University Of California-Irvine

NIH Research Projects · FY 2025 · 2022-09

Project Summary Polycyclic terpenoid natural products are endowed with a broad range of medicinally relevant biological activities. Taxol and artemisinin are two premier examples of life-saving terpenoids; the former is used clinically to treat several different cancers, while the latter is a critical antimalarial agent used worldwide. Chemical synthesis approaches to natural products provide opportunities to make compounds that might be scarcely available from nature, to generate analogues that are only available by total synthesis, and to make probe molecules for increased understanding of the underlying biology. A balance of innovative strategy and new chemical methodology promises efficient syntheses that can serve to answer important biological questions that often can only be probed with small molecules. As part of our laboratory’s long-term goal to enhance efficiency in the synthesis of complex natural products to facilitate important studies in biology, the objective of the proposed research program is to develop concise and creative synthesis designs and empowering methodological advances to permit access to many bioactive diterpenoid natural products. The rationale for this work is that synthetic chemistry is critical to the development of natural product “hit molecules” into legitimate preclinical lead compounds by analogue production, by identification of structure-activity relationships, by the synthesis of chemical probe molecules for mechanism of action studies, and more. An efficient total synthesis of targeted natural products provides a platform from which to address each of these key areas of research. The proposed research is significant because chemical synthesis will provide access to a broad range of biologically important secondary metabolites and analogues with which to interrogate key processes; at the same time, the underlying synthesis designs and methodologies will lead to vertical advancement of the field of organic chemistry. These contributions are innovative by virtue of the chemistry-driven, multi-faceted investigations into the biological activity of diterpenoids with immunosuppressive, antiviral, antibiotic, and neurological activity; the development of new stereocontrolled polyene cyclization strategies to access polycyclic diterpenoid natural products; and the elaboration of new radical bicyclization strategies to make complex architectures relevant to bioactive natural products.

