY 2024 · 2015-09

The second phase of NIH Common Fund 4D Nucleome Network Organizational Hub Abstract The second phase of the 4DN program requires an efficient organizational center to synergize the activities of all the funded teams and integrate the research products. Accordingly, the 4DN Organizational Hub is proposed to: (1) coordinate and integrate the efforts of all the funded projects, (2) build an efficient consortium infrastructure to serve as a center for the collaborative efforts, and (3) provide the 4DN web portal as a central resource gateway to access all the 4DN-Network generated tools, policies, guidelines, protocols, reagents, cell lines, and as a central hub for the outreach activities. Our major deliverables are: (1) an organizational structure composed of a steering committee and problem-solving working groups with a clear report chain to enable effective communications and decision-making processes, establish co-organizers and schedule, clarify action items, and ensure execution of the action items; (2) 4DN Web Portal (https://www.4dnucleome.org/), which is the always up-to-date community-wide resource and point of access for all data, protocols, reagents, resources, and methods; (3) 4DN internal wiki, the central organized resource for all teams, centers, and working groups to document internal progresses. (4) 4DN annual meetings, including the "kick-off" meeting in winter 2020 and the subsequent annual meetings and 4DN-ASCB (American Society of Cell Biology) satellite meetings; (5) 4DN outreach workshops at Keystone symposia and American Society of Human Genetics (ASHG) meetings, and on 4DN YouTube channel.

NIH Research Projects · FY 2024 · 2015-08

PROJECT SUMMARY/ABSTRACT The Prevention of Lower Urinary Tract Symptoms (PLUS) Consortium was established to lay the foundation necessary to study bladder health (BH) in women and girls, including the identification of risk and protective factors for BH using a longitudinal cohort. Factors identified across the spectrum of the PLUS conceptual model, from societal to biologic, will establish the evidence base for intervention studies designed by PLUS researchers and others in the field. The objective of this application is to present the UC San Diego Clinical Research Center (CRC) as a diverse, experienced, committed, and invaluable contributor to the Consortium. We will continue to provide the unique transdisciplinary expertise necessary for the Consortium to advance the study and promotion of BH. Members of our team have extensive experience in transdisciplinary research, epidemiology, longitudinal cohort studies, prevention science, public health, health education, cross cultural research, qualitative research, primary care (Pediatrics, Obstetrics & Gynecology), clinical care of women with LUTS, and are world renown for population-based microbiome research. We boast a history of strong leadership and critical contributions to the foundational work of the Consortium, including leadership in the development and validation of instruments for BH and qualitative research on the Study of Habits, Attitudes, Realities and Experiences (SHARE) and related knowledge, attitudes and beliefs (KAB). UC San Diego is a well-established research institution, funded by the National Institutes of Health, including a sponsored Clinical & Translational Science Award which provides a full spectrum of resources and personnel for the conduct of basic science, translational, epidemiologic, behavioral, and clinical research across the full age spectrum among racially and ethnically diverse communities. In addition to the foundational work of validating the bladder health instrument (BHI) for use in a culturally diverse population, our proposal incorporates cutting edge translational research aimed at identifying a healthy urogenital microbiome. The unique strengths of our application include: a) robust contributions to the foundational work of the Consortium including two team members actively engaged in the design of the planned longitudinal cohort study; b) a history of strong recruitment and retention of ethnically diverse participants for qualitative (SHARE), quantitative clinical trials and longitudinal cohort studies; c) a conceptual model centered around the notion that BH may be mediated through societal, institutional, interpersonal, behavioral and biologic factors via the urogenital microbiome; d) a feasibility pilot study of collection and analysis of voided urine specimens from a longitudinal cohort, which will foster investigations of prevention studies that manipulate the urogenital microbiome (directly or indirectly) through changes in modifiable risk factors across the PLUS conceptual framework; and e) the development of new measures for use in linguistically and culturally diverse populations starting with an adapted and validated Hispanic (English and Spanish language) BHI and KAB questionnaire.

