University Of California, San Diego

NIH Research Projects · FY 2026 · 2021-04

PROJECT SUMMARY/ABSTRACT RNA structure is involved in all aspects of the central dogma of molecular biology. However, there are relatively few unique RNA structures determined at high resolution that would give detailed insight into functional mechanisms. We are focused on studying RNA structure/function relationships via two major themes: Theme #1: Determine the architectures of protein-free non-coding RNAs at high resolution to gain insight into in vivo function. Cryo-EM of protein-free RNAs is difficult with only a handful of structures determined to better than 4 angstroms. We have developed a scaffolding methodology that enables us to determine cryo-EM structures of protein-free RNAs to 2.5 angstroms resolution routinely. This now enables us to delineate precise structure function relationships at the individual nucleotide level for non-coding RNAs. Using this pipeline, we are targeting bacterial and human non-coding RNAs that have significant biological impacts. A bacterial non-coding RNA known as raiA is necessary for sporulation and biofilm formation. We are using structural biology, in vitro biochemistry, and in vivo biology to interrogate its involvement in these processes. Our scaffolding methodology is also being applied to the untranslated regions (UTRs) of human RNAs encoding proteins. As part of our preliminary studies, we have determined structures of human UTRs that have significant effects upon gene expression. These pilot studies show that human RNAs contain highly structured domains that are important for gene regulation. Theme #2: Investigate mechanisms that are part of the central dogma of molecular biology, such as translation, splicing, and cDNA synthesis. We are studying the molecular mechanism of peptide bond formation in the ribosome. We have discovered structural evidence for metal ion catalysis in the ribosome with mechanistic and active site similarities to RNA splicing. We will use this metal ion model as a guide for the design of biochemical experiments to probe the mechanism of peptide bond formation in the ribosome. In addition, consistent with our interests in retrotransposition, we are studying novel priming mechanisms in reverse transcriptases involved in phage defense. In summary, we are investigating the biological and biochemical significance of RNA structure in cellular processes across the different kingdoms of life.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Ewing Sarcoma Family of Tumors (ESFT) are bone and soft tissue cancers that affect children and adolescents. Surgery, radiation and multi-agent chemotherapy have improved patient prognosis but a therapeutic plateau has been reached for both localized and metastatic cases. Hence, there is an urgent need for novel and targeted therapeutic strategies. Towards that goal, this Multi-PI collaborative research project will investigate the hypothesis that ESFT cells are dependent on FEN1, an endonuclease that processes the 5’ flaps of Okazaki fragments during lagging strand DNA synthesis, for viability. This hypothesis is supported by: (a) Four genome- scale CRISPR library screens have independently found that ESFT cell lines are highly sensitive to FEN1- CRISPRs; (b) ESFT cells were reported to be phenotypically BRCA1-deficient, and we have shown that BRCA1/2-deficient cells are hypersensitive to FEN1 inhibition; and (c) we have found several ESFT cell lines to be sensitive to FEN1-CRISPRs, FEN1-siRNAs and two small molecular FEN1-inhibitors. A team of three principal investigators will jointly direct this project: RD Kolodner is a geneticist and an inventor of FEN1-inhibitors in documented patent applications, JYJ Wang is a cancer biologist with expertise in DNA damage response, and SH Choo is a pediatric oncologist with an ongoing IRB to conduct research with ESFT patient samples. Together, we will pursue four specific aims to investigate the ESFT-dependency on FEN1. AIM-1: To demonstrate and to quantify FEN1-essentiality in a panel of 10 ESFT cell lines by genetic ablation of FEN1 with validated siRNAs and CRISPR/CAS9, respectively. AIM-2: To determine the cytotoxic effects of small molecule FEN1 inhibitors on 10 ESFT cell lines by short-term growth and death assays and longer-term colony formation assays in 2D and 3D cultures. We will edit FEN1 in ESFT cells to express a drug-resistant FEN1R enzyme so as to demonstrate on-target effects. We will evaluate the potential synergistic interactions between FEN1-inhibitors and the chemotherapeutic drugs currently used in the clinic to treat ESFT patients. AIM-3: To investigate the mechanisms underlying ESFT dependency on FEN1 by addressing three mechanistic questions on whether (a) the EWS-FLI1 oncoprotein of ESFT induces FEN1-dependency, (b) FEN1-inhibitors cause irreversible blockade of DNA replication in ESFT cells, and (c) FEN1-inhibitors cause DNA breakage and mitotic catastrophe in ESFT cells. AIM-4: To determine the effects of FEN1 inhibitors on ESFT growth in mice. We have found that a FEN1- inhibitor (SMD154) reduced the growth of orthotopic xenografts from an ESFT cell line in athymic nude mice. The pharmacokinetics (PK) of SMD154 support its testing on orthotopic xenografts from two ESFT cell lines that show sufficiently low in vitro IC50 for this compound. We will construct 4-6 ESFT patient-derived xenografts (PDX) from biopsies and resected tumors. We will test SMD154 and future FEN1-inhibitors with optimized PKs on cell line- and patient-derived xenografts. This comprehensive preclinical study will produce a detailed understanding of the ESFT-dependency on FEN1 and pave the way towards developing FEN1-inhibitors to treat ESFT.

NIH Research Projects · FY 2024 · 2021-04

In 2017 the International Diabetes Federation estimated that the worldwide prevalence of diabetes would increase from 415 million in 2015 to 642 million in 2040. Approximately 40% of individuals with diabetes develop diabetic nephropathy (DN). Twenty percent of these individuals do not follow the typical path toward chronic kidney disease, which is a slow multi-decade increase in albuminuria and serum creatinine, the current standard-of-care for surveillance of chronic kidney disease (KD). Consequently, there is an unmet clinical need for routine surveillance during the first decade of chronic KD. We propose external imaging of mesangial cell function as a biomarker for diabetic nephropathy. Our reasoning is based on the following. Mesangial cell matrix (MCM) expansion is a histologic hallmark of diabetic nephropathy, which precedes the reduction of a patient’s glomerular filtration rate or increase in albuminuria. Additionally, all the clinical manifestations of diabetic nephropathy are highly correlated with MCM expansion. There currently does not exist an imaging, serum, or urine biomarker that is sensitive to mesangial cell function. Current imaging agents and biomarkers are only sensitive to glomerular filtration, effective renal plasma flow, or albuminuria, which are altered late in the disease when therapeutic intervention is not effective. We propose a Phase 1 clinical trial of Tc-99m-tilmanocept, which accumulates in the liver and kidneys. The molecular mechanism is binding to CD206, which resides on the cell surface of fixed macrophages within the liver and mesangial cells within the kidney. We present preliminary data consisting of human SPECT/CT and rat microPET images of renal cortex. Additionally, we present evidence of sensitivity to MCM expansion via Tc- 99m-tilmanocept dynamic imaging of db/db mice, an accepted disease model of diabetic nephropathy. We propose an open-label study to investigate the biodistribution at two dose levels (2.0 & 20 nmol) of Tc- 99m-tilmanocept. We will study 5 groups at each dose (10 subjects each): 1) Advance DN, 2) early DN, 3) diabetes with no kidney disease, 4) advanced hypertension (HTN) with KD, and 5) HTN without KD. The study will include a 30-min dynamic followed be a 30-min kidney SPECT/CT, and periodic blood and urine sampling. Dynamic imaging will yield plasma clearance half-lifes, and liver and kidney accumulation rates; SPECT/CT will yield SUVs for the heart, liver, renal cortex, renal medulla. We will also calculate urinary bladder accumulation. We expect the renograms and biodistribution data to reflect the following pathology: Group 1, severe MCM expansion; G2, mild MCM expansion; G3 & G5, no MCM expansion; and G4, low MCM expansion. This study is the necessary first step toward FDA-approval of Tc-99m-tilmancoept as a kidney imaging agent. The study will also provide evidence of imaging sensitivity to MCM expansion in DN patients, and insensitivity to patients with HTN. This senerio will be required if Tc-99m-tilmanocept renograms as “first-line” diagnostic test for diabetic patients.