NIH Research Projects · FY 2025 · 2022-08

Alzheimer's disease and related dementias (ADRDs) are complex multifactorial disorders characterized by progressive memory loss, confusion, and impaired cognitive abilities in older adults. In addition to genetic variants, studies have reported that certain epigenetic, network, and genome organizational perturbations, and their complex interplay, contribute to ADRD progression, informing new cellular etiologies. The recent single-cell revolution, especially multimodal genomic profiling, makes it possible to scrutinize multi-scale dysregulations in ADRDs at the finest possible resolution. However, few methods have been developed to address this critical yet challenging task due to the high missingness, dimensionality, and complex feature interactions in single-cell data. In this project, we will develop interpretable deep learning methods and software tools to highlight multi-scale dysregulations contributing to ADRDs, including genetic, epigenetic, network, and chromatin structural alterations at a single-cell resolution. Distinct from previous efforts reporting a set of one-dimensional (1D) functional cis-regulatory elements (CREs) from only one genome and applying it to all samples, we aim to construct personal, compact, gene-centric, and cell-type-specific brain regulome from sc-multiome data. Specifically, we will first propose a scalable multimodal deep generative model to integrate large-scale, heterogeneous ADRD single-cell data with single-, multi-, and hybrid modalities. Distinct to existing methods, we will include an invariant representation learning scheme to derive latent cell representations uncorrelated with confounding factors (e.g., age, gender, read depth, and batch effects) for bias-free transcriptome and epigenome reconstruction (Aim 1). Then, we will go beyond the 1D genome annotation by deciphering the multi-scale gene regulation code (Aim 2), including cell-type- specific chromatin compartmentation, CREs and their target genes for functional interpretation, and transcription factor (TF) regulatory networks (TRNs). Lastly, we will develop interpretable deep learning models to link multi-scale dysregulations to ADRD with mechanistic explanation (Aim 3). This proposal is built on an existing multi-year collaboration among the Zhang, Won, and Gerstein labs that originated from the ENCODE and PsychENCODE projects, with diverse expertise in computer science, neuroscience, and genomics. Upon completion, our proposal will significantly accelerate research in a broader scientific community by providing essential tools to investigate functional regions in the genome and prioritize multi-scale risk factors for ADRD.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Substantial research effort has uncovered a critical role for chronic inflammation in the development of insulin resistance, a hallmark of Type 2 Diabetes (T2D). Even with this discovery, no successful anti-inflammatory therapeutics have been developed, and research has stagnated with a focus on characterizing adipose tissue inflammation in mouse models of T2D. Importantly, the initiator of chronic inflammation in T2D remains unknown. Type 2 Diabetes presents with autoantibodies against a wide array of self-proteins, but protein screens for antibody reactivity have not discovered initiators of T2D inflammation. Remarkably, not all autoantigens in T2D are protein. We have identified lipid autoantigens in T2D that have been completely overlooked, likely due to the proteins which present lipid antigen, Group 1 CD1 molecules, not being expressed in mice. This lack of expression has left a gaping hole in our understanding of lipid antigens in immunological homeostasis and disease, especially T2D. To remedy this issue, we will use transgenic mice which express human CD1 molecules and in vitro models of human immune responses to understand lipid-mediated immune function in T2D. The long-term goal of this proposal is to establish a new scientific direction with the broad long-term purpose of understanding the role of lipids in pro-inflammatory responses, T2D, and autoimmune diseases. This proposal tests the overarching hypothesis that lipid antigen presentation is a key mechanism by which chronic inflammation in T2D is initiated. This innovative and novel investigation of lipid antigen presentation in T2D combines use of human blood samples, transgenic and humanized mice, single cell RNA sequencing, multiparameter cytokine analysis and flow cytometry, coupled with computational methods that allow for a systems biology approach to antigen discovery and hypotheses testing. In research Area 1, we will assess lipid antigen dependent activation of T cell responses. We will use humanized mice and human blood samples to interrogate the impact of these lipid antigens on T cell function in vitro (CD1 tetramer loading) and in vivo (high fat diet). In research Area 2, we will target lipid antigen presentation to prevent and treat T2D. The capacity of neutralizing antibodies targeted against group 1 CD1 molecules and lipid autoantigens to inhibit pro-inflammatory cytokines secreted from human immune cells and to prevent and treat disease in transgenic mice will be assessed. In research Area 3. Lipid autoantigen discovery in T2D will be performed with a novel 3D printed lipid microarray for antibody profiling. This proposal has the potential to redefine T2D as an autoimmune disease driven by lipid antigen presentation and completely change the strategies for management of disease and therapeutic development. Outcomes of this proposal will allow for the identification of specific inflammatory pathways that can be targeted for disease treatment.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY / ABSTRACT This is a DP2 NIAID New Innovators Award proposal to harness cell-to-cell variability to understand viral infection outcomes at the single cell level. Infected cells differ in the outcome of infection (abortive/productive), the timing and level of viral gene expression and the number of progeny they produce. Two striking phenomena revealed by studying infection at the single cell level are (1) the ability of some cells to abort infection after viral gene expression has commenced and (2) the fact most infected cells release very few progeny while a small fraction of cells release thousands (here termed “super producers”). While it is now accepted that cell-to-cell variability affects infection outcome, we currently lack a molecular understating of the determinants of infection outcome in single cells, mainly due to the lack of appropriate tools and methodologies. Understanding these determinants is likely to lead to the discovery of novel cellular anti-viral modalities, better design of therapeutics and deepen our understanding of the viral life cycle. In this proposal I will apply cutting-edge technologies to investigate virus-host interactions at the single cell level and gain mechanistic insights as to the host factors that control the opposing phenotypes of abortive infection and super producers. My central hypothesis, supported by my past work and preliminary data using Herpes Simplex virus 1 (HSV-1) as a model system, is that infection outcome at the single cell level is dependent on the cell’s state at the time of infection and on the activation of specific cellular transcriptional programs. To test this hypothesis I will develop new approaches to interrogate virus-host interactions by combining viral genetics, single-cell RNA-sequencing, custom-made microfluidic devices, live- cell imaging and machine learning approaches. My background in performing cross-disciplinary research through the combination of classical virology, cell-biology, microfluidics and systems biology to study virus-host interactions uniquely positions me to successfully tackle these important questions. My results will identify the host factors that determine infection outcome and define a new paradigm, using cell-to-cell variability to understand virus-host interactions. The tools and technologies will be developed using HSV-1 as a model system, and then applied to investigate other important viral pathogens, in collaboration with leading virologists. This “high-risk high-reward” project requires a considerable investment of time and resources and the construction of new tools, making it a perfect fit for the DP2 program. The award will allow me to pursue cutting-edge science at the interface of virology and single cell biology and establish a long-term research plan to decipher the cellular mechanisms that control infection outcome.