University Of California, San Diego

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Peripheral insulin administration and impaired insulin clearance lead to hyperinsulinemia and insulin resistance in individuals with type 1 diabetes (T1D). As independent risk factors for cardiovascular disease (CVD), hyperinsulinemia and insulin resistance contribute to the high burden of CVD experienced by this population. The long-term goal is to develop approaches to reduce the excess risk of CVD in T1D. As the next step toward this goal, the overall objective of this proposal is to quantify the ability of combination adjunct-to-insulin therapy to reduce hyperinsulinemia and insulin resistance in T1D. The central hypothesis is that combination adjunctive therapy will (1) mitigate peripheral hyperinsulinemia via decreased insulin dosing and increased insulin clearance and (2) improve insulin resistance in muscle, adipose, and liver tissues. The rationale for this work is that gaining insight into the relationship between hyperinsulinemia and insulin resistance will inform new treatment strategies to mitigate the risk of CVD in T1D. The central hypothesis will be tested in a double-blind, placebo-controlled, crossover study in which participants with T1D will receive combination adjunct-to-insulin therapy and double placebo treatment, in random-order. The following specific aims will be pursued: (1) Quantify the reduction in hyperinsulinemia induced by combination adjunctive therapy and (2) Quantify the effect of reducing hyperinsulinemia on tissue-specific insulin resistance. For the first aim, ambulatory insulin dosing and circulating insulin concentrations will be measured, and the metabolic clearance rate of insulin will be calculated to determine the relative contribution of insulin dosing and insulin clearance in reducing hyperinsulinemia. For the second aim, insulin sensitivity will be measured in adipose, hepatic, and skeletal muscle tissue using hyperinsulinemic-euglycemic clamps with stable isotope tracer. This research is innovative because it leverages a unique therapeutic intervention (combination adjunct-to-insulin therapy) to achieve meaningful improvements in insulin clearance, hyperinsulinemia, and insulin resistance in T1D. This research is significant because it is expected to demonstrate a novel treatment approach and justify the study of adjunctive therapies to mitigate hyperinsulinemia, insulin resistance, and potentially CVD risk in T1D. The proposed studies will also provide a framework for mentored research training. The principal investigator (PI) seeks to gain expertise in advanced human metabolic research techniques to study insulin clearance, insulin resistance, and CVD in T1D. The PI has established a comprehensive training plan and a multidisciplinary mentorship team with diverse but complementary expertise to achieve the research and career development goals and transition to independence. This training will prepare the PI to pursue high impact research using novel therapies to mitigate hormonal and metabolic imbalances in T1D, with the mission of improving the health and wellbeing of people living with the disease.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY The airway epithelium serves as a barrier between the aerosol environment and the underlying submucosa. Inhaled air often carries noxious agents and pathogens that can injure the lung. Severe lung injury can lead to persistent inflammation, dysplastic repair, and permanent loss of gas exchange surface area. Injury from common respiratory viral infection, such as influenza and SARS-CoV-2, can lead to chronic lung disease or exacerbation of lung conditions. Airway cells contribute to regeneration following lung injury, although the heterogeneity of airway cell identity following lung repair is incompletely understood. My research centers on rare airway cell types and their function in the post-viral lung. We have previously studied the differentiation of an unexpected airway cell type, tuft cells, during repair. Using single nucleus expression and accessible chromatin sequencing to survey airway cells following influenza injury, we identify a rare airway cell present in the post-viral lung, Microfold (M) cells. M cells have not been studied in the lung but in other contexts associate with lymphoid follicles, where they function to capture and deliver luminal antigens and secrete chemokines. The proposed research will identify pulmonary M cell progenitors and determine mechanisms required for M cell differentiation. This proposal uncovers M cells as part of a follicle associated epithelium (FAE) overlying induced bronchus associated lymphoid tissue (iBALT), implying M cells function to promote mucosal immune responses. The proposed aims will define the role of M cells in lung immune surveillance. My future research program will test the role of M cells in secondary bacterial infection, as much of the morbidity of pulmonary influenza infections can be attributed to secondary bacterial infection. Examining interactions between M cells and the immune compartment will contribute to our understanding of immune regulation during post-viral chronic inflammation. A second outcome from my focus on airway repair following severe influenza infection is an effort to promote the resolution of basal-like scar tissue into normal alveolar epithelium. Bronchiolization of the distal airway is a hallmark of multiple human lung diseases, and providing regenerative therapies will require detailed understanding of the regulation of basal-like cells. I propose defining a genetic mouse model that promotes differentiation of alveolar basal-like cells into alveolar epithelial cell fates. My primary mentor is Dr. Xin Sun, a leader in the field of lung biology who has made fundamental discoveries in lung development and disease. I will receive guidance from my mentorship committee, a group with expertise in lung biology and physiology, immunology and epigenetics. The proposed experiments and training plan will further my skills in bioinformatics and immunology. This research will be conducted at University of California San Diego, a leading research institution with necessary resources and a collaborative scientific community.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY Black women (BW) in the US are disproportionately affected by HIV. Concurrently, Black women face sociostructural inequities including structural racism, HIV stigma, and medical mistrust, in addition to the syndemic effects of substance use, and intimate partner violence, and adverse mental health. These factors act as barriers to health seeking behaviors that contribute to poor HIV outcomes. BW’s participation in HIV clinical research remains historically low and BW’s participation in HIV research (i.e., prevention, treatment, cure), which remains historically low, is contextualized within a system that does not consider their intersectional identities (i.e., race, gender), while routinely excluding women due to biological (e.g., reproductive potential, menstruation, pregnancy), social (e.g., restrictive contraception requirements, provider bias, perceived as “hard to reach”), and structural factors (e.g., lack of welcoming research spaces including extended hours and childcare support). While studies have identified factors that limit women’s participation in HIV research, there is limited data on perceptions of HIV research participation among BW. The prevalence of illicit substance use (e.g., cocaine, amphetamines) among Black women is higher than the national average. Consequently, there is a significant risk of BW not benefitting from HIV related scientific advances (i.e., access to HIV medications, curative therapies) derived from research. Guided by syndemic theory and intersectionality framework, this explanatory sequential mixed methods study will utilize a case control approach to elucidate the effects of sociostructural inequities and past-year substance use, and past-year intimate partner violence, and adverse mental health on participation in HIV research among BW (Aim 1). For the case-control portion of the study, we will recruit, screen, and match participants 1:1 across case and control groups (n=280, cases (n=140): participated in research; controls (n=140): not yet participated in HIV research) along factors of (a) HIV serostatus and (b) recruitment source (sociobehavioral, clinical, vaccine, biomedical prevention study and non-research efforts. Understanding these effects on BW participation in HIV research may elucidate aspects for intervention (e.g., policy changes regarding stopping rules, minimum participant thresholds) to ensure more diverse study populations in HIV research. The proposed study will include three groups: a) BW enrolled in ongoing sociobehavioral research studies by members of the mentoring team, LinkPositively (R34MH122014; PI: Stockman) and Women Shine; (R01MH125785 PI Stockman) who agreed to be contacted about future research opportunities (n=40), b) BW with histories of research participation who agreed to be contacted about future research opportunities, and those seeking care at AIDS Clinical Trials Group clinical research sites without a history of research participation (n=65), c) participated in LinkPositively (Grant #R34MH122014; PI: Stockman; n=70), an mHealth HIV intervention for BWH in California and Oklahoma affected by interpersonal violence, c) Participants of the Enhanced Peer Involvement in Care Amplifying the Voices of BW (EPIC) program embedded within existing peer navigation programs in community-based organizations located in Ending the HIV Epidemic jurisdictions in the Southern United States, and d) BW without HIV and with histories of research participation who agreed to be contacted about future research opportunities and those seeking care at an HIV Vaccine Trials Network clinical research site without a history of research participation (n=140). Next, we will qualitatively explore perceptions of and facilitators and barriers to participation in HIV research among BW with and without HIV (Aim 2).The proposed study, which is in response to PAR-22-172, will support the scientific investigation by underrepresented pre- doctoral students, and is the first to attempt to quantify and contextualize the impacts of sociostructural inequities and syndemics on BW’s willingness to participate in HIV research to inform multilevel efforts and interventions to conduct equitable and diverse research.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Ovarian cancer is the most lethal gynecologic malignancy for women; standard of care surgery and chemotherapy treatment can provide a short period of remission but cannot eradicate the disease and prevent recurrence. Cancer immunotherapy has shown great potential in treating cancers, but no clinical success has been reported for ovarian cancer. One major hurdle for cancer immunotherapy that is needed to overcome is to convert the immunosuppressive tumor microenvironment (TME). The goal of this proposal is to develop effective nanomedicines that are capable of reprogramming and converting the suppressive TME for ovarian cancer treatment. To achieve this goal, I will utilize viral nanoparticles (VNPs) to incorporate various functionalities targeting different aspects of the TME through bioengineering approaches. The two VNPs that will be used in this proposal are cowpea mosaic virus (CPMV) and hepatitis B virus capsid (HBVc), which both have well-characterized and stable structures for in vitro bioengineering. Toll-like receptor (TLR) agonists have been demonstrated to be potent to activate the innate immune system and modulate the TME. CPMV is a triple TLR 2, 4, and 7 agonist and is effective to reprogram the TME of ovarian cancer. In the mentored K99 phase, I will focus on using CPMV as a triple TLR agonist to develop an adjuvant and antigen combination in-situ vaccine for ovarian cancer treatment (Aim 1) and developing multi-TLR agonists to investigate the mechanism of action (MOA) of CPMV and multi-TLRs activation in cancer treatment to design potent TLR agonists combination for downstream applications (Aim 2). During my independent R00 phase, I will use HBVc as a nanotechnology platform to develop multiple functional therapeutic nanomaterials aiming to reprogram the suppressive TME to treat ovarian cancer and investigate the MOA. First, I will develop HBVc-based TLR agonist and pro-inflammatory cytokine combination therapies, which can exert the functions of reprograming the TME and killing cancer cells concurrently (Aim 3). Secondly, I will develop HBVc into a “smart” nanoparticle that functions as a TLR agonist and targets and converts the pro-tumor M2 macrophages into anti-tumor M1 macrophages (Aim 4). During my graduate study, I have been trained in manipulating HBVc in vitro assembly and genetic engineering of HBVc to design novel structures. In the past two and a half years as a postdoc in Dr. Steinmetz’s lab at UCSD, I have been trained systematically in the bioengineering of VNPs and the application of engineered VNPs for cancer treatment. A further two years of training in Dr. Steinmetz’s lab will allow me to enrich my background in cancer immunology, immune-oncology, and tumor modeling. With the help and guidance from my advisory committee, by the end of my mentored phase, I will be able to secure a tenure-track faculty position in a top-tier research institute to establish my independent research program focusing on using HBVc as a nanotechnology platform to develop novel and effective multi-functional nanomedicines for cancer patients.