NIH Research Projects · FY 2026 · 2021-04

Project Summary Despite the advent of aggressive cervical cancer screening programs, cervical cancer remains one of the most common cancers affecting women under age 35, and the fourth most common cause of cancer death worldwide. The standard of care for early stage (≥IB) cervical cancer is hysterectomy or radiation. Unfortunately, the consequences of radical treatment include fertility loss, nerve injury causing bladder and bowel dysfunction, and pelvic pain. There is a critical need to reduce cervical cancer mortality, while minimizing the potential morbidities of treatment. To achieve this end requires refined approaches for diagnosis and evaluation of response to treatment using noninvasive biomarkers to differentiate indolent from clinically significant disease at the earliest possible time-point. PET/CT is currently the mainstay in evaluating response to treatment and is highly confounded by post-treatment changes such as edema. Magnetic resonance imaging (MRI) with advanced diffusion-weighted imaging may offer an alternative approach to evaluate treatment response, with additional advantages of being a radiation-free and contrast media-free exam. The overall objective in the current and parent grant is to develop and evaluate a robust advanced diffusion-weighted imaging technique that provides a highly sensitive and specific reflection of cervical cancer tumor burden and treatment response at the earliest possible time point. Our parent grant hypothesis is that restriction spectrum imaging (RSI), an advanced diffusion imaging technique, is as sensitive and specific as standard of care post-treatment PET/CT for evaluation of treatment efficacy of cervical cancer and can be performed 3 months earlier than standard of care PET/CT. In the parent grant we developed a cervical cancer-specific diffusion imaging model capable of differentiating cancer signal from edema both before and during treatment. However, in contrast to cancers in other organs, 73% of the cervical cancers were not visible at higher b-values and 31% of cancer lesions were undetectable with DWI at the current spatial resolution (voxel size 1.56×1.56×3mm3). Thus, we hypothesize that the sensitivity of RSI in detecting, evaluating and staging cervical cancer will significantly improve by developing an optimized high-resolution multi-shell DWI protocol. With a higher resolution protocol than our prior study, we will aim to increase sensitivity to smaller residual active disease/new disease. The aims of the new proposal are: (1) Develop and optimize a high resolution (HR) multi-shell DWI protocol for improved sensitivity to small cervical cancer lesions. (2) Evaluate the updated cervical cancer-specific RSI framework in an independent cohort using the high resolution multi-shell DWI protocol.