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY: Lung cancer (LC), the most frequent cause of cancer deaths for men and women, is estimated to lead to 228,820 new cases and 135,720 deaths in the United States in 2020. In recent years inhibition of immune checkpoints, such as programmed cell death-1 (PD-1) and programmed cell death ligand- 1 (PD-L1), has been shown to provide survival benefits to patients with LC. However, most patients demonstrate either primary resistance or experience tumor recurrence and die of their disease. Our group has demonstrated that cancer cells upregulate PD-L1 on natural killer (NK) cells, immune cells that can target malignancies without the necessity of chimeric antigen receptors or prior antigen exposure and do not require matching to recipient's human leukocyte antigen for potential activity. Upregulation of PD- L1resulted in enhanced NK-cell function. Furthermore, the PD-L1 inhibitor atezolizumab (AZ) resulted in enhanced leukemic cell killing against myeloid leukemia lacking PD-L1 expression, and mice treated with selective cytokines (IL-12, IL-15, and IL-18) in combination with AZ showed a significant improvement in survival even in the absence of PD-L1 expression in their tumor tissue. We were able to express soluble IL-15 (sIL-15) tagged with a truncated epithelial growth factor receptor in umbilical cord NK cells in vitro, while upregulating endogenous PD-L1 expression on the NK cells. These transduced NK cells maintained greater than 30% antigen-specific tumor lysis compared to mock-transduced NK cells and demonstrated cytotoxicity against A549 Non-Small-Cell LC (NSCLC) cells. Human A549 NSCLC cells were subsequently injected in non-syngeneic mice and followed with treatment with these “enhanced” cordon blood CB NK cells. In comparison to treatment with mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation, the enhanced CB NK cells induced substantial reduction in tumor volume. We also performed safety/toxicity in vivo studies of this approach and compared with AZ alone. Our data suggest that anti-PD-L1 mAb therapy has a unique therapeutic role in treating PD-L1 negative cancer, acting through PD-L1(+) NK cells. This activity is achieved independent of PD-1 activity and in the presence of NK-activating cytokines. We hypothesize that cytokine “enhanced” NK cells will provide clinical benefit to NSCLC patients and that the antitumor activity of this approach will be further enhanced by co- administration of AZ. To test this hypothesis and document the safety of this strategy, we propose additional in vivo safety and efficacy studies followed by a phase 1 study in which CB NK cells, genetically modified to express sIL-15, followed by ex-vivo expansion in the presence of IL-2, IL-18, and IL-12 will be administered either by themselves or combined with AZ, following lymphocyte depletion, to NSCLC patients whose tumor has previously progressed on or after treatment with PD-1/PD-L1 inhibitors.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Our goal is to test a novel hypothesis in humans about the impact of chronic stress and stress-related biobehavioral processes on stem/progenitor cell biology. Although substantial progress has been made in understanding how stress becomes biologically embedded to produce long-term effects, crucial knowledge gaps remain. The processes implicated in biological embed- ding have been described primarily at the level of differentiated cells types that form tissues and organ systems. Based on the consideration that the long-term effects of stress can extend well beyond the lifespan of most differentiated cells, whose replenishment does not occur from already-differentiated ‘parent’ cells, but occurs from stem/progenitor cells, we advance the hypothesis that biological embedding of the effects of chronic stress may extend all the way down to the level of stem cells, to define fundamental aspects of their biology that determine the earliest vulnerabilities for common stress- and age-related disorders. We underscore the importance of studying fetal (newborn) stem cells, focus specifically on hematopoietic (HSCs) and mesenchymal stem/progenitor cells (MSCs), and on the functional capacity of their telomere and mitochondrial systems as our primary outcomes. We operationalize chronic stress using a composite biological measure of maternal allostatic load that incorporates the principal biomarkers of the gestational stress transmission pathway. Because stress responsivity is a key modulator of chronic stress effects, we additionally propose to characterize this phenotype in HSCs and MSCs via an in vitro oxidative stress [H2O2] challenge. We will conduct the proposed study in N=300 mother-child dyads; isolate and culture newborn HSCs and MSCs from umbilical cord blood and cord tissue, respectively; and perform cellular telomerase activity and high-resolution respirometry experiments to characterize telomere and mitochondrial functional capacity. Aim 1 will test the hypothesis that chronic stress exposure (allostatic load) is prospectively associated with reduced functional capacity of newborn HSC and MSC telomere and mitochondrial systems. Aim 2A will test the hypothesis that chronic stress exposure primes the stress responsivity phenotype of newborn HSCs and MSCs, and Aim 2B will determine whether antioxidant (resveratrol) pretreatment attenuates this effect. Both aims will include tests for effect modification by sex and key covariates of telomere and mitochondrial function. Aim 3 will elucidate the maternal sociodemographic, psychosocial, behavioral and biophysical determinants of variation in components of allostatic load that impact newborn HSC/MSC biology using state-of-the-art machine learning and prediction approaches. Aim 4 will establish a shared Biobank repository of HSC, MSC, cord blood, cord and placental tissue samples for future studies of molecular mechanisms (gene expression, epigenetic profiles) and in vitro differen- tiation. Significance and Impact: Our project will 1) define novel measures (and their norms) in human newborn stem cells that profile the earliest vulnerabilities for cell health and risk of age-related disorders; 2) broaden under- standing of novel cellular targets and molecular mechanisms underlying biological embedding of stress, that, in turn, may inform the development of personalized interventions; and 3) provide shared resources (human newborn stem cell, placenta, cord, and cord blood biobank).