NIH Research Projects · FY 2025 · 2024-05

Project Summary Depression affects 1-in-5 individuals in the United States and has a tremendous cost burden for our economy at $210 billion per year. Depression is hard to treat given that it is a heterogeneous illness associated with affective, cognitive and behavioral dysregulation. Around 33% of depressed individuals will not respond to the first two treatments offered. In this context, there is increasing evidence that cognitive functioning in major depression may be an independent factor predicting treatment response, quality of life, disability and suicide, and cognitive control (CC) deficits may persist even when other depression symptoms remit. For instance, FDA-approved therapies for treatment-resistant-depression (TRD) such as repetitive transcranial magnetic stimulation (rTMS) target the dorsolateral prefrontal cortex (DLPFC) brain region that is crucial for CC. But rTMS studies show remission rates of only ~30%, and there is no evidence that rTMS for depression improves CC. This suggests that for individuals with TRD, synergistically targeting CC alongside treatments like rTMS may augment depression treatment response, improve quality of life and potentially reduce morbidity and mortality. While multiple options may exist for targeting CC, based on our preliminary evidence, here we propose to use a digital breath-focused attention training paradigm paired with DLPFC rTMS. We hypothesize that this multimodal neurotherapy will significantly improve CC, and result in better antidepressant treatment response than observed with extant rTMS treatment, particularly by engaging the neural target of default mode network (DMN) activity suppression. Our rationale for pairing digital breath attention training with rTMS is that mindfulness-based interventions that have a core foundation in training attention to internal sensations such as breathing, can improve CC and also ameliorate ruminative symptoms of depression. Digital training, compared to therapist delivered, allows for greater scalability, immediate feedback on performance and application of algorithmic closed-loop training methods tailored to each subject’s performance. From a cognitive neuroscience viewpoint, the CC deficits that occur in depression are linked with impaired top-down control of DMN activity. Both DLPFC rTMS and mindfulness training are hypothesized to work, in part, by enhancing efficacy of top-down/prefrontal suppression of DMN, suggesting potential synergy of these distinct approaches. We therefore hypothesize that our multimodal neurotherapeutic strategy of pairing a digital breath attention training that is akin to mindfulness training, with rTMS will result in improved CC, as well as enhanced antidepressant effects. The R61 phase of the project will focus on dose response of the multimodal therapy for neural target engagement, i.e. DMN activity suppression, while the R33 phase will replicate neural target engagement and also aim to show improvement in CC and greater depressive symptom response to treatment in a randomized controlled trial.