NIH Research Projects · FY 2025 · 2021-03

Project Summary This is an application for a K01 award for Dr. Iris Broce-Diaz, a neuroimaging genetics postdoctoral fellow at the University of California, San Diego and University of California, San Francisco. Dr. Broce-Diaz is establishing herself as a young imaging geneticist conducting clinical research on neurodegenerative disease. This K01 will provide Dr. Broce-Diaz with the support necessary to accomplish the following goals: (1) gain proficiency in machine learning and computational modeling techniques, (2) gain proficiency in clinical and genetic research methodology for cognitive aging and complex spectrum of neurodegenerative diseases, including clinical characterization of frontotemporal dementia (FTD) and other Alzheimer’s Disease-Related Dementias, differential diagnosis, risk prediction, and biomarker development, and (3) develop an independent research career. To achieve these goals, Dr. Broce-Diaz has assembled an expert mentoring team, including her primary mentors: Dr Anders Dale (renowned computational neuroimaging genetics scientist) and co-primary mentor Bruce Miller (internationally recognized behavioral neurologist and leader in FTD), co-mentors: Drs. Jennifer Yokoyama (expert in FTD genetics) and Chun Chieh Fan (expert in epidemiology/biostatistics), and two collaborators: Drs. Adam Boxer (leader in clinical trials for FTD-spectrum disorders) and Wesley Thompson (expert in advanced statistics). The goal of the proposed project is to develop novel imaging genetics biomarkers for predicting individuals at risk of developing sporadic (non-familial) FTD and improving classification accuracy of sporadic FTD. Dr. Broce- Diaz will achieve this goal through the following specific aims: (1a) utilize a polygenic hazard approach to develop and validate a novel genetic biomarker for predicting age-specific risk of sporadic FTD; (1b) leverage pleiotropic information to increase accuracy of the genetic risk scores and derive biologically-based genetic risk scores; (2) use machine learning approaches to reliably and accurately classify FTD clinical subtypes and obtain personalized atrophy scores from these brain maps; and (3) improve FTD classification by integrating atrophy scores with genetic risk scores. This proposed study uses highly innovative methodological approaches for informing FTD prognosis, diagnosis, and, ultimately, clinical trial design. If validated, these biomarkers will make significant contributions by assisting clinicians in identifying patients at elevated risk for sporadic FTD and assisting in diagnosing sporadic FTD in its earliest stages—reducing diagnostic delays, accelerating the discovery of novel treatments, and improving recruitment accuracy in clinical trials. This K01 research project will provide Dr. Broce-Diaz with the protected research time and opportunity to train with leaders in the field she needs to master the skills required to establish an independent, patient-oriented, imaging genetics and biomarker development clinical research program that will inform diagnosis, prognosis, and guide treatments of FTD and other neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT. While significant changes in cannabis potency, methods of use (e.g., flower, edibles) and policy have occurred, high levels of use by youth remain relatively constant. In addition, rates of secondhand cannabis exposure in children is increasing. The cannabis plant contains over 120 cannabinoid constituents, with delta-9-tetrahydrocannabinol (THC), a psychoactive constituent, most associated with deficits in verbal memory, attention, and working memory; however, the persistence of these effects remains controvertible. Differences in frequency, method of use, and potency may produce variation in measures of cannabis metabolized. Research to-date has primarily relied on self-report, despite potential mis- reporting by participants and reliance on episodic (rather than dose or patterns) of cannabis use, and almost no research investigating secondhand exposure. Such limitations may explain prior inconsistencies in the cannabis-cognition literature. The primary aim of this K08 proposal is to facilitate interdisciplinary expertise in toxicology to examine cannabinoid analyte levels using a robust biosample (hair) to assess cognitive correlates. The use of hair allows for improved methodological investigation of the relationship between cannabis use, secondhand smoke exposure, and potential cognitive impact. Expansion of hair analysis in the Adolescent Brain Cognitive Development (ABCD) study will add novel and important knowledge to the scientific body of literature regarding the cannabis-cognition link, clarifying prior discrepant results of both negative and null cognitive function changes associated with cannabis using adolescents, and examining the influence of second-hand cannabis smoke. Causal inference models will be used to determine the influence of THC and its metabolite levels from hair on cross-sectional and longitudinal cognitive function among a subsample of the ABCD cohort. Cognition and substance use will be measured annually for the length of the proposed project, when ABCD participants are between the ages of 13-14 and 17-18 years. The aims of this project are consistent with NIDA's strategic funding plans, as this work would measure behavioral sequelae of environmental and direct exposure of cannabis in a vulnerable, young population. The additional training afforded to Dr. Wade, particularly in cannabis toxicology, secondhand smoke exposure, early adolescence, and statistics, would complement her prior experience in substance use, neurocognition, and emerging adults. A mentorship team of experts will bridge these unique fields to improve our understanding of the effects of THC exposure (personal use or environmental exposure) on adolescent cognitive development. At the conclusion of this award, Dr. Wade will meet her goal of career independence as an interdisciplinary clinical scientist with expertise in cannabis toxicology and cognition, exemplified by submission of an R01. Finally, the funding of analysis of additional hair samples will also benefit the open-science model of ABCD, as all data collected will be available to researchers worldwide through the annual curated ABCD data release.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY Autism-spectrum disorders impact millions of individuals worldwide, representing a heavy toll on affected children, their families, and the health care system. Pitt–Hopkins Syndrome (PTHS) is an ASD caused by de novo mutations in the TCF4 gene. PTHS is characterized by severe intellectual disability, pronounced developmental and motor delays, absence of speech, repetitive behaviors, peculiar facial gestalt, and gastrointestinal manifestations. While the genetic etiology of PTHS is well established, the cellular and neural phenotypic alterations in human patients are still not fully understood, nor is it clear how TCF4 mutations cause such abnormalities. Lack of understanding about PTHS's molecular and cellular mechanisms is a problem because, until this information becomes available, specific altered pathways cannot be therapeutically targeted. Moreover, without neuropathological knowledge, it is impossible to treat and eventually cure PTHS by directly correcting the mutation in the genome. Our long-term goal is to understand how specific genetic defects and altered pathways in the brain result in the debilitating phenotypes exhibited by autistic children. The objectives of this application are to: (a) use human models of neural development in vitro to define the cellular and neural pathological consequences of clinically relevant TCF4 mutations in PTHS; and (b) provide proof-of-concept that correctional molecular strategies can be used to fix TCF4 expression, an approach that could eventually be used as gene therapy for PTHS. Our central hypothesis is that TCF4 mutations cause aberrant phenotypes in specific cell types of the nervous system, leading to the patients' neurological symptoms. We postulated that patient-derived in vitro models of PTHS can better recapitulate the pathophysiology than mouse models, because brain structure, genome architecture and development vary greatly between rodents and humans, and current PTHS animal models do not closely mimic all the disease's clinically relevant aspects. In preliminary experiments, we obtained patient-derived brain organoids and cultured neural cell types in vitro and used them as human models to show that PTHS neural progenitor cells exhibit senescence and decreased proliferation, accompanied by downregulation of Wnt signaling and SOX3 expression. Moreover, we observed that PTHS brain organoids fail to develop normal anatomically organized progenitor structures and that PTHS neurons display severely impaired firing properties. Our anticipated results/deliverables include the identification and manipulation of specific altered molecular pathways and neural cell types and the testing of genetic correctional strategies for the disease, which could propel future research on pharmacological and gene therapy for PTHS.

NIH Research Projects · FY 2026 · 2021-02

Project Summary/Abstract Nicotinic acetylcholine (ACh) receptors mediate fast chemical neurotransmission and serve as therapeutic targets for neurological disorders, addiction, and autoimmune diseases. These pentameric ligand-gated ion channels assemble in diverse subunit compositions, enabling regulation of essential physiological functions such as cognition, muscle contraction, and inflammation. Despite significant progress, fundamental gaps remain in understanding the molecular basis of ACh receptor-associated diseases and the precise subunit assemblies in the brain. Building on our prior work, this project investigates three key areas of ACh receptor biology. Aim 1 focuses on myasthenia gravis (MG), an autoimmune disorder where autoantibodies target the muscle ACh receptor at the neuromuscular junction. Using human receptor preparations, we will employ cryo- electron microscopy (cryo-EM), electrophysiology, and functional assays to define pathogenic epitopes and elucidate how autoantibodies mediate receptor-based disease mechanisms. Aim 2 examines congenital myasthenic syndromes (CMS), which arise from inherited receptor mutations that alter channel function. We will determine high-resolution structures of wild-type and mutant receptors in the presence and absence of therapeutic modulators, linking structural defects to disease phenotypes and drug mechanisms. Aim 3 addresses a major unanswered question in the field: the native subunit compositions of neuronal ACh receptors. Leveraging our expertise in structural biology, we will isolate and determine the structures of receptors from mammalian brain tissue, providing fundamental insights into their physiological roles and pharmacological potential. The findings will advance understanding of ACh receptor function in health and disease, paving the way for novel therapeutic interventions for MG, CMS, and neurodegenerative disorders.