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) nearly doubles progression-free survival and increases overall survival when added to endocrine therapy in hormone receptor-positive (HR+), human epidermal growth factor receptor 2–negative (HER2-) metastatic breast cancer (MBC). The growing incidence of MBC among younger women highlights the need to optimize adherence to complex medication regimens proven to increase survival. The complex dosing schedule of CDK4/6is along with high cost is thought to contribute to non-adherence. There is limited evidence on the impact of CDK4/6i nonadherence on survival, symptom burden, or quality of life. As healthcare transitions to a greater dependence on telemedicine, and with the increasing penetration of text-enabled mobile phones across every segment of the population, we propose to test an innovative personalized multilevel mHealth intervention to improve CDK4/6i adherence by conducting a randomized controlled trial of a CONnected CUstomized Treatment Platform (CONCURxP) versus enhanced usual care (EUC). The CONCURxP trial will recruit 390 English- or Spanish- speaking adult women with HR+ HER2- MBC and a new prescription for a CDK4/6i, who have a mobile phone with text messaging and are treated in one of the participating NCI Community Oncology Research Program (NCORP) practices. To objectively measure CDK4/6i adherence, all enrolled patients will receive a smart pillbox. CONCURxP arm patients in addition to usual care will: (1) receive automated text reminders for missed or extra doses that are signaled by the smart pillbox; (2) respond to text messages citing the reason for each incident of CDK4/6i non- adherence; and (3) can view dosage history on a study web-portal. Missed or double doses beyond a predefined threshold will trigger an email notification to a designated member of the oncology provider team, who will be prompted to contact the patient to address the issue to close the communication loop. If nonadherence is due to cost, providers will be trained to refer patients to the Patient Advocate Foundation, a national non-profit financial navigation program. EUC patients will have access to educational materials to improve side effect management. Patients will receive the interventions for 12 months and complete surveys at baseline, 3, 6, and 12 months after enrollment. Our goals are to: (1) compare CDK4/6i adherence; and (2) patient-reported outcomes (PROs) including symptom burden, quality of life, patient-provider communication, self-efficacy for managing symptoms, and financial worry at 12 months between CONCURxP and EUC arms; (3) use mixed methods to describe the patient and provider experience with the CONCURxP intervention; and (4) explore healthcare utilization, progression-free, and overall survival at 12 months in CONCURxP patients compared to EUC. The primary endpoint is adherence at 12 months, defined as the proportion of days patients took CDK4/6i according to the prescribed frequency, measured by pillbox. Our novel multilevel mHealth intervention will provide valuable and actionable results to improve health outcomes and patient experience.