NIH Research Projects · FY 2026 · 2015-08

Breast tumors are often identified by manual palpation due to their apparent “hardness” compared to normal tissue. The presence of a fibrotic focus in breast tumors is associated with a 10-50-fold increase in tissue stiffness and correlates with distant metastasis and poor outcome. Recent studies show that increasing matrix stiffness can induce a malignant phenotype in cultured human mammary organoids, suggesting that mechanical properties of extracellular matrix directly regulate tumor metastasis. However, how mechanical forces are translated into biochemical signals to promote tumor invasion and metastasis is largely unknown. Our preliminary studies found that rigid matrix stiffness activates a novel mechanotransduction pathway to induce Epithelial-Mesenchymal Transition (EMT) and promote tumor metastasis. We therefore hypothesize that mechanical forces activates the LYN kinase to allow the EMT-inducing transcription factor TWIST1 to promote tumor invasion and metastasis. To test this hypothesis, we plan to 1) To elucidate the molecular mechanism by which high tissue stiffness activates a novel mechanotransduction cascade to promote TWIST1 nuclear translocation and EMT; 2) To elucidate the novel molecular mechanism by which soft matrix stiffness prevents TWIST1 nuclear translocation and inhibit EMT; 3) To determine the involvement of the Twist1 mechanotransduction pathway in promoting metastasis in vivo and in predicting human breast cancer progression.

NIH Research Projects · FY 2025 · 2015-08

Project summary: Essential regulation of the cellular machinery is achieved by a network of highly dynamic signaling molecules, which, when dysregulated, allow cancer cells to misinterpret or ignore signals that normally tell cells to stop dividing or begin apoptosis, leading to uncontrolled tumor growth. For the last 18 years, my laboratory has been at the forefront of applying a native biochemistry approach to cell signaling and cancer research. We have developed enabling technologies, and established spatiotemporal regulation as a fundamental paradigm in cell signaling, and discovered that its alteration leads to uncontrolled cell growth. This NCI R35-supported research program seeks to establish a new conceptual framework to specifically understand the cellular organization of molecular activities. We hypothesize that cellular biochemical activities are spatially organized into an “activity architecture” and dysregulated driver molecules can re-organize and re-structure this activity architecture, leading to loss of control over cell growth, division and death. In the past 6 years, we published 51 peer-reviewed articles, and made significant advances in establishing this framework. We developed first-in-class technologies for imaging protein-protein interactions and enzymatic activities in living cells at molecular length-scales and first kinase biosensor that achieved high- resolution imaging in awake mice, which have provided evidence for the biochemical activity architecture across different scales. We also made a breakthrough discovery that a regulatory subunit of Protein Kinase A (PKA), RIα, undergoes liquid-liquid phase-separation (LLPS) to enable the dynamic buffering and spatial compartmentalization of a ubiquitous second messenger, cAMP, providing an answer to a long standing question. We further showed that the oncogenic fusion in fibrolamellar carcinoma (FLC) potently inhibits RIα LLPS and induces aberrant cAMP signaling, which leads to increased cell proliferation and cell transformation. We have also discovered novel regulation in the Ras/ERK pathway and developed a novel Ras biosensor. In the proposed research, we will have three focuses. First, we will develop innovative technologies including super-resolution activity imaging to illuminate the biochemical activity architecture across different scales. Secondly, we will elucidate how the disorganized cAMP-PKA activity architecture leads to tumorigenesis in FLC, and further discover novel, cancer-relevant biomolecular condensates. Thirdly, we will investigate the spatiotemporal regulation of ERK that is critical for its physiological functions and identify the vulnerable connections in the re-organized cancer-driving architecture in pancreatic cancer, which is a deadly cancer that is addicted to the Ras-ERK pathway. We will also facilitate the development of new therapeutics by developing novel assays for evaluating Ras inhibitors and measuring target engagement.