NIH Research Projects · FY 2026 · 2024-05

Project Summary Depression affects 1-in-5 individuals in the United States and has a tremendous cost burden for our economy at $210 billion per year. Depression is hard to treat given that it is a heterogeneous illness associated with affective, cognitive and behavioral dysregulation. Around 33% of depressed individuals will not respond to the first two treatments offered. In this context, there is increasing evidence that cognitive functioning in major depression may be an independent factor predicting treatment response, quality of life, disability and suicide, and cognitive control (CC) deficits may persist even when other depression symptoms remit. For instance, FDA-approved therapies for treatment-resistant-depression (TRD) such as repetitive transcranial magnetic stimulation (rTMS) target the dorsolateral prefrontal cortex (DLPFC) brain region that is crucial for CC. But rTMS studies show remission rates of only ~30%, and there is no evidence that rTMS for depression improves CC. This suggests that for individuals with TRD, synergistically targeting CC alongside treatments like rTMS may augment depression treatment response, improve quality of life and potentially reduce morbidity and mortality. While multiple options may exist for targeting CC, based on our preliminary evidence, here we propose to use a digital breath-focused attention training paradigm paired with DLPFC rTMS. We hypothesize that this multimodal neurotherapy will significantly improve CC, and result in better antidepressant treatment response than observed with extant rTMS treatment, particularly by engaging the neural target of default mode network (DMN) activity suppression. Our rationale for pairing digital breath attention training with rTMS is that mindfulness-based interventions that have a core foundation in training attention to internal sensations such as breathing, can improve CC and also ameliorate ruminative symptoms of depression. Digital training, compared to therapist delivered, allows for greater scalability, immediate feedback on performance and application of algorithmic closed-loop training methods tailored to each subject’s performance. From a cognitive neuroscience viewpoint, the CC deficits that occur in depression are linked with impaired top-down control of DMN activity. Both DLPFC rTMS and mindfulness training are hypothesized to work, in part, by enhancing efficacy of top-down/prefrontal suppression of DMN, suggesting potential synergy of these distinct approaches. We therefore hypothesize that our multimodal neurotherapeutic strategy of pairing a digital breath attention training that is akin to mindfulness training, with rTMS will result in improved CC, as well as enhanced antidepressant effects. The R61 phase of the project will focus on dose response of the multimodal therapy for neural target engagement, i.e. DMN activity suppression, while the R33 phase will replicate neural target engagement and also aim to show improvement in CC and greater depressive symptom response to treatment in a randomized controlled trial.

NIH Research Projects · FY 2025 · 2024-04

Project Summary The anterior lobe of the cerebellum has unique sensitivities in developmental and degenerative disorders. How anterior and posterior compartments are differentiated developmentally are not adequately understood. The classical mouse mutation meander tail (mea) causes both the kinked tail for which it is named and a compartment-specific disorganization of the anterior cerebellum. Three independent alleles have been reported. Positional cloning efforts more than a decade ago identified a genomic interval but failed to identify causal variants due to technical limitations. Data from that project and from genome-scale mutagenesis projects highly suggest a recurrent regulatory mutation involving one or more structural genes adjacent to the mapped location. This exploratory proposal will identify mutations underlying the two extant mea alleles and test the molecular sequelae of mea mutation on anterior compartment identity and development using cutting-edge genomic tools. The mea locus has been studied for nearly 50 years, but molecular identification eluded efforts with earlier technology. We will resolve this genetic puzzle using de novo long read sequencing, layered onto previously unpublished mapping data. We propose that mea mutations are regulatory in nature and affect compartmentation or compartment-specific identity with respect in the anterior cerebellar lobe. This predicts either discrete or graded changes in expression signatures within or across compartments. We will test this family of hypotheses using single nucleus sequencing of RNA and transposon-accessible chromatin sites from mutant and control littermates in a highly congenic background. Together these aims will resolve a longstanding mystery in the genetics of cerebellum development and point to plausible mechanisms for pleiotropic effects on vertebral development.