NIH Research Projects · FY 2025 · 2021-02

ABSTRACT Plasmacytoid dendritic cells (pDCs) are a unique subset of innate immune cells capable of multiple functions essential for antiviral responses, including type I interferon production, antigen-presentation and T cell activation. The mechanisms that govern these distinct pDC functions remain poorly defined; however, they could be mediated by distinct subpopulations. Using high-dimensional single-cell proteomic and transcriptomic approaches, we and others recently discovered a novel human dendritic cell (DC) population that is captured within traditional pDC definitions. These cells harbor phenotypic features of both pDCs and conventional DC subsets (cDCs); thus, we called them transitional DCs or tDCs. We have now performed an integrated multidimensional comparison that resulted in the identification of the mouse homolog of human tDCs. The discovery that tDCs occur in both human and mouse suggests they have an evolutionarily conserved role during immune responses. However, tDC function has never been investigated. Similarly, the developmental origin of tDCs has not yet been analyzed. This represents a fundamental gap in our understanding of the cellular components that mediate innate immune responses against viruses and poses an impediment to the development of therapeutics. Based on our preliminary data generated in mouse, we hypothesize that tDCs and pDCs form a distinct developmental lineage that cooperates at the site of viral infection to modulate immune responses. In three specific aims, we propose to query tDC origin, function and relationship with pDCs. To achieve these aims, we will take advantage of high-dimensional approaches already established in our lab, in vitro and in vivo differentiation assays, and novel lineage tracing and cell-specific depletion mouse models. We anticipate that findings from this proposal will enhance our current understanding of innate cellular pathways that result in the positive outcome of viral infection. Importantly, our integrated approach will incorporate analyses of both mouse and human tDCs; thus, it has the potential to reveal features of the innate immune compartment that are conserved between species. This proposal has the potential to impact the rational design of future therapeutic strategies.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT This project aims to disseminate validated technologies and resources of the National Center for Microscopy and Imaging Research (NCMIR) at UC San Diego to advance the completion of strategic goals of the BRAIN Initiative. The proposed technology integration and dissemination resource will provide the neuroscience community with technical help to obtain and manage large scale data, broadening the access to and incorporation of high throughput multiscale imaging tools and leading-edge analysis strategies in their studies. Richly integrated resources will be offered, including molecular probes, imaging platforms and data analysis tools certain to enable and hasten brain research. The types of research projects we will support, include: 1) investigations requiring the traversal of spatiotemporal scales to reveal new insight and understanding of specific neural populations; 2) investigations which aim to mark and track neuronal processes and/or visualize targeted connectomes of local circuits within large volumes of nervous tissue in multiple animal models; 3) projects which seek to perform nano-histological assessment of cellular and subcellular level alterations associated with learning, physiological state, or disease; and 4) projects performing higher resolution 3D morphometric analysis of subcellular underpinnings of function, with particular emphasis on synaptic function as influenced by subcellular constituents. This new center will leverage an administrative framework and information technology cyberinfrastructure which is already in place and has been refined over many years. In addition to providing a management structure, this framework includes a community outreach, project review, selection, and onboarding process refined and long practiced by the PI (and the assembled team). Additional mechanisms for tool/technology dissemination and training, data management and sharing, and mechanisms for cost-recovery and long-term sustainability will also be leveraged and expanded so as to maximize the reach and impact of our technologies and resources.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract This project is directly aligned with the NIA’s strategic goals of 1) developing improved approaches for the early detection and diagnosis of disabling illnesses and age-related debilitating conditions and 2) identifying appropriate strategies for disease, illness, and disability prevention and healthy aging among the underserved. As the older adult population continues to grow, it is expected that an increasing number of seniors will be living with Alzheimer’s disease and related dementias. As such, it is imperative to identify early risk markers of cognitive decline prior to symptom manifestation. Although Hispanics/Latinos (henceforth referred to as Hispanics) are at increased risk for mild cognitive impairment compared to non-Hispanic Whites, research investigating early risk markers in this growing and underserved segment of the United States (U.S.) population is lacking. One potential early risk marker of Alzheimer’s disease is subjective cognitive decline (SCD), which is used to describe self- reported perceived changes in cognitive function compared to a previous state. Although the expression, reporting, and predictive value of SCD may vary due to factors such as cultural/ethnic background, acculturation, and education level, little research has been conducted outside of non-Hispanic White cohorts. In fact, most existing SCD research with Hispanics has been conducted in Spain, whose population is very culturally different than Hispanics living in the U.S. The proposed study will help advance SCD research by characterizing the cognitive and biomarker correlates of SCD in U.S. Hispanics cross-sectionally, and by establishing its predictive value for cognitive change over three years. To achieve this, we will prospectively administer a validated SCD questionnaire, a culturally sensitive cognitive test battery, mood questionnaires (i.e., depression), and culturally- relevant measures that may influence SCD to older Hispanics with normal cognition (N=100) or mild cognitive impairment (N=100). We will also obtain SCD reports from participant’s informants to determine its differential ability to predict cognitive decline. Participant recruitment will leverage on existing cohorts at two sites: The University of California San Diego Shiley-Marcos Alzheimer’s Disease Research Center (ADRC) and the 1Florida ADRC. We will investigate if self and informant SCD reports are associated with concurrent, objective cognitive function (adjusting for relevant covariates) and examine if baseline SCD reports predict change in cognition over 3 years. Moreover, we will use existing biomarkers collected by the ADRCs, as well as novel blood-based biomarkers, to investigate if SCD is associated with amyloid-β and apolipoprotein E ε4 allelic status. Furthermore, we will investigate if acculturation, health literacy, country of origin, and language of testing, as well as demographic variables (age, sex, years of education) influence SCD reporting. Findings will characterize the cognitive and biomarker profile and predictive value of SCD in U.S. Hispanics, help refine SCD measurement, and identify individual differences in SCD reporting that may confer greater risk for decline.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is a disease of the pulmonary arterial vasculature and its remodeling, in which patient mortality is significantly associated with impaired RV function. The prognosis of patients with PAH is very poor with five-year survival <50%, and there are no available therapies to prevent right heart failure in PAH. Recent studies by the applicant’s group in animal models of PAH and clinical studies have shown that altered RV diastolic stiffness to be an important feature of PAH pathogenesis. Here we propose to investigate the structural mechanisms by which remodeling of RV extracellular matrix (ECM) alters their mechanical properties and the cellular mechanisms by which changes in RV mechanical loading and material properties in turn regulate the phenotype and pro-fibrotic signaling of cardiac fibroblasts. Using a comprehensive time-course in a well-established animal model of PAH progression, we will conduct detailed in-vivo physiological studies and biaxial tissue biomechanical testing of intact and decellularized RV samples together with microstructural mathematical modeling to determine how ECM remodeling alters RV myocardial mechanic and diastolic function. We will then recapitulate these alterations in RV ECM structure and mechanics in a novel in-vitro model to investigate how altered ECM structure and loading conditions in PAH regulate RV cardiac fibroblasts (CFB) differentiation, activation, and pro-fibrotic ECM expression. Finally, we will use these new in-vitro measurements to extend and validate a mathematical model of mechano- regulated CBF cell signaling. The specific aims of this proposal will determine the time course of changes in RV geometry, contractility and diastolic material properties that compensate for altered hemodynamic loads during PAH and determine how these mechanisms become maladaptive (Aim 1); the changes in RV myocardial structure and mechanics during adaptive and maladaptive RV ECM remodeling, and identify the biomechanical stimuli driving these changes in PAH (Aim 2); and the mechanobiological mechanisms regulating adaptive and maladaptive RV ECM remodeling during PAH (Aim 3). The overall outcome of the proposed research will be the discovery of quantitative biological principles of the RV extracellular matrix (ECM) remodeling that contribute to the changes in diastolic function that occur during the progression of PAH and the transition to decompensated RV dysfunction.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Nucleic acids and their building blocks play central roles in all cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. Studying such events is complicated by the non-emissive nature of the natural nucleobases, which frequently deprives researchers from the use of modern fluorescence-based techniques. Faithful minimally perturbing emissive nucleoside surrogates can thus facilitate the monitoring of nucleoside, nucleotides and nucleic acids-based transformations at nucleoside/tide- “resolution”, and advance basic research, diagnostic tools and drug discovery efforts. The goal of the proposed program is to design and synthesize new isomorphic emissive nucleoside and nucleotide analogs and implement them as probes for monitoring nucleoside- and nucleotide-based transformations as well as nucleic acids function, structure, dynamics and recognition. Specifically, major contemporary challenges will be tackled in an attempt to bridge major gaps, among them: (a) Powerful biophysical techniques, such as Fluorescence-Detected Circular Dichroism (FDCD), introduced nearly five decades ago, remains practically unexplored; (b) Multiphoton, imaging and single molecule spectroscopy- based experiments, using native or minimally perturbed oligonucleotides or nucleotide cofactors, are severely underutilized; (c) Similarly, single molecule enzymology of nucleoside/tide processing enzymes has not advanced; (d) Probes for real time exploration of fundamental processes such as peptidyl transferase, phase separated membrane-less organelle formation and mRNA decay are lacking; (e) Nucleoside/tide-based metabolic processes and nucleotide-based signaling events cannot be directly monitored; and (f) High throughput screening for nucleosides and nucleosides processing enzymes cannot be performed in real-time and in a high throughput manner without the use of faithful emissive surrogate substrates. Capitalizing on several useful families of emissive nucleoside surrogates developed in our laboratory, we will further refine our “designer” emissive and isomorphic nucleosides/tides and apply them to advance solutions to the challenges articulated above. We will pursue the advancement of new physical and biochemical methods, as well as effective real-time screening and diagnostic tools. These efforts will expand the community's arsenal of emissive functional probes, driving future strides into discovery and imaging applications. These innovations, in turn, will further fundamental understanding of key biological processes related to disease development and will have long-term impact on improving human health.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY Somatic mutations accumulate daily in every cell of the human body. These mutations originate from mutational processes due to environmental exposures, lifestyle choices, defective cellular machineries, and even normal cellular activities. Each mutational process imprints a characteristic pattern of mutations on the genome of somatic cells, termed “mutational signature”. Since somatic mutations are retained in the genomes of cells and their progenies, the presence of mutational signatures in a somatic genome serves as an “archaeological imprint” of the activities of the mutational processes that were operative during a person’s lifetime. Recent developments of computational tools have allowed identifying mutational signatures from the DNA sequences of cancer samples and quantifying the activities of different mutational processes in individual cancer patients. Analysis of many thousands of cancer patients across the world has now revealed almost 80 distinct mutational signatures. Importantly, for each of these patients, we now know the mutational processes that have caused their cancers and, for many of these patients, we could identify potential strategies to reduce environmental exposures and prevent their cancers. However, an effective and timely cancer prevention requires knowing the mutational processes operating in a healthy individual and eliminating or reducing the activities of these processes before that individual develops cancer. Unfortunately, currently, there are no approaches that allow quantifying mutational signatures of environmental exposures in a healthy individual and, thus, many opportunities for personalize cancer prevention are missed. Here, we propose to develop a novel computational approach that will allow noninvasive monitoring of mutational signatures in easily accessible normal somatic tissues of healthy individuals. Our approach will perform a direct detection of somatic mutational signatures from low coverage single-cell DNA sequencing data without relying on prior identification of somatic mutations. The approach will be optimized and validated using single-cell DNA sequencing data from: (i) in vitro cell lines exposed to environmental mutagens; (ii) an in vivo mouse model consuming water contaminated with a strong chemical mutagen; (iii) healthy individuals with established exposures to known environmental mutagens. Overall, this project will transform our ability to monitor the activities of the mutational processes in normal tissues of healthy individuals and it will open a plethora of opportunities for personalized cancer prevention through possible targeted interventions that reduce mutagenic exposures from environment agents and lifestyle choices.