NIH Research Projects · FY 2025 · 2015-07

This is a renewal application for a K24 Midcareer Investigator Award in Patient-Oriented Research to support my ability (1) to attract scientists to patient-oriented research at the intersection of neuroHIV and substance use, (2) to prepare a new crop of investigators to tackle complex research questions that require translational approaches and interdisciplinary collaboration, and (3) to foster creativity, insights, and critical thinking in problem-solving approaches to untangle drug effects in neuroHIV. Mentoring and training will be integrally tied to my own research and to the major projects within the HIV Neurobehavioral Research Program (HNRP) at the University of California, San Diego, which are rich sources of data, specimens, and research infrastructure. Mentees will also have access to didactics made available by training programs in which I participate, such as the R25 Interdisciplinary Research Fellowship in NeuroAIDS, which I direct, and a T32 program for Training in Research on Addictions in Interdisciplinary NeuroAIDS, where I am a faculty mentor. I plan to involve mentees in developing new research directions in medical applications of cannabinoids for neuroHIV and explore predictors of central nervous system outcomes in people with HIV who use sedatives and other substances. I will also involve mentees in my continued work on individual differences in vulnerability to CNS dysfunction associated with stimulant use in the context of HIV. I propose additionally to pursue my own professional development to continue to improve my mentoring skills, as well as to enhance my leadership skill set. The superb research environment of the HNRP, along with the larger UCSD HIV and substance use research community, affords opportunities for mentoring and fostering the advancement of early career clinically oriented scientists.

NIH Research Projects · FY 2025 · 2015-05

Applying ethical standards and norms to research both improves its quality and advances the research, in addition to protecting the rights of research participants. Arabic-speaking countries in the Middle East and North Africa suffer from a low level or non-existent availability of training in research ethics despite a growing research profile and level of activities in most of these countries. Our program, The Research Ethics Education Program in Jordan, was able in the past 5-year cycle to make an impressive impact on addressing this lack of training by achieving its proposed goals and more. There were 118 faculty and researchers trained during this period in the Responsible Conduct of Research (RCR) from across 25 institutions in 12 different countries in the region. Those who graduated are strongly committed to continue their career in this field. In addition, more than 2,175 other graduate students, researchers, and faculty across the MENA region received training in RCR by our fellows and instructors through 45 different workshops during this period. Fellows have also led the publication of 40 peer-reviewed papers. In at least five academic institutions fellows are leading the establishment of ethics committees for the first time, and many are now members or leading existing ethics committees. We also achieved a three-fold higher representation of women among our fellows than currently found in academia in Jordan and many of the MENA countries, with 4 of our female fellows assuming administrative academic positions as deans and one as department chair. Our goal is to continue these achievements and take this program to the next level by establishing the first Master of Bioethics for the MENA which would be provided locally by the host research-intensive University, the Jordan University of Science and Technology (JUST). JUST will also establish a comprehensive Center for Research Ethics that will carry on the program’s training at multiple levels and offer certification and workshops in RCR. Arabic language certification in RCR will also be made available to universities in the MENA. If funded for renewal we will be able to transition the activities of the current program from the US-based-institution to the LMIC-based institution represented by JUST. Both the master’s, the proposed center, and the Arabic language RCR certification will provide vital capacity building in the region. It is being offered by a local university to graduate highly qualified trainees ready to lead the region in bioethics and responsible conduct of research, and expand the breadth of RCR training at the individual and institutional levels in the MENA. We have created a paradigm shift in the social norms associated with research and research ethics among our fellows, and at JUST. In this next phase we will expand on our success by adding more in-depth training offered through the Master of Bioethics and increase the breadth of dissemination of our activities through a greater number of MENA research and academic institutions.