NIH Research Projects · FY 2026 · 2024-04

Project Summary American Indian communities have been greatly affected by the opioid epidemic with many Tribes being overwhelmed by opioid use and overdose. Among all racial/ethnic groups in the U.S., American Indians and Alaska Natives have the highest rate of overdose fatalities from all opioids. Within Southern California, areas encompassing American Indian Tribes have experienced some of the highest age-adjusted rates of opioid overdose deaths. In March 2023, the Harm Reduction Coalition of San Diego (HRCSD) formed strategic partnerships with Southern California Tribes to install overdose prevention vending machines (ODPVM) that freely dispense naloxone, with the goal of reducing opioid-related overdose deaths on tribal reservations. To our knowledge, these are the first ODPVM to be established on Tribal Sovereign Land in the U.S. With the current or soon-to-be placement of ODPVMs on several tribal reservations, our research team, led by two American Indian Principal Investigators, is in a unique position to conduct timely applied research that is responsive to tribal leadership's call for community-based prevention activities aimed at reducing overdose deaths. Through a collaborative community-based and participatory research (CBPR) approach we will conduct a bi-phasic, milestone-driven R61/R33 study over a 3-year period. Our study will focus on the implementation process of ODPVM on tribal lands using the RE-AIM framework (Reach, Effectiveness, Adoption, Implementation and Maintenance), with a focus on health equity. During year 1, the exploratory R61 phase, the study will be guided by the following specific aims: 1. develop the CBPR infrastructure by identifying and working with tribal and community representatives who will aid in the development of a culturally appropriate study design to examine the implementation of ODPVM on tribal lands and 2. identify the initial reach of ODPVM during the first year of vending machine placement. During years 2-3, the developmental R33 Phase, our specific aims will address the following: 3. describe the implementation process and identify implementation strategies to increase utilization of ODPVMs within tribal reservations and 4. compare the impact of ODPVMs by examining the statistical and spatial distribution of fatal overdose rates on tribal reservations vs San Diego County, California. Opportunities to evaluate harm reduction strategies on tribal lands are rare but urgently needed for American Indians. Timely research to evaluate the implementation of ODPVM in real-time will allow key questions to be addressed about approaches, activities, and processes that lead to ODPVM uptake. The knowledge gained from this work will provide crucial preliminary hypothesis- generating data on facilitators to ODPVM uptake and inform a larger scale study to test implementation strategies for effectively implementing ODPVMs within Tribal Sovereign Nations.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY/ABSTRACT Hematopoietic stem cells (HSCs) give rise to all blood and immune cells throughout life. Aging HSCs exhibit diminished regenerative function, myeloid-biased differentiation, and clonal outgrowth, which contribute to compromised immunity and increased incidence of bone marrow (BM) failure, and hematological malignancies in the aged population. Recent findings demonstrated that HSCs require strict regulation of protein homeostasis (proteostasis) to maintain self-renewal potential, but this declines with age. Translation is the most error prone step in gene expression and is a prominent source of proteostasis dysfunction. Previous studies demonstrated that HSCs depend on lower protein synthesis rates to preserve proteostasis, and increasing protein synthesis diminishes proteome quality and impairs HSC function. This raises the possibility that HSCs depend on enhanced translation fidelity, and that increasing fidelity could mitigate age-related changes in proteostasis and declines in HSC function. Our central hypothesis is that young adult HSCs have higher translation fidelity compared to restricted progenitors and old adult HSCs, and interventions that enhance translation fidelity will improve HSC fitness and longevity. To directly test this hypothesis, I generated a fluorescence-based translation fidelity reporter mouse, and in Aim 1 I will compare translation fidelity in young and old HSCs and progenitors in vivo. To examine the impact of enhancing fidelity, I generated a mouse model with high-fidelity ribosomes (Rps23K60R/K60R). Using this model, I will test if enhancing translation fidelity protects HSCs from the proteostasis disrupting effects associated with increased protein synthesis in a Pten-deficient mouse model in vivo. In Aim 2, I will test if enhancing translation fidelity improves age-associated declines in HSC function by performing comprehensive hematopoietic analyses and competitive transplantation assays. I will also determine if enhancing translation fidelity extends HSC longevity in serial transplantation assays. Finally, I will extend preliminary RNA-sequencing data to confirm that enhancing translation fidelity promotes improved proteostasis maintenance in aging HSCs using a suite of single cell assays to quantify misfolded and unfolded protein. Collectively, these studies will determine if translation fidelity varies across cell types within the blood and is altered upon aging or in the presence of HSC disrupting pathogenic mutations. Research outcomes will reveal if modulating translation fidelity is a potential therapeutic strategy to enhance stem cell fitness in older adults.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY/ABSTRACT Rotator cuff (RC) tears are common, affecting over 20% of the general population with prevalence increasing with age. A common treatment method is arthroscopic RC repair surgery, where one of many different fixation techniques are utilized to mechanically attach the torn rotator cuff tendon to the bone. However, there is still a high incidence of retear – ranging between 30% to 94% - leading to instability of the joint, pain, potential repeat repair surgery, and depending on severity of the tear, highly invasive total shoulder arthroplasty. Several causes of RC tear repair failure exist including stress risers in the tendon due to sutures, insufficient reattachment of and healing between the tendon and bone, mechanical instability, and poor quality of tissue. There is a need to innovate in this field in order to develop new ways to mitigate the risk of tendon tear after arthroscopic RC repair surgery. One potential solution is to design a system that targets improving upon the most common identified failure modes. 3D bioprinting has emerged as a novel technique for fabricating structures with precise geometry and user defined mechanical and biochemical properties. One limitation to the current class of handheld 3D bioprinters being used for biomedical applications is that in their current form, they require direct access to the tissue that is being treated, which necessitates a highly invasive, open procedure. Arthroscopic 3D bioprinting is a potential high impact medical treatment, where structures with user defined geometry, mechanical properties, and biologic components can be precisely applied to a damaged tissue in a minimally invasive manner. Using an arthroscopic 3D printing system, a tendon can be “spot-welded” back down to the bone to provide a continuous mechanical interface between the tendon and bone using a previously described Janus Tough Adhesive (JTA) biomaterial. The objective of this proposal is to develop an arthroscopic 3D bioprinter to precisely deposit bioinks to enhance the attachment of torn RC tendons to the bone. In Aim #1 we will construct an arthroscopic 3D bioprinter with the following key design features: 1) JTAs will be deposited via extrusion, 2) light will be focused at the tip of the nozzle to facilitate photopolymerization, 3) control of extrusion and polymerization on the device handle, 4) replaceable cartridges, 5) sterilizable, and 6) no more than 7mm in diameter (standard arthroscope geometry). We will evaluate the effect of printing parameters and JTA concentrations on 3D printed structures. We will also perform a proof of principle demonstration in a cadaver. In Aim #2 we will evaluate how and arthroscopic 3D bioprinted RC tear repair can enhance mechanical properties of the tendon-bone interface in a rabbit model. An arthroscopic 3D printer will be used to “spot weld” the tendon back to the bone after a traditional RC repair in a rabbit model to reduce the point loading of the tendon where the sutures are placed. Load to failure of the repaired RC will be evaluated and compared to the gold standard (suture repair). These are the foundational studies necessary to develop a new surgical tool to allow surgeons to incorporate minimally invasive 3D printing strategies for augmenting mechanical and biologic properties in RC tear repair.