NIH Research Projects · FY 2025 · 2021-01

Abstract The goal of cancer immunotherapy is to utilize the patient’s immune system to reject the invading “foreign” tumor. However, the pancreatic cancer microenvironment is characterized by an abundance of immunosuppressive cells and a dense stroma that prevents infiltration of anti-tumor immune cells. Electroporation is a technique that has been utilized for decades in the laboratory; electrical voltage is applied to cells to make holes for delivery of DNA and RNA. Irreversible electroporation (IRE) is a technique now being used clinically for ablation of localized tumors that cannot be removed surgically (locally advanced tumors). Our objective is to use IRE as an "in situ vaccine" to help the host recognize foreign tumor proteins (neoantigens) and generate anti-tumor immune responses that will decrease recurrence rates. We have utilized mouse models of pancreatic cancer to show that IRE generates anti-tumor immune cells that prevent growth of new tumors (prophylactic immunity). We hypothesize that combining IRE with agents that augment the immune response will result in inhibition of established, distant tumors (therapeutic immunity or “abscopal” effects). In Aim 1, we will use mouse models to compare the effects of IRE to radiation therapy (XRT), as this is the most relevant clinical comparator. Both methods are used clinically for the ablation (killing) of locally advanced pancreatic cancer but have been shown stimulate systemic immune responses in preclinical models. We hypothesize that IRE will induce stronger immune responses because XRT causes fibrosis (scarring) that will inhibit immune cell infiltration. In Aim 2, we will combine local ablation with local delivery of agents that stimulate the innate immune system in mouse models of metastatic pancreatic cancer. In Aim 3, we will use a novel model in which human tumors and their associated immune cells are implanted into immunocompromised mice in order to create a “humanized” immune system. We will use this model to study the effects of IRE on human tumors. We have assembled a multi-disciplinary team that encompasses broad expertise in IRE, mouse tumor models, stromal biology, immunotherapy, clinical trials, and clinical care of patients with pancreatic cancer. We envision that the combination of IRE with immunotherapy will be first beneficial to patients with locally advanced pancreatic cancer. However, if effective, this approach may also be beneficial to patients with metastatic disease. Since the IRE technique is already in use clinically, a clinical trial in which one or more of the agents to be studied is delivered during or after IRE as adjuvant therapy would likely be feasible in the near future. We will use data from the proposed research to design such a study.