NIH Research Projects · FY 2025 · 2015-04

PROJECT SUMMARY / ABSTRACT The overall objective of the NEI Mentored Clinical Scientist Development Program Award at UCSD is to develop outstanding clinician scientists to successfully compete for NIH grants and emerge as leaders within academic ophthalmology. Through this institutional career development award, we will continue to mentor clinician scientists toward independent academic appointments in ophthalmology. Since 1991, the UC San Diego Shiley Eye Institute has excelled in mentoring the career development of clinician scientists throughout ophthalmology. Many of these individuals now have successful research and clinical careers around the world; they include Department Chairs, Division Chiefs, and impactful academicians. With continued funding, we will amplify these strengths through a structured program of mentoring, research, and education to identify and mentor the next generation of academic clinician scientists, across all subspecialty areas of ophthalmology. With an emphasis on recruiting strong applications from women and minority applicants, Scholars will be selected each year after their completion of a post-residency clinical fellowship in ophthalmology. The Scholar will undertake these activities in the area of basic and/or clinical sciences appropriate to their scientific focus, and by partnering with mentor(s) to facilitate career development. Each Scholar will be trained in clinically relevant research, statistics, scientific writing, grant preparation, ethics, leadership, and the responsible conduct of research. Additional aspects of the curriculum of each Scholar will be individualized by the Program Director (PI), Research Directors, Education Director, Executive Committee, Mentoring Group, and lead mentor based upon the Scholar's selected primary research track and his/her skills, background, and career goals. Each Scholar will create an original research program and through this Award develop an independent academic career. We propose six primary research tracks: (1) Visual Neuroscience, (2) Genomics and Proteomics, (3) Bioengineering, (4) Stem Cell Biology, (5) Artificial Intelligence and Informatics, and (6) Clinical (human subjects) Research. In the first K12 funding period, we trained and recruited four clinician scientists in the subspecialty areas of retina (2), glaucoma (1), and pediatric ophthalmology (1). Two of these Scholars already have received NIH K08 awards, and the other two have submitted K08 proposals that are under consideration.

NIH Research Projects · FY 2025 · 2015-02

Project Summary/Abstract Background: The prevailing model of epidermal differentiation is that transcription factors recruit RNA Polymerase II to differentiation gene promoters only upon the induction of differentiation. It is possible that mechanisms downstream of transcription initiation such as transcriptional elongation can have major impacts on regulating epidermal growth and differentiation. We have shown that specific elongation factors such as ELL and ELL2 are necessary for epidermal growth while SPT6 is essential for differentiation through the control of transcription elongation. Objective/hypothesis: This proposal seeks to understand the regulation of epidermal stem and progenitor cell self-renewal and differentiation through transcription elongation. We have found that up to 30% of induced epidermal differentiation genes contain promoter proximal paused RNA polymerase II in stem and progenitor cells. Upon differentiation, specific elongation factors such as SPT6 are necessary to promote the transcriptional elongation of these paused differentiation genes. In stem and progenitor cells there are also elongation factors that promote the transcription of growth and proliferation genes to allow for epidermal renewal. Specific Aims: (1) The role of transcription elongation factors on epidermal growth and differentiation (2) Mechanisms on how elongation factors control epidermal growth and differentiation. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we generate 3-dimensionally intact human skin, containing human epidermal cells (that have been permanently knocked down for transcription elongation factors) in the context of human dermal stroma and basement membrane, regenerated on immune compromised mice. By using this model, we can perform loss of function experiments on the transcription elongation factors in regenerated human skin to characterize their role in epidermal growth and differentiation. We will also use chromatin immunoprecipitations followed by next generation sequencing to determine which genes the elongation factors bind and regulate.

NIH Research Projects · FY 2024 · 2015-02

ABSTRACT Immune stimulatory pathways are dysregulated during chronic viral infections, preventing timely T cell and antibody mediated immunity. Human persistent viruses, including human immunodeficiency virus (HIV), hepatitis C and B viruses (HCV and HBV) cause tremendous disease burden worldwide but are restricted to human and nonhuman primates, which in turn poses great limitations for experimental-based research. Importantly, lymphocytic choriomeningitis virus (LCMV) in its natural rodent host has been successfully used as a model system to uncover common principles underlying the regulation of immune responses during persistent viral infections, often highlighting basic mechanisms that were later extended into humans. We have found that interleukin-6 (IL-6) and IL-27 are essential for optimal CD4 T cell differentiation and CD4 T cell survival, respectively, while they redundantly induce IL-21, a critical factor for CD8 T cell and antibody responses, late after infection. Consistently, both IL-6 and IL-27 were absolutely required to control chronic (but not acute) LCMV infection. More recent data indicate that late IL-27 derived from B cells is crucial for viral control and it is regulated by host microbiota during chronic LCMV infection. The overall goal of the current proposal is to fully dissect the mechanisms underlying the antiviral effects of B-cell-derived IL-27 as well as its regulation by host microbiota and related metabolites during persistent infection. To accomplish this goal, we propose three specific aims. In Aim 1 we will investigate the relationship between B-cell-derived IL-27 and antiviral responses during chronic LCMV infection. We will test the hypothesis that B cell-derived-IL-27 is both a dependent and a driver of CD4 T cell responses during chronic infection, and indirectly enhances antibodies and CD8 T cells, promoting viral control late after infection. In Aim 2 we will identify the microbiome commensals that enhance IL-27 production by B cells and the impact for antiviral responses and viral control during chronic LCMV infection. We will investigate the possibility that Segmented Filamentous Bacteria (SFB), a commensal that associates with accelerated viral control, enhances IL-27 production by B cells in small intestine as well as T cell responses both locally and at distal sites, overriding the need for IL-6. Finally, in Aim 3 we will identify microbiota-related metabolites that modulate IL-27 production by B cells and the impact for antiviral responses and viral control during chronic LCMV infection. We will investigate the metabolites that associate with the microbiota driving IL-27 production by B cells and evaluate their effects on IL-27 expression (in mouse and human B cells) as well as T cell responses and viral control in LCMV chronically infected mice. The knowledge gained from this study will not only enhance our understanding of the basic biology of key immune regulators (i.e. IL-27, microbiome and related metabolites) but may also be valuable for therapeutically manipulating these factors during infections and perhaps other immune diseases.