NIH Research Projects · FY 2025 · 2022-08

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to human genetic disease, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The control of alternative splicing is a highly combinatorial process, where many inputs dictate the splicing outcome for each exon. A critical feature of these regulatory mechanisms is the specific interaction of trans-acting splicing factors with cis-acting RNA elements. We use a highly integrated approach to investigate the molecular mechanisms that regulate pre-mRNA splicing. This includes knockout and knock-in tissue culture models, reconstitution assays using radioligands, transcriptomics, bioinformatics, kinetics, structure- function and biochemical techniques. In the next five years, we aim to address several outstanding challenges in the field, pursuing the following novel research directions. (1) Our demonstration that splicing regulatory proteins display highly position-dependent activities that negatively or positively influence splice site choice changed the way we think about the classical splicing activators (SR proteins) and the classical splicing repressors (hnRNPs). It is now appreciated that the context-dependent activation or repression of U1 snRNP serves as a gateway to allow the abundant U1 snRNP to fulfill its splicing function and its role to protect the pre-mRNA from premature degradation. However, it is not understood how splicing regulators achieve activation or repression of U1snRNP at the 5’ splice site. We aim to dissect the mechanisms of splicing repression by embracing multi-system approaches and by understanding the role of U1 snRNP conformers in mediating spliceosomal assembly. (2) Intron retention is an important alternative splicing pathway that has eluded extensive study. Thus, its regulation is not well-understood. The existence of inefficiently spliced introns within coding exons (exitrons) further highlights the biological importance of understanding when introns are removed efficiently and when they are not. We will decipher the rules of efficient intron removal and investigate the impact of cis-acting elements in this process using synthetic biology approaches. The argument is that the depth of the sequence variation tested in massively parallel reporter assays is far greater than the testing landscape that the human genome offers. Here, we will take advantage of our expertise in experimental molecular biology and bioinformatics. (3) It has become widely appreciated that gene expression events are highly integrated, with evidence suggesting that most pre-mRNA processing occurs co- transcriptionally. Defects in any one of these steps has been linked to disease. However, most published studies evaluate only steady-state levels of gene expression or focus only on a single step. This ignores the dynamics of gene expression steps that collectively contribute to the generation of proteins from mRNAs. Thus, it is unclear how the kinetics of RNA processing and mRNA stability translate into an endpoint gene expression signature. We have established a reliable method to metabolically label nascent RNA, which allows us to track transcripts from synthesis to degradation. Work in this project will probe how steady state mRNA levels are established, how the splicing and translation regulator SRSF1 influences mRNA dynamics and how these processes adjust as a cell undergoes transformation. The goals of our research program are to obtain a better understanding of exon recognition and alternative splicing. The new mechanistic insights will be leveraged to improve strategies to therapeutically target this essential gene expression step.

NIH Research Projects · FY 2025 · 2022-08

Project Summary This K08 proposal will address a critical gap in our fundamental knowledge of the effects of insufficient sleep and depression on longitudinal sleep-dependent memory development and will prepare this applicant, Katharine Simon, PhD, to become an independent translational investigator with expertise in sleep, memory, and psychopathology across development. Depression in adolescence is a growing public health concern, with symptoms beginning during the transition to adolescence (9 -12 years)1 and conferring heightened risk for long-term deleterious psychological and cognitive outcomes.20-21 In contrast to other clinical disorders, depression is associated with specific deficits in hippocampal-dependent (HcDep) memory (e.g., declarative memory).9-11 Although the brain mechanisms underlying depression-related memory deficits are not understood, one potential risk factor may be insufficient sleep (e.g., short sleep duration, variable timing, or poor quality) given the critical role of sleep in supporting new hippocampal-dependent memory formation (sleep dependent memory [Hc-SDM]).12 The transition to adolescence is a particularly vulnerable time that involves increasingly dramatic changes to sleep patterns.2,3 Although a few nights of insufficient sleep does not consistently affect HcDep memory performance in adolescents18,50-53; typical insufficient weekly sleep is associated with reduced hippocampal volume.19 At present, it is clear there is a fundamental gap in our knowledge of the individual trajectories of insufficient sleep, depression, and Hc-SDM across the adolescent transition and the longitudinal associations between these factors. To address this question, 27 pre-adolescents (9 to 12 years) will be assessed using a repeated measurement-burst design with sampling at quarterly intervals over a year using a personalized, mobile health (mHealth) platform. At each quarterly evaluation, for 7 consecutive days, participants will complete daily sleep diaries, ecological momentary assessments, and Hc-SDM tasks. The hypothesize is that insufficient sleep and depression symptoms across the adolescent transition will predict long-term Hc-SDM performance trajectory deficits. This Mentored Clinical Scientist Research Career Development Award will support Dr. Simon’s training goals to acquire skills and knowledge in 1) gaining expertise in naturalistic, longitudinal cognitive and clinical assessment using mHealth and wearable devices, 2) training in clinical pediatric research, and 3) intensive repeated-measure analyses. These training aims will build on Dr. Simon’s prior training in cognitive neuroscience and clinical psychology and expertise in short-term experimental sleep-dependent memory research. Furthermore, the training Dr. Simon will gain from this K08 is an essential step to become an independent developmental translational researcher and will directly lead to my future R01 application to further evaluate the longitudinal trajectories of insufficient sleep and depressive symptoms on older adolescents’ Hc-SDM memory and identify key windows in which sleep-based interventions can support cognitive trajectories in depressed adolescents.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Although bariatric surgery is the most effective treatment for severe obesity, a large proportion of patients experience significant weight regain with longer follow-up. Because weight regain is associated with re- emergence of weight-related comorbidities, increased health care costs, and impaired quality of life, it is imperative to effectively manage this problem. Diet, exercise, and behavior therapy have demonstrated minimal efficacy in reversing weight regain. Pharmacotherapy could be useful to manage this clinical problem. However, there are no published randomized controlled trials (RCTs) of pharmacotherapy for reversal of weight regain. This is a major therapeutic gap, which the proposed project will aim to fill. In this well-powered meticulously designed double-blind RCT employing a Sequential Multiple Assignment Randomized Trial (SMART) design, a total of 120 subjects with weight regain after bariatric surgery, will be initially randomized in a 3:3:2 ratio to daily treatment with topiramate (TPM) 50 mg or phentermine (PHEN) 7.5 mg or placebo. After 4 months, responders (those with ≥5% weight loss) will continue the same treatment, while nonresponders will be re-randomized to a higher dose of the same drug or phentermine/topiramate 7.5/50 mg combination (PHEN/TPM) during Months 5-12. The placebo group will receive placebo for the full 12 months. All subjects will receive diet and lifestyle counseling throughout the study. Aim 1. To determine whether pharmacotherapy can reverse post-bariatric surgery weight regain. We hypothesize that, compared to placebo, all three active drug therapies – TPM, PHEN, and PHEN/TPM will lead to greater percent weight loss at Month 12. Aim 2. To examine change in energy intake objectively assessed with the Intake-Balance Method using doubly labeled water and DXA. We hypothesize that active drug therapy will lead to greater reduction in energy intake at Month 12. Aim 3. To examine changes in the most common maladaptive eating behaviors in the post-bariatric surgery patients - grazing, loss-of-control eating, and binge eating. We hypothesize that, compared to placebo, all three drug therapies will lead to decreased maladaptive eating behaviors. A battery of carefully chosen exploratory outcomes including other likely modulators of changes in energy balance (appetite, night eating behavior, dietary restraint, thyroid hormones, leptin, and physical activity), additional efficacy (ALT, AST, glycemia, lipid levels, inflammatory markers, quality of life, and psychological wellbeing), and safety (heart rate, BP, anxiety, depressive symptoms) will be investigated. Additional efficacy questions including the best initial treatment, dose escalation vs combination strategy, and each adaptive intervention will be examined. The findings of this highly rigorous study will provide high-quality evidence for the efficacy and safety of pharmacotherapy in reversing weight regain after bariatric surgery. Study results could lead to a change in clinical practice paradigms and enhance long-term outcomes for patients after bariatric surgery. Because the SMART design mimics clinical practice, the findings of this RCT are directly translatable to clinical practice.