NIH Research Projects · FY 2026 · 2021-01

PROJECT SUMMARY/ABSTRACT The overall goal of this MIRA and its preceding R01 grant has been to describe in molecular detail the mechanism of action and specificity of physiologically important human forms of phospholipase A2 (PLA2). We have demonstrated that the association of a water-soluble PLA2 enzyme with the membrane or micelle interface containing its phospholipid substrate causes a specific conformational change in each enzyme resulting in its allosteric activation. We also showed that each type of PLA2 exhibits a unique specificity for distinct phospholipids as each can discriminate exquisitely between different fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse free polyunsaturated fatty acids (PUFA), some of which are converted to potent downstream inflammatory mediators including eicosanoids and related oxylipins. We have now determined each PLA2’s general specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a critical role in which specific phospholipids are hydrolyzed by each enzyme type. This renewal application will extend our current studies on the pure recombinant human cytosolic Group IVA cPLA2, secretory Group V sPLA2, Ca2+-independent Group VIA iPLA2, and lipoprotein-associated Group VIIA LpPLA2 focused on how each of them interacts with their relevant membrane surface and determine their specificity in their natural environment in intact macrophage cells in cell culture. During the renewal period, we will focus on three new directions. First, we will explore the functioning and physiological role of the three intracellular PLA2s in macrophages, where the specificity will depend on the proximity and availability of optimal phospholipid molecular species as substrate. We developed a new platform for measuring PLA2 specificity and inhibition ex vivo in macrophages by identifying the major phospholipid molecular species and quantifying the amounts of each remaining after specifically activating each enzyme. We will now employ this platform to identify the specific double-bond positions separately in the sn-1 and sn-2 fatty acids in each of these phospholipid molecular species. Second, during the current grant, we discovered specific allosteric sites for PIP2 on cPLA2 and ATP on iPLA2 and we will explore their role in how the regulatory domain of each of these key enzymes associate with the membrane and will extend our molecular dynamics (MD) simulations with relevant intracellular macrophage membranes and enhanced sampling MD methods. Third, we will further apply what we learned with phospholipases to a triglyceride lipase PNPLA3, which is homologous to iPLA2. A natural mutation (I148M), enriched in Hispanics, leads to an increase in fatty liver disease and decreased hydrolysis of lipid droplets. We will generate important widely applicable novel information on how physiologically important phospholipases and triacylglycerol lipases interact with the lipid-water interfaces of membranes, micelles, lipoproteins, and lipid droplets to compete physiologically in selecting their substrates. We hope to fully explain and integrate at a structural level the resulting specificity of the PLA2 superfamily acting on the specific lipid molecular species.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT Fibrosis, defined by the deposition of collagen I, is a devastating pathological event that occurs in many organs including the heart, kidney, liver and lung in response to injury and inflammation. This fibrotic response inhibits recovery inflammation and can even lead to organ failure. Despite the potential importance, very little is known about whether there is a fibrotic response in the central nervous system (CNS) following neuroinflammation that occurs in diseases such as multiple sclerosis, neuromyelitis optica, stroke and CNS infections, and how this response affects repair and recovery. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of neuroinflammation, we have identified that a robust collagen I- based fibrotic scar forms covering the neuroinflammatory lesion and we hypothesize that this fibrotic scar inhibits the ability of reparative cells to enter the lesion. In preliminary studies using lineage tracing and single cell sequencing, we have identified that this fibrotic scar is formed by the activation and proliferation of fibroblasts. We have further generated methods to isolate and culture CNS fibroblasts providing an in vitro model to study the proliferation, migration and collagen 1 production from these cells. In this proposal we aim to determine whether the fibrotic scar is helpful or harmful for recovery following neuroinflammation and to further study the mechanisms that regulate fibrotic scar formation. We will first determine whether inhibition of fibrotic scar formation can lead to an increased recovery from EAE. We will then examine whether TGFβ and PDGFR signaling pathways regulate fibrotic scar formation. We hypothesize that TGFβ signaling drives the proliferation and collagen I production by the fibroblasts and that PDGFR signaling regulates the migration of the fibroblasts to the lesion. Our goal is to determine whether modulating the fibrotic scar is a potential therapeutic target to aid in recovery for patients with neuroinflammatory diseases.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY/ABSTRACT The prevalence of Type 2 diabetes mellitus (T2DM) is rising dramatically on a global basis and has now reached epidemic proportions. Current anti-diabetic therapeutics are available, but are inadequate to fully control T2DM in most patients, resulting in a great deal of morbidity and mortality. Insulin resistance is a major etiologic cause underlying T2DM. However, there are no clinical options to directly improve insulin sensitivity, except for thiazolidinediones, which are infrequently used due to unwanted side effects. Obesity is the most common cause of insulin resistance, and accumulating evidence indicates that obesity-induced inflammation (or metaflammation) is an important cause of insulin resistance and obesity- associated metabolic complications. Therefore, there are huge unmet medical needs for the prevention and treatment of metaflammation. However, it is not known how chronic metaflammation is initiated and propagates during the development of obesity, and why it is not resolved. Macrophages are the major immune cell type mediating metaflammation, and evidence suggest that changes in macrophage mitochondria activity can promote pro-inflammatory activation and M1-like polarization of macrophages. However, how mitochondrial activity is regulated during the course of M1- or M2-like macrophage polarization, especially in the obese/energy-excess adipose tissue microenvironment is not clearly understood. We will tackle the question of obesity-induced metaflammation from a new angle, focusing on mitochondrial biology with a novel target and hypothesis. Our preliminary suggest that ANT2 is a mitochondrial sensor of FFAs in macrophages to induces mitochondria remodeling favoring pro- inflammatory activation. Since the major source of increased plasma FFAs in obesity is adipocytes, FFA- induced ANT2 activation can provide an adipose tissue-specific pro-inflammatory macrophage activation mechanism in obesity. Interestingly, the effect of macrophage ANT2 KO includes the changes in mitochondrial number, mass, and capacity, whereas adipocyte or myocyte ANT2 KO does not cause these changes. This suggest that macrophage ANT2 mediates mitochondria remodeling through a cell type-specific mechanism that is distinct from ANT2 effects in adipocytes and myocytes. We will explore whether macrophage ANT2 mediates the initiation and/or maintenance of metaflammation in obese adipose tissue. We will then assess how ANT2 regulates mitochondrial capacity, dynamics, and metabolism to support the pro-inflammatory activation of macrophages, and how macrophage ANT2 activation induces insulin resistance. If successful, our studies will provide a novel mechanism for how obesity induces tissue-selective metaflammation and insulin resistance. Successful completion of the proposed study will identify ANT2 as a potential target for novel therapeutics to prevent metaflammation, insulin resistance, and glucose intolerance.