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Synthetic biology offers transformative technologies for constructing or rewiring gene networks to create novel biological functions. However, the rational engineering of complex biological traits remains very challenging. To address this, we propose to integrate synthetic biology and computational modeling to engineer cellular aging, an intricate, multifaceted biological process crucially related to various diseases, including cancers and neurodegenerative disorders. In this proposal, we choose to focus on replicative aging of budding yeast Saccharomyces cerevisiae, a genetically tractable model for aging of mitotic cells in mammals, such as stem cell. We previously found that genetically-identical yeast cells age through two distinct routes with different molecular and cellular changes. About half of the cells age with chromatin silencing loss, leading to nucleolar decline, whereas the other half age with heme depletion, resulting in mitochondrial deterioration. We further revealed that the fate decision and commitment to the two aging paths are governed by a core gene circuit comprised of the lysine deacetylase Sir2 and heme-activated protein (HAP) transcriptional complex. Sir2 and HAP inhibit each other, resembling a toggle switch to drive the divergence in aging. Very recently, we used computational modeling to guide our network engineering and rewired the Sir2-HAP circuit to create a synthetic oscillator that dramatically slows aging by avoiding the cell’s commitment to either of the two natural aging paths. Building on these results, we hypothesize that engineered oscillations in pro-longevity factors (e.g. Sir2 and HAP) can be a general strategy for enabling dynamic homeostasis to slow cell deterioration and promote longevity. To test this, we will systematically tune the amplitude and period of the Sir2-HAP oscillator to optimize its effect on longevity (Aim 1). We will then apply the same design principles to engineer oscillations in the proteasome (Aim 2) and energy sensing systems (Aim 3), both of which are considered as pro-longevity factors, but their constitutive overexpression/over-activation negatively impact cell physiology and lifespan. We will systematically evaluate the effects of oscillations on damage accumulation, aging phenotypes/hallmarks, as well as the lifespan, and will evaluate the mechanisms underlying their effects. We will construct computational modeling to guide the design of synthetic gene networks and to quantitatively analyze the dynamics and effects of engineered oscillations to advance our mechanistic understanding of aging processes. If successful, the proposed research will not only further our basic understanding of aging in natural systems but also establish a general strategy for promoting longevity applicable to diverse aging processes and organisms.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Post-translational targeting and insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane is a process mediated by the conserved transmembrane domain recognition complex (TRC)/guided entry of TA protein (GET) pathway. Cooperating with the chaperone SGTA and the TRC35-UBL4A-BAG6 pre- targeting complex, ASNA1 (ArsA arsenite transporter, ATP-binding, homolog 1) executes selective recognition of its TA protein substrates, inserting them into the ER membrane via interaction with the ER-bound TRC receptor. Patients carrying V163A/C289W,Q305* compound heterozygous mutations in ASNA1 develop dilated cardiomyopathy (DCM) and death in early infancy, with no extra-cardiac abnormalities. The C289W,Q305* mutation is a nonsense mutation/null allele, while the V163A mutation is a missense mutation. Individuals carrying only one mutation, C289W,Q305* or V163A, do not exhibit DCM. While heterozygous null Asna1 mice do not display any phenotypes, homozygous null Asna1 mice die embryonically. Despite its clinical relevance and functional importance, little is known as to specific roles of ASNA1 in cardiomyocytes (CMs), and molecular mechanisms by which compound V163A/C289W,Q305* mutations lead to DCM. To address this, we generated constitutive (cKO) and inducible (icKO) Asna1 CM-specific knockout mice. We are also generating ASNA1 V163A homozygous and V163A/C289W,Q305* compound mutant mice. In addition, we will utilize human induced pluripotent stem cell (iPSC)-derived CM models of homozygous V163A and compound V163A/null mutations to address their impact on ASNA1 function in human CMs. In preliminary studies, Asna1 cKO mice exhibited embryonic lethality and cardiac developmental defects. Inducible ablation of Asna1 in adult CMs of 8-week-old icKO mice resulted in DCM with 100% lethality within 90 days post-induction. Components of the pre-targeting complex, including SGTA, BAG6, and UBL4A, were downregulated in both Asna1 cKO and icKO hearts, suggesting an unrevealed function of ASNA1 in the stability of the pre-targeting complex in CMs. Together, the foregoing lead us to the hypothesis that ASNA1 plays an essential role in cardiac development and adult heart structure and function by regulating membrane targeting of critical TA proteins and/or the stability of the pre-targeting complex in CMs, and that compound V163A/C289W,Q305* mutations in ASNA1 impair specific aspects of ASNA1 function leading to cardiomyopathy. Accordingly, our Specific Aims are: 1, To determine the role of ASNA1 in developing and adult myocardium by analyzing cKO and icKO Asna1 CM- specific knockout mice for heart morphogenesis, structure and function, and the progression of cardiomyopathy; and 2, To elucidate mechanisms by which the ASNA1 V163A mutation leads to loss of function, and how individuals with ASNA1 compound V163A/C289W,Q305* heterozygous alleles develop pediatric DCM, by analysis of ASNA1 V163A/V163A and compound V163A/C289W,Q305* knock-in mutant mice and human iPSC-derived CMs containing ASNA1 V163A/V163A or compound V163A/null mutations.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY The immune system is a significant barrier to successful clinical organ transplantation, requiring life-long immunosuppression to prevent allograft rejection. Current immunosuppressive strategies rely primarily on broad pharmacologic inhibition of lymphocyte function. This approach is limited by susceptibility to breakthrough immune responses causing acute rejection episodes, with simultaneous unwanted impairment of protective immune responses against infection and tumors. Thus, new approaches for specific and durable allograft- specific immunosuppression are critically needed. We propose to utilize a pMHCII-based allogeneic 5-module chimeric antigen receptor-cytotoxic T cell (5MCAR-CTL) system to target and eliminate alloreactive CD4+ T cells. These investigations represent a conceptual advance in proposing to utilize cutting-edge cellular engineering with 5MCAR technology to develop a novel approach aimed at specific and durable immunosuppression in transplantation. Successful completion of the proposed experiments will generate tools and data to support refinement and development of this clinically-translatable cell engineering approach.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY ‘Binding’ refers to the process whereby elements encoded by multiple brain areas are integrated into a coherent whole during perception, cognition, and action. The elements may be perceptual (such as the color, shape, location, motion, and texture of a visual object), but they may also include executive function and the integration of meaning, instructions, and movements. Disruption in cortical neural binding may also be implicated in the pathophysiology of many forms of neuropsychiatric disease, however, the mechanisms by which highly diverse neural information is integrated in the human cortex are largely unknown. Current explanations often rely on hierarchical multimodal convergence, or on synchronized high frequency oscillations, but these remain largely untested in the human brain. Several properties of recently observed human cortical ripples are consistent with ‘binding-by-synchrony’, including their broad anatomical and behavioral distribution, phase-modulation of local firing, and especially their co-occurrence and phase-locking between widely separated locations. Thus far, our group and others have demonstrated that co-rippling is enhanced during memory recall in waking, and in NREM sleep, possibly associated with memory consolidation. The proposed study will test if cortical ripples possess other properties crucial to a possible role in binding. Aim 1 will test if cortical neurons in different areas interact more strongly when their locations engage in co-occurring ripples by recording populations of single neurons in different segments of the cortex during spontaneous behavior using established microelectrode technologies. Aim 2 asks if cortical rippling and co-rippling occur in behavioral contexts that elicit binding beyond memory recall during waking, and consolidation during NREM. To achieve these aims, this proposal includes the acquisition of novel human intracranial recording data and the analysis of a large collection of human electrophysiology data that has grown over the last decade. This unique dataset includes multi-hour and multi-day continuous recordings of local field potentials and single neuron firing in multiple cortical areas in humans, using two patient groups and several micro-arrays. Together, these aims will test if widespread co-occurring and phase-locked cortical ripples may serve as a substrate for one of the foundational functions of the human brain: to bind disparate content into a unified experience. Ultimately, illuminating the interactions between neurons during cortical ripples may be critical for understanding the temporal organization of neuronal network activity, and may provide the foundation to help better understand human cognitive disfunction.