NIH Research Projects · FY 2026 · 2015-01

Patient-Directed Computational Analysis of Atrial Fibrillation Project Summary Atrial fibrillation (AF) poses a significant and rapidly escalating health challenge, impacting over 5 million Americans and contributing to heightened risks of stroke, heart failure, and mortality. Despite extensive research efforts spanning decades, the precise mechanisms underlying AF remain incompletely understood, leading to suboptimal therapeutic interventions. Recent investigations, however, have shed light on the role of rotational or focal activity in driving AF, with targeted ablation of these sources showing promise for long-term outcomes. Yet, due to the complexity of AF and limitations in visualization, targeted ablation approaches do not consistently yield successful results. This project aims to advance our understanding of AF mechanisms and enhance therapeutic strategies for AF management. A key focus will be the development of a novel analysis technique capable of generating easily interpretable visual maps depicting the electrical activation patterns in AF patients. Leveraging a large and unique database of AF patient data, this technique will retrospectively analyze AF activity to elucidate how it is organized in these individuals. Additionally, extensive simulations will be conducted using patient-specific anatomical models to explore how atrial anatomy influences AF dynamics. The significance of this project lies in its potential to increase our understanding of AF and uncover novel mechanisms underlying AF maintenance. The insights gained can directly inform clinical practice and facilitate the development of more effective treatment options, ultimately improving outcomes for individuals with AF.

NIH Research Projects · FY 2025 · 2014-12

PROJECT SUMMARY/ABSTRACT: One-third of women in the U.S. have bacterial vaginosis (BV), a condition associated with greater risks of reproductive tract infections and preterm birth. BV is characterized by loss of lactobacilli and polymicrobial overgrowth of diverse potential pathogens, including Gardnerella vaginalis. Vaginal sialidase activity is found in nearly all women with BV and is independently associated with adverse outcomes. However, little is known regarding how sialidase supports dysbiosis and disease. This proposal examines how the sialidase produced by certain subtypes of G. vaginalis participates in the molecular pathogenesis of vaginal and intrauterine infection and encourages overgrowth of diverse BV bacteria. We have identified the genetic basis of G.v. sialidase activity and found that 80% of BV cases have G.v. encoding the sialidase genes nanH2 or nanH3, and 80% of women with nanH2-positive or H3-positive vaginal microbiota have BV. Our data suggests that G.v. sialidase fundamentally transforms the cellular glycan landscape in BV and shows that glycan degradation encourages pathogen colonization. In three specific aims, this proposal will use human vaginal samples, diverse G.v. isolates, a mouse model that replicates relevant features of BV, new wild-type and paired nanH3 mutant G.v. strains, and novel ex vivo models employing native human vaginal communities to test several hypotheses about the molecular mechanism of G. vaginalis sialidase in the causes and complications of BV. In Aim 1, we will examine the relationship between G.v. clades and biochemical and cellular phenotypes in human clinical samples and compare sialidase positive and negative G.v. clades and a G.v. sialidase mutant in our mouse vaginal colonization model. In Aim 2, we will use our repository of live native vaginal communities in vitro to test how the availability of free sialic acid and exposed glycans, both consequences of sialidase activity, influence BV community composition and also examine the relationship between nanH2/H3-positive G.v. and bacterial burden and diversity in clinical specimens. Finally, Aim 3 we will evaluate samples from a longitudinal cohort of pregnant women to determine whether women who went on to experience preterm birth have higher levels of vaginal nanH2/H3-positive G.v., larger numbers of exfoliated epithelial cells, degraded mucus and epithelial glycans, and more diverse microbiomes with higher vaginal bacterial burden compared to term controls. The successful completion of these aims will have an important positive impact on this field by identifying G.v. subtypes that drive pathophysiology, defining mechanisms by which G.v. sialidase supports the development of dysbiosis and disease, and identifying biomarkers that might be used in clinical surveillance efforts to prevent preterm birth.