NIH Research Projects · FY 2026 · 2020-12

Project Summary/Abstract An increasing number of undergraduates (~30% in the University of California system) enter research universities after 2+ years in community colleges. Although many transfer students express interest in research, they have little to no access to such experiences at community colleges, and very few continue onto the PhD. Soon after matriculating at universities, they tend to opt out of scientific research careers, particularly the idea of continuing to graduate school. In response to this need, we have developed a program to provide transfer students with the skills, mentorship, and research experience to improve their chances of success in a research career. Here, we propose to continue the BP-ENDURE STARTneuro program, which for four years has brought cohorts of transfer students to campus in the summer before matriculating to UC San Diego. Via partnerships with local community colleges and in collaboration with multiple campus offices (e.g., the Triton Transfer Hub), the STARTneuro program begins by identifying interested students before they apply to UC San Diego. Accepted participants then engage in an 8-week summer bridge program, with intensive and immersive full-day lab training in neuroscience techniques, from physiology to gene expression and function. Each student also designs and implements an independent research project based on the summer modules. During the academic year, students meet regularly with faculty, are shepherded into funded lab internships, and are mentored in applying for additional research opportunities and ultimately graduate programs. A core group of faculty who have demonstrated success mentoring undergraduates will provide the research experience and stewardship necessary to ensure that participants can succeed in scientific research beyond college. Even in our first four years, we have observed improved outcomes for our STARTneuro scholars, with all of them persisting in STEM and several en route to a Ph.D. We are requesting a renewed grant to continue building on this foundation so that we may increase our reach into more community colleges, propelling more of these transfer students into neuroscience career paths.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY/ABSTRACT Communication between neurons and their targets depends on proper synaptic growth and activity. The microtubule cytoskeleton plays a central role in synaptic terminal development, and microtubule dysfunction is associated with many neurological disorders. Neurons contain stable and dynamic microtubules, and these two populations must be properly balanced for synapses to grow and form stable connections. In this proposal, we use a synergistic combination of in vivo genetic analyses and cell-free in vitro biophysical approaches to elucidate the mechanisms by which microtubule dynamics and stability are balanced. We leverage a novel α- tubulin mutant that alters the normal microtubule balance and perturbs synaptic growth. This tubulin mutation disrupts a highly conserved, essential α-tubulin site that is acetylated. Post-translational modifications (PTMs), such as acetylation, have the potential to directly and specifically regulate microtubule stability and dynamics to shape synaptic morphogenesis, yet relatively few microtubule PTMs have been studied. Our preliminary data implicate this previously uncharacterized α-tubulin site in regulating the addition of tubulin dimers to growing microtubule ends, which suggests a novel acetylation-based mechanism to control microtubule dynamics. Based on our preliminary findings, we will test the hypothesis that microtubule dynamics and stability are balanced by α-tubulin acetylation and other known regulators to shape synaptic terminal morphogenesis (Aim 1). We will use the Drosophila neuromuscular junction as a model and investigate the effects of manipulating microtubule dynamics and stability on two different motor neuron types, called type Ib and type Is, whose synaptic terminals have distinct morphologies and transmission properties. Our preliminary data indicate that altering the microtubule cytoskeleton has strikingly different effects on the growth of type Ib and Is synaptic terminals. We will test the hypothesis that stable and dynamic microtubules are uniquely balanced in different neuron types to establish distinct neuron-specific synaptic structures and activities (Aim 2). Combined, our studies will reveal novel mechanisms that regulate synaptic microtubule networks and provide fundamental new insight into the central role that microtubules play in creating diverse synaptic morphologies and functions.

NIH Research Projects · FY 2025 · 2020-12

Central nervous system (CNS) complications are common among people with HIV (PWH), even those who are taking antiretroviral therapy (ART). The spectrum of CNS complications is broad, ranging from mild cognitive deficits that do not affect daily functioning to life-threatening encephalitis. Cognitive and mood disorders are among the most common CNS diseases that affect PWH and share a common risk factor, inflammation. Inflammation persists in effectively treated PWH for multiple reasons, including production of HIV RNA and proteins and gut dysbiosis and microbial translocation. CHARTER is a multisite, U.S.-based, neuroHIV cohort study that is funded by NIH and that has investigated CNS disorders in PWH for nearly two decades, during which it has completed more than 6,000 assessments generating more than 10 million data points. CHARTER has made important contributions to understanding the frequency, risk factors, and pathogenesis of these disorders. In recent years, new questions have arisen about the heterogeneity, biological basis, clinical impact, and management of CNS disorders. This debate has highlighted the need to create new classifications of CNS disorders in PWH that are more biology-based. We propose to use methods such as machine learning and an agnostic approach to categorize CHARTER’s high-dimensional neurobehavioral, neuromedical, psychiatric, substance use, and imaging data. Such analyses would yield not just cognitive phenotypes but biopsychosocial (BPS) phenotypes that could identify new mechanisms that lead to clinically useful diagnostic tests, new therapies, and better management of CNS disorders in PWH. Our overarching goal is to leverage prior investment in CHARTER by using its data and stored specimens to a) better understand cognitive and BPS phenotypes in PWH and b) relate them to biological mechanisms. To accomplish this, we will use unsupervised and supervised machine learning methods to analyze data from CHARTER’s comprehensive assessments with the goal of identifying new cognitive and BPS phenotypes (Aim 1). We will then compare these new phenotypes to high-dimensional data from CHARTER’s completed genomewide association study as well as new data we will generate on inflammation (45-plex bead-based array) and highly novel assays of the microbiome and the metabolome in blood and CSF (Aim 2). Our analyses will include a specific focus how sex affects the observed relationships. To determine if this novel approach relates more strongly to biology than prior methods, we will also compare the performance of the new phenotypes to those defined by the 2007 HAND criteria. This highly innovative application is supported by strong preliminary data, responds well to Office of AIDS Research priorities, and will address key gaps in the field, including the need to better understand the pathogenesis of comorbid disease.

NIH Research Projects · FY 2025 · 2020-12

Project Summary Human cognition is a product of vastly complex interactions between different cellular networks that continues to elude a mechanistic link to neurobiology. Our understanding of the brain basis for cognition has benefitted from studying human conditions in which cognitive disability is linked to single gene mutations that disrupt healthy cortical development early in life. One such condition is Rett syndrome (RTT), a severe neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 that presents a variety of clinical symptoms including transient autistic-like behavioral deficits, severe cognitive impairments, and abnormal scalp electrophysiology (EEG) activity. However, it is unclear how abnormal cellular network formation gives rise to reported patient EEG abnormalities and associated cognitive disability. Our understanding is severely limited by a fundamental lack of knowledge of any convergent mechanisms relating gene mutations and cortical activity in the developing human brain to cognition within the same individual. This proposal aims to test the hypothesis that there is a convergent mechanism for aberrant cortical activity and cognitive disability housed in the patient transcriptome that can be identified across electrophysiological scales, ranging from in vitro brain organoid electrophysiology to non-invasive human EEG. By using RTT as a genetic model for human neurodevelopment, this proposal aims to leverage multimodal brain data from the same individual to advance our understanding of the neurobiological mechanisms underlying cognition in health and disease. To do so, this proposal will identify biomarkers of cortical function via parallel in vivo and in vitro analytical approaches relating electrophysiological and genetic features to cognitive ability and disease severity. This research will use state-of-the-art analytical tools to parameterize electrophysiological periodic and aperiodic features of scalp EEG from RTT and healthy participants and correlate them to genetic and electrophysiological features of brain organoids derived from the same human participants’ induced pluripotent stem cells. Project findings will define novel electrophysiological and molecular biomarkers for cognitive ability across brain data modalities. These results are expected to have a positive impact in the development of preclinical models of neurodevelopmental disorders as they will provide a strong evidence-based proof of principle for using human-derived tissue cultures to test potential new, personalized therapies in a safe and high-throughput manner.