NIH Research Projects · FY 2026 · 2024-03

Project Summary Mutations are the ultimate source of genetic diversity upon which other evolutionary forces—natural se- lection, genetic drift, recombination—act. As such, mutations impose fundamental constraints on what phenotypes can evolve, but we lack a theory for understanding these constraints. Two overarching goals of my lab’s research are to (i) develop a theory for predicting the phenotypic and fitness effects of new mutations across genotypes and environments and (ii) understand how evolution proceeds on the emerging “fitness landscapes”. We take three complementary approaches to achieve these goals. Our first approach is theoretical. Even the simplest unicellular organisms are complex biochemical machines, but it is unclear how this biochemical architecture constrains the structure of the resulting fitness landscapes and the rules by which mutations “move” the organism on this landscape. We are developing mathematical theory for understanding these constraints, focusing initially on fitness landscapes that emerge in complex metabolic networks. Our second approach is empirical. While we construct the theory from first principles, we will also use tractable experimental systems, such as the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli to directly measure the effects of mutations on some cellular phenotypes that are im- portant for microbial fitness, e.g., the transcriptome. Our goal here is to identify broad and hopefully general statistical patterns in how the effects of mutations vary across genetic backgrounds and environ- ments that will help us narrow down and parameterize our theory. One practically important outcome of this work that we hope to achieve is the ability to predict the evolution of collateral antibiotic resistance. Our third approach is exploratory. If our theoretical and empirical efforts mentioned above are successful, they will give us a handle on predicting evolutionary trajectories and outcomes in controlled laboratory conditions. However, by necessity, these conditions lack many of the complexities of natural environ- ments. We would like to understand how the evolutionary process in such more complex environments differs from what we can observe and potentially predict in simple controlled laboratory conditions. To this end, we will apply some of the cutting-edge experimental tools, such as whole-genome time-course sequencing, to observe evolutionary dynamics in microbial systems of intermediate complexity, in partic- ular, in a two-species microbial community that we already developed and in a semi-natural marine mi- crobial system that we plan to develop with our collaborators.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Solid organ transplantation (SOT) has transformed the survival and quality of life of patients with end-stage organ failure. In 2019, 39,719 transplant surgeries were conducted in the US. However, allograft rejection represents a significant cause of morbidity with an estimated 25-50% of transplant recipients experiencing some of form of acute or chronic cellular rejection. Effective immunosuppression management is therefore essential to improve organ transplant recipient outcomes. Clinically used immunosuppression regimens are overall effective in reducing rejection, but come with potentially life threatening side effects including cardiotoxicity, nephrotoxicity and diabetes. Regulatory T cell (Treg) infusion, alongside SOT, is an emerging strategy which focuses on rebalancing the Treg ratio in the transplanted organ to prevent rejection or loss. However, developing effective therapeutics using live cells necessitates means to determine their in vivo biodistribution, persistence, and efficacy after administration. Clinicians do not know the fate of injected cells, thus, interpreting cases of non-responding patients remains a barrier to wider use. We posit that developing imaging-guided biomarker-based therapeutic approaches capable of visualizing Treg homing to tissue targets in vivo would be invaluable for assessing of expected therapeutic activity following infusion. Magnetic resonance imaging (MRI) is the only technique that is radiation-free, clinically translatable, and enables direct visualization of labeled cells in vivo. Due to the clinical translation potential, MRI applications and contrast agent development continue to see remarkable growth, especially in the cell tracking sphere. Labeling generally occurs by co-incubation with iron oxide or fluorinated moieties in vitro, resulting in internalization of the contrast agent. Safe and efficient labeling of non-phagocytic cell types, such as T cells, remains challenging and often requires use of transfection agents, which are not U.S. FDA approved. Indeed, increased cell manipulations exacerbate the risk of cell contamination, transformation and viability impairment. Here, we propose a fundamentally different approach. The microbeads used in the process of T cell purification from human blood leukapheresis will directly label the target cells, producing proton contrast. This new labeling approach, recently published and patented by our lab with CD25 microbeads, does not involve any additional manipulation compared to clinical T cell isolation protocols. Our goal is to develop a one-stop-shop Treg labeling protocol with cell sorting microbeads to track Treg distribution and homing for clinical applications. If successful, this project will address the clinical bottleneck of in vivo cell surveillance, providing rapid and quantitative methods to tailor specific treatment regimens to individual patients and improve overall outcomes by preventing potentially life-threatening toxicities.

NIH Research Projects · FY 2026 · 2024-03

The overarching goal of the proposed Scripps Center for Oceans and Human Health (SCOHH) is to advance the science and community engagement surrounding seafood pollutants in the United States. The project brings together a multidisciplinary team of biomedical and oceanographic researchers with broad expertise in fish ecology, microbiology, marine chemistry, technology development, bioaccumulation, genomics, toxicology, and public health. This team will study and track the distribution of essential micronutrients and harmful contaminants in marine food webs to the three billion people who consume seafood globally, the roles that the marine microbiome play in their production and transport, and the developmental toxicity of seafood pollutants and their interactions with human drug transporters. The Center’s scientific goals and focus are guided by the needs of society, established through bidirectional community engagement led by a proven community engagement team. The proposed research program of SCOHH spans four main areas which address the objectives of the NIEHS and NSF sponsored COHH4 program RFA: 1. Assessing seafood composition to advance dietary health in the U.S. 2. The marine microbiome as a source for the synthesis, transformation, and distribution of seafood contaminants. 3. Mechanisms of bioaccumulation and developmental toxicity of seafood pollutants. 4. Seafood risks and benefits – Science, literacy, and engagement. We expect the overall outcome of SCOHH to better inform policies, consumption guidelines, and individual decisions to lower risk and enhance greater benefits associated with seafood consumption by bridging science discovery, environmental health literacy, and community engagement.

NIH Research Projects · FY 2026 · 2024-03

Abstract / Summary Staphylococcus aureus (SA) is a leading cause of infection worldwide and a major driver of antibiotic resistance. Although there have been close to thirty vaccine trials targeting the pathogen, all successful pre-clinical vaccines taken to human trials have failed for unclear reasons. Recently, we provided evidence that SA vaccine failures occurred because of routine and frequent human exposure to SA compared to laboratory mice. We showed that prior exposure of mice to SA leads to the development of anti-SA antibodies with increased Fc sialylation, incapable of supporting opsonophagocytic killing of the pathogen. Staphylococcal vaccination of these pathogen-exposed mice recalls the non-protective antibody response and leads to vaccine failure. Using this model, we have successfully explained the failure of all clinical SA vaccines we have tested to date. To understand the mechanistic basis for the vaccine failures, we now show that SA induces abundant IL10 that enhances Fc sialylation of non- protective antibodies. IL10 also blocks anti-SA cellular immunity by antagonizing TH17 generation. Taken together, we hypothesize that non-protective immune imprints develop as a result of IL10 induced by the pathogen, and IL10 underlies the development of non-protective responses by subsequently administered T and B vaccines targeting SA. To test our hypothesis, 1) we propose to investigate the molecular link between IL10 and Fc glycosylation, and the mechanism whereby Fc glycosylation leads to aborted opsonic killing of SA. 2) We will seek to identify the bacterial source of IL10 induction and address the mechanism of IL10+T cell expansion and IL10-mediated suppression of TH17 development. 3) Finally, we will address the practical strategic implications of these findings on vaccine development. Overall, our proposal aims to understand the interaction between SA, the host, and the vaccine to provide new insights on anti-SA vaccine approaches. Our findings has the potential to shed light on why vaccines targeting other pathogens also failed.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT Gastrointestinal stromal tumor (GIST) is often effectively treated with FDA-approved first-line therapies such as imatinib and avapritinib. However, this strategy is ineffective in a particular subset of GIST patients—those with a GIST caused by inherited loss-of-function mutations or sporadic epigenetic silencing of genes encoding the succinate dehydrogenase (SDH) complex. These SDH-deficient (SDH-def) GISTs exhibit quite variable biology, ranging from indolent tumors to rapidly progressive disease that quickly leads to death, often among adolescents and young adults. We recently reported that treatment of SDH-def GISTs with temozolomide (TMZ) resulted in favorable responses in a small patient cohort, and these findings led to a multicenter phase II trial to investigate the efficacy of TMZ in SDH-def GIST patients. We also developed the first and only patient-derived SDH-def GIST cell culture biorepository, which we utilized to discover that TMZ induces expression of death receptor 5 (DR5), a TRAIL receptor. Inhibrx Inc. in La Jolla, CA has developed an entirely new therapeutic agonist that binds to DR5 receptors, and collaboratively, we have used these together with TMZ to initiate apoptosis and reduce cell viability in vitro. This initial finding, together with major new preliminary data we now present, suggests that a synergistic strategy, combining DR5 agonism with TMZ, may lead to more effective treatment outcomes for SDH-def GIST patients, and thus address this unmet medical need. The overall objective of this proposed U01 is to unite NIH intramural and extramural SDH-def GIST clinicians and researchers with expertise in adult and pediatric medical oncology, surgical oncology, basic and translational science, patient advocates, and an industry partner to address two long-term goals: (1) to accurately distinguish SDH-def GIST patients who will have indolent biology from those who will have aggressive biology; and (2) to identify and test safe, effective therapeutic strategies for treating SDH-def GIST patients, especially those with more aggressive forms of this cancer who will otherwise die. Supported by our preliminary data, we hypothesize that SDH-def GISTs possess underappreciated tumor heterogeneity and hence correspondingly complex biology that will require combination therapies to achieve disease control. By accomplishing our aims, we will better understand the biology of these tumors and identify an improved treatment strategy for this disease. We propose to leverage our existing National Succinate Dehydrogenase-Deficient GIST Translational Research and Clinical Trial Consortium along with unique NIH Clinical Center resources to: (1) evaluate the safety and efficacy of TMZ with INBRX-109 (a potent DR5 agonist) among patients with progressive SDH-def GIST in a Phase I/II clinical trial; (2) create a centralized biobank of well-annotated SDH-def GISTs; (3) develop a network of research laboratories to develop improved preclinical models to investigate diverse SDH-def GIST biology; and (4) develop clinical tools to predict SDH- def GIST prognosis. Our project has the potential for immediate clinical impact to manage and treat SDH-def GIST patients, and will provide much-needed near-term hope for these patients, their families, and caregivers.