NIH Research Projects · FY 2025 · 2014-09

ABSTRACT: Cholestatic fibrosis is the outcome of chronic liver diseases, including primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), secondary biliary cirrhosis (SBC). It is characterized by extensive deposition of extracellular matrix (ECM), including collagen Type I. Activated hepatic stellate cells (aHSCs) and portal fibroblasts (aPFs) are the major source of the fibrous scar in the liver. aPFs have been implicated in liver fibrosis caused by cholestatic liver injury. (AIM1) Here we propose to study the role of Msln- Muc16-Thy-1 signaling in the pathogenesis of cholestatic fibrosis in Mdr2-/- mice. For this purpose, Mdr2-/- mice are crossed to Msln-/- mice, Muc16-/- mice, or Thy-1-/- mice. To dissect the relationship between Msln-Muc16- Thy-1, we generated Mdr2-/- mice devoid of both Msln and Muc16 (triple knockout Mdr2-/-Msln-/-Muc16-/- mice) or Msln and Thy-1 (Mdr2-/-Msln-/-Thy-1-/- mice). The time course comparison of cholestatic fibrosis in Mdr2-/- and BDL-injured mice will reveal similarities of aPF activation in both models. The paracrine signaling between aPFs and cholangiocytes will be evaluated. (AIM2) We will investigate the unique mechanisms, common mediators, and molecular factors that mediate activation of aPFs. We hypothesize that Msln induces a non- canonical TGFb/TGFbRI-Smad2/3/4-activation of aPFs. Additional potential pathways of Msln signaling in aPFs (such as FGFR-Msln-Akt/ERK and JAK2/STAT3) will also be evaluated. To dissect the mechanism by which Msln-Thy-1 pathway regulates aPF functions, Col-GFP+Thy-1+Msln+ aPFs will be sort purified, and analyzed by RNA-Seq and mass spectrometry. To elucidate novel signaling pathways, gene expression profiles and binding partners of WT and KO aPFs will be compared. (AIM3) To translate our findings from mice to humans, the role of human MSLN-THY-1 will be studied in human aPFs in vitro and in vivo. We have already isolated and characterized human aPFs from 6 livers of patients with cholestasis. Human aPFs are identified by expression of the “signature genes” MSLN, CA125, THY-1, BNC1, UPK1β, CALCA, GPC3. 2 selected human aPF cell lines will be analyzed using shRNA-knockdown ± TGFb1, and RNA-Seq. Binding partners of human MSLN will be identified by mass spectrometry using anti-human MSLN Ab. (AIM4) Our central hypothesis is that MSLN- MUC16-THY-1 pathway plays an important role in activation of human aPFs in response to cholestatic injury, and therefore serves as a target for anti-fibrotic therapy. We will test if ablation of MSLN with anti-MSLN Ab- immunotoxins can suppress development of cholestatic fibrosis in “humanized” MSLN mice (in which mouse msln gene was replaced with human MSLN gene) or liver xenograft Rag2-/-gc-/- mice (generated by adoptive transplantation of GFP-labeled human MSLN+ aPFs into the livers of Rag2-/-gc-/- mice, this technique is developed in our laboratory). Effectiveness of immunotherapy will be estimated in these mice by disappearance of MSLN+ aPFs, and suppression of cholestatic fibrosis. We anticipate that anti-MSLN Ab-immunotoxins will improve cholestatic fibrosis.