NIH Research Projects · FY 2025 · 2020-12

Project Summary – Overall: Developmental Mechanisms of Human Meningomyelocele The central goal of this Program Project application is to understand mechanisms of Meningomyelocele (MM), the most severe neural tube defect (NTD) compatible with survival, a condition in which folic acid (FA) fortification has had a major impact on disease risk. This PPG is designed to advance biomedical knowledge and make a high impact on our understanding of the molecular genetics of MM across the evolutionary scale, with the purpose of advancing our ability to determine disease risk, and establish mechanisms by which FA alters risk. MM is the most common birth defect of the central nervous system, affecting 3.7 per 10,000 live births, and is one of the high impact conditions prioritized by the NIH for research. In our preliminary data we have: 1] Constructed a cohort of over 1500 human trios with MM, stratified by whether the child was conceived in a FA-supplemented geography. 2] Established Xenopus laevis as a high-throughput model to assess human mutant alleles, gene-gene interactions, and FA exposure. 3] Established a number of murine NTD models with measured effect of FA on penetrance and expressivity. 4] Demonstrated a proven track record of applying these tools to study mechanisms of disease. As a result of the extensive preliminary data presented below, we have formulated this PPG with a two-fold thrust: 1] By taking advantage of the technical revolution in next generation sequencing and CRISPR genetic engineering, we will uncover and functionally assess new MM risk factors. 2] By comparing phenotypes across the evolutionary timescale, we will enhance our understanding of the basic mechanisms of NTDs and the impact of FA. The central theme running throughout the application is Gene-Environment Interaction (GXE), because of the important role FA has on MM risk in human, mouse and frog, and because the theme applies to all three Projects and Cores. Three Cores will carry out essential functions and benefit each Project. 1] Administrative Core to facilitate communication and provide opportunities for scientific collaboration. 2] Epigenomics Sequencing Core to provide essential functions in assessing FA-dependent DNA methylation and other impacts on chromatin and transcription. 3] Bioinformatics Core to provide essential functions in data processing and harmonization, mutation identification, and custom computational solutions. Specific Aims of the PPG are: 1] To uncover a host of new developmental causes of MM from this unique human cohort, as well as from mouse and frog models. 2] To explore mechanisms by which FA reduces disease incidence in human, mouse and frog. 3] To utilize mechanisms uncovered in mouse and frog NTD models to inform gene prioritization in human MM. We believe that this PPG will have a major impact on our understanding of the cellular and molecular mechanisms underlying NTDs, taking advantage of new breakthrough technology, and will set the stage for improved diagnosis and ultimately prevention of disease.

NIH Research Projects · FY 2026 · 2020-12

PROJECT SUMMARY/ABSTRACT About 12 million individuals over the age of 12 have a substance use disorder (SUD). Cocaine is involved in about 1 in 5 overdoses in the United States and is abused by both men and women. Women are more vulnerable to the rewarding effects of cocaine and more rapidly progress from initial use to dependence than men but the neurobiological mechanisms that contribute to these differences are yet to be determined. A limitation of the current work investigating sex differences in preclinical research is that the research is primarily focused on hormonal contributions that may be driving sex differences. There is little understanding of how sex steroids and other neurobiological mechanisms contribute to cocaine dependence. The overall goal of this project is to identify the effects of a history of cocaine self-administration on sex steroids and nicotinic acetylcholine receptor (nAChRs) subunit expression and to test the hypothesis that decreased nAChRs alpha 5 (ɑ5) in the dorsal hippocampus (dHIPP) produced by decreased progesterone levels promote cocaine self-administration behavior. Aim 1 will use single-cell transcriptomics to identify sex differences in the dysregulated transcriptional networks in rats with a history of compulsive-like cocaine self-administration using brain (dHIPP), adrenal, pituitary, reproductive organ samples. Aim 2 will causally investigate the role of the nAChR ɑ5 in the dHIPP and progesterone on cocaine self-administration behavior using an AAV-ShRNA to knock down nAChRs ɑ5 in the dHIPP and pharmacology to block the progesterone receptor. These projects will be the first to look at associations between sex steroids and nAChR ɑ5 in the dHIPP on cocaine selfadministration in both sexes. My Sponsor, Dr. Olivier George (UCSD) is an expert on the neurobiology of drugs of abuse and addiction and is recognized as an innovative leader in the field of drug abuse and, therefore, the perfect fit to mentor me for the proposed research. The proposed Research and Training Plans will deepen my knowledge of transcriptomics, the cholinergic system, the hippocampus, and neuroendocrinology in addictionlike phenotypes and will expand my expertise in cocaine dependency. The training I will receive in single-cell transcriptomics, ELISAs, bioinformatics, intrajugular catheter surgeries, and advanced operant paradigms will give me the skills necessary to answer questions focused on cell-specific differences in males and females that may be driving dysregulation of reward circuitry pre- and post-drug exposure. The training I have received in the F99 phase and the training I will receive in the K00 phase will allow me to establish a lab where I can research my long-term research goal of investigating the mechanisms underlying maladaptive addiction behaviors and how these behaviors differentiate by sex.

NIH Research Projects · FY 2025 · 2020-12

Charge matters: Pursuing the most common, and least understood molecular interactions in cells PROJECT SUMMARY / ABSTRACT The long-term objective of the Süel lab is to determine and understand how ion fluxes and electrostatic interactions regulate fundamental biological processes that promote stress tolerance in bacteria. The central problem to be addressed: The vast majority of molecular interactions that occur within any living cell have remained obscure. How can this be? Nearly all molecular interactions that have been studied to date, and on which our current understanding of biology is based on, are covalent interactions. These interactions are strong, making them suitable for experimental measurements. However, the vast majority of interactions among molecules within the cell are non-covalent interactions that are based on electrostatics. Electrostatic interactions can be weak and short-lived, and thus their measurements pose a great technical challenge. Consequently, how such interactions are regulated and what functions they play in cells remains largely unknown. To bridge this gap, we propose a research program to develop new devices, techniques, and a theoretical framework to investigate the functional roles of electrostatic interactions, specifically in bacterial cells and biofilm communities. Impact: The proposed work aims to investigate the regulation of ionic interactions and their functional roles in bacteria, to better understand and control their tolerance to antibiotics. The resulting findings will determine how changes in ionic strength and composition affect cell physiology. We will thus begin to characterize the dynamics of the prokaryotic “metallome”. We will also integrate quantitative experiments with physics-based theoretical approaches to identify general principles governing electrostatic interactions that can be applied beyond our bacterial model systems. Given the tremendous number of ionic interactions within any given cell, it is very likely that our work will uncover a new layer of molecular regulation of fundamental biological processes. Specifically, we postulate the hypothesis of “ionic allostery”, where we propose that cells regulate their cytoplasmic ion composition to modulate electrostatic interactions, and thereby globally regulate transcription and translation. In particular, ionic interactions may play a crucial role in bacterial cell fate decisions, such as entry into, and exit from dormancy, which is the major cause of antibiotic resistance. Our work will thus reveal whether “the central dogma of biology” is modulated by changes in the ionic composition and strength of the cytoplasm and provide a new paradigm for understanding and controlling the regulation of fundamental stress responses in bacteria.