NIH Research Projects · FY 2025 · 2024-03

Project Summary. Until recently, sphingolipids within the microbial environment have been attributed to host association, as the conventional wisdom was that bacteria did not contain their own biosynthetic pathways to produce them. Recently, members of our team uncovered a new bacterial sphingolipid pathway involving an acyl carrier protein similar to that of fatty acid synthase (FAS). This new acyl carrier protein, AcpSP, appears to play a role distinct from its FAS progenitor, AcpP, whose primary purpose lies in de novo fatty acid synthesis. Remarkably, AcpSP shares less than 30% sequence identity to AcpP, suggesting a unique activity and interaction landscape than that of AcpP. We have identified AcpSP and serine palmitoyltransferase (SPT) within in many pathogenic bacteria, including Neisseria gonorrhoeae, Escherichia coli (studied herein), Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa, indicating that this pathway may be involved in pathogenesis. In this program, our team applies a suite of chemical and structural biology tools originally developed for the study of AcpP in order to explore the interaction of AcpSP with its partner enzymes. Using a combination of chemical probes and structure-based methods, we will characterize the interface between AcpSP and SPT, the first key step in sphingolipid biosynthesis. To further explore the cryptic interactions between sphingolipid biosynthesis and FAS, we will deploy a systems-wide structural analysis to detail AcpSP interactions with de novo FAS enzymes. This program provides an essential step toward understanding bacterial sphingolipid biosynthesis and its role in pathogenesis and will likely provide important new targets for future drug discovery.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY The blood-brain barrier (BBB) is characterized by a series of properties that tightly regulate the transport of ions, molecules and cells between the blood vessel lumen and the brain parenchyma. This BBB is critical to control the environment of the brain to allow for proper neuronal function, and to protect the brain from injury and disease. Dysregulation of the BBB has been implicated in a variety of disorders including stroke, Alzheimer’s disease, multiple sclerosis, and epilepsy. In addition, the BBB is a key obstacle to central nervous system (CNS) drug delivery. The BBB has been extensively studied in a small number of model organisms (mouse, zebrafish) in which endothelial cells form this barrier. Interestingly, several species have a BBB formed instead by glial cells including invertebrates, elasmobranchs (sharks, rays and relatives) and sturgeons, but very little is known about the molecular mechanisms of BBB function in these species. Furthermore, a lack of correspondence between phylogenetic relationships and endothelial versus glial BBB suggests that the BBB evolved independently several times. Hypotheses that may explain these observations include an ancestral vertebrate with a glial BBB and independent evolution of the endothelial BBB at least six times (hypothesis A), an ancestral vertebrate with an endothelial BBB and independent re-evolution of the glial BBB at least twice (hypothesis B), or an ancestral vertebrate in which endothelial and glial cells each accomplished aspects of BBB function, with current BBB diversity a result of a “push-and-pull” toward either extreme (hypothesis C). Detailed molecular knowledge of the BBB in non-model organisms is required to better understand the mechanisms underlying BBB function in vertebrates and their evolution. To achieve this we will employ single nucleus RNA-sequencing (snRNA-seq) of brain tissue in fish species with an endothelial BBB (hagfish, trout, zebrafish, and lungfish) and fish with a glial BBB (shark, ray, and sturgeon). We will also profile mouse and octopus as outgroup comparators. This will allow us to define the molecular characteristics of endothelial and glial cells in each species and perform a cross-species comparison of BBB-forming cell types. Furthermore, we will examine BBB function and ultrastructure by injecting horseradish peroxidase (HRP) systemically into each species and visualizing HRP localization with electron microscopy. Together, the proposed study will provide unprecedented new information related to the molecular foundations of BBB function across life and will yield new information on the evolution of the BBB. The resulting information may also reveal novel molecular targets for CNS drug delivery and for treating BBB dysfunction in CNS disorders.

NIH Research Projects · FY 2025 · 2024-03

Project Summary Membrane tension governs endocytosis, exocytosis, cell migration, mitosis, meiosis, membrane transportation, and many other biological phenomena, but there are no tools to map and measure membrane tension in vivo and in real time. Here, we seek funds to demonstrate the feasibility of a novel small molecule imaging probe/contrast agent that produces changes in both fluorescence intensity and photoacoustic intensity as a function of membrane tension. We will compare our probe and methods to the current state-of- the-art: fluorescence lifetime and micropipette/tethering analysis. The technical advance provided by photoacoustic imaging is in vivo imaging of membrane tension: Ultrasound waves are not absorbed/scattered as much as photons and thus imaging through 3-5 cm of tissue is routine. The technical advance of fluorescence intensity is ease of use: Unlike fluorescence lifetime, fluorescence intensity can be done with most common microscopes. Our innovation is grounded in the use of novel conjugation switching chemistry and novel photoacoustic imaging for in vivo imaging. Indeed, a probe that offers in vivo imaging of membrane tension could facilitate fascinating new questions about disease and therapy to be addressed in subsequent proposals using this probe: “How is membrane tension distributed across a 3D organ?; How does an organ’s membrane tension change when it encounters a therapeutic?; and How does an organ’s membrane tension change across the lifespan or with stressors?” This exploratory work will use the following aims to test the feasibility of the contrast agent and gain new technical knowledge about its quantitative advances over the state-of-the-art lifetime- and micropipette-based approaches. Aim 1 will synthesize and characterize the probe using a logical yet innovative organic chemistry workflow. Aim 2 will use giant unilamellar vesicles with tunable membrane tension to test the probe versus a commercially available fluorescence lifetime probe. We will use a micropipette to establish the ground truth tension values and then compare the sensitivity of fluorescence lifetime (gold standard) versus fluorescence intensity and photoacoustic intensity via five different vesicle populations with unique membrane tension values. The imaging method with the steepest slope will be the most sensitive—we expect that our probe will be more sensitive than lifetime because of its activatable nature. Aim 3 will test the novel probe with cultured cells. We will control the membrane tension via osmotic pressure and compare the imaging data of cells at hypo, hyper, and isotonic conditions. To validate the in vivo utility of photoacoustic imaging, we will image cells locked into high or low membrane tension states beneath increasingly thick pieces of tissue-mimicking materials—this experiment will be a proof of concept of in vivo imaging of tension differences. This work is feasible because of Jokerst’s expertise in contrast agent development and in vivo imaging. All needed tools and personnel are in place, and the work is ideally responsive to RFA-22-126’s call for conceptual studies in technology development.

NIH Research Projects · FY 2026 · 2024-03

Project Summary This research will significantly expand the PI’s unique research program in quantitative bacterial physiology from the exclusive focus on the model organism Escherichia coli during the past decade to a variety of species across the bacterial phylogeny, including Vibrio, Pseudomonas, Bacteroides, Mycobacterium, and Bacillus. Transcriptomic, proteomic, and metabolomic approaches will be applied to these bacterial species for a spectrum of conditions with emphasis on cementing absolute abundances of the measured quantities as well as the rate of synthesis and turnover. For each species investigated, we will replicate previous studies for E. coli to derive both coarse-grained phenomenological models as well as establish relation to molecular regulation. We will also develop methods for inter-species comparisons at the quantitative level. These studies will not only generate a vast amount of useful quantitative data and models for bacteria of

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY/ABSTRACT Research Project: Dengue virus (DENV) is a medically important pathogen posing a major public health threat. DENV infection can result in potentially fatal cases of severe dengue associated with vascular leak as a result of endothelial dysfunction, but the triggers of these pathologies were unknown. We and others have defined a direct role for the DENV non-structural protein 1 (NS1) in mediating endothelial dysfunction in vitro and vascular leak in vivo, independently from viral infection, via direct interactions with endothelial cells. Our preliminary data indicate that NS1 binding to endothelial cells and uptake via clathrin-mediated endocytosis are distinct steps that are both critical for NS1-mediated endothelial dysfunction, but the NS1 receptors on endothelial cells that mediate uptake of NS1 are unknown. My preliminary data identified a role for beta-2 adrenergic receptor (β2AR) and epidermal growth factor receptor (EGFR) in NS1-mediated endothelial dysfunction. Further, activation of β2AR has been reported to transactivate EGFR, triggering endocytosis. Thus, I hypothesize that β2AR and EGFR serve as a NS1 receptor complex where NS1 functions as a β2AR agonist to trigger EGFR- mediated endocytosis. This proposal will test this hypothesis by investigating the capacity of NS1 to activate β2AR and EGFR signaling pathways in Aim 1. Aim 2 will investigate the capacity of β2AR and EGFR to serve as NS1 receptors. The proposal holds the potential to characterize a fundamental and novel step in dengue virus pathogenesis via identification of NS1 receptors which also serve as therapeutic targets for treatment of dengue. Candidate and Career Goals: The candidate for this K22 proposal has a strong track record investigating host- pathogen interactions from his work studying entry mechanisms of Nipah virus as an undergraduate student in the labs of Dr. Benhur Lee and Dr. Hector Aguilar-Carreno at the University of California, Los Angeles, investigating innate immune mechanisms by which interferons control viral infection as a graduate student in Dr. Seungmin Hwang’s lab at the University of Chicago, and investigating flavivirus NS1-mediated pathogenesis as a postdoctoral scholar in Dr. Eva Harris’s lab at the University of California, Berkeley. In Dr. Harris’s lab, Dr. Biering recently identified candidate NS1 receptors on endothelial cells and will investigate their role in NS1 pathogenesis in this proposal. Dr. Biering is committed to establishing his own research group at a major research university where he will investigate mechanisms by which viral pathogens cause disease. Career Development Plans and Environment: Dr. Biering will work with his mentor (Dr. Eva Harris) and mentoring committee (Dr. Britt Glaunsinger, Dr. Michael Diamond, Dr. Kamil Godula, Dr. Suzanne Fleiszig, and Dr. P. Robert Beatty) to acquire additional training in biochemistry, advanced-microcopy, glycobiology, and in vivo models of virus infection which will enhance his current research proposal and the research in his future lab. This, coupled with the additional courses and trainings outlined in his training plan, will prepare Dr. Biering for a successful transition to a primary investigator role able to prepare data for a competitive R01 proposal.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY/ABSTRACT Mural cells, encompassing pericytes and vascular smooth muscle cells, line blood vessels and regulate vessel development, stability, and tone. In the central nervous system (CNS), mural cells have additional, specialized functions: they regulate the blood-brain barrier, mediate neurovascular coupling, produce neurotrophic factors, and contribute to molecular transport and metabolism. CNS mural cells are dysfunctional in neurological disorders including Alzheimer’s disease, cerebral small vessel diseases, and diabetic retinopathy. Reflective of their functional specializations, CNS mural cells have gene expression profiles distinct from mural cells of other organs, a phenomenon termed organotypicity. Despite their importance to CNS homeostasis, we have virtually no knowledge of how these specialized properties arise during CNS mural cell development. CNS mural cells have two distinct developmental origins (neural crest in retina and forebrain, mesoderm in other regions), but converge on a remarkably similar molecular profile. This observation suggests that extrinsic signals from neural tissue control the development of organotypic properties. Further, a novel analysis of transcriptional and epigenomic data suggest that Wnt/β-catenin signaling and the transcription factors ZIC1 and ZIC4 are key regulators of this process. Therefore, the proposed project will test the hypothesis that neural tissue-derived Wnt/β-catenin signaling drives expression of ZIC transcription factors, which activates a CNS-specific gene regulatory network in mural cells that mediates their specialization. In Aim 1, genetic mouse models will be used to test whether Wnt/β-catenin signaling is necessary and sufficient to achieve CNS-specific molecular and functional properties in mural cells. Similarly, in Aim 2, genetic mouse models will be used to evaluate whether ZIC1 and ZIC4 are required for development of organotypic properties in CNS mural cells. In Aim 3, transcription factor CUT&RUN and chromatin accessibility profiling will be used to define the gene regulatory network and cis-regulatory elements controlling CNS mural organotypicity. Together, the proposed work will advance our understanding of the molecules and signaling networks that govern CNS mural cell development, which could ultimately be leveraged to develop therapeutic strategies facilitating CNS mural cell survival or regeneration in pathological conditions. The proposed research project also serves as the basis for a postdoctoral fellowship training plan, through which Dr. Benjamin Gastfriend (applicant) will receive mentored training from Dr. Richard Daneman (sponsor) and Dr. Christopher Glass (co-sponsor) at the University of California, San Diego. Training in rigorous research using genetic mouse models, epigenomic techniques, and in skills necessary to lead research in an academic setting will synergize with the applicant’s graduate research and training, and prepare Dr. Gastfriend to make future contributions to biomedical research.