NIH Research Projects · FY 2025 · 2022-08

SUMMARY/ABSTRACT (30 lines) Relevance to public health. Polymorphonuclear leukocytes (PMN, neutrophils) release reactive oxygen species (ROS) to combat infection, but this inflammatory response can also initiate and propa- gate lung damage. Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS) that is fatal in 40% of patients, are characterized by accumulation of albumin-rich fluid in the pulmonary air spaces. Drug therapies focused on downstream cytokine actions have failed to improve morbidity or mortality; we hypothesize, and offer evidence, that targeting the human voltage-gated pro- ton channel (hHv1) at early steps can be more effective. We propose to target hHv1 because (i) the chan- nel in PMN initiates and sustains the inflammatory response, (ii) C6, a unique blocker of hHv1 sup- presses human PMN ROS production, and (iii) C6 suppresses lung compromise in an ALI mouse model. Brief background. This application builds on advances in the last period when we created the first high-affinity and specific direct blocker of hHv1 (C6 peptide) and used it to show, first, that human sperm require hHv1-mediated H+ efflux to initiate capacitation, allowing the acrosomal reaction, and oocyte fertilization and, second, that hHv1 in human PMN is required to produce and sustain release of inflammatory agents, including ROS and proteases, during the innate immune inflammatory response. Unique features and innovation. Our pilot data reveal a second target in the pathway: albumin (Alb) is required to activate hHv1 in human PMN and we describe a peptide (L*) that blocks Alb-activa- tion and ROS production. Supporting our driving hypothesis, we show here that both C6 and L* inhibit hHv1 in human PMN, decreasing ROS production, and that C6 protects in an ALI mouse model, restor- ing lung compliance, and decreasing ROS, proinflammatory cytokines, protein, and PMN in bron- choalveolar lavage fluid. We employ our novel membrane tethered (T-peptide) method to speed struc- ture-function studies and peptide design, show a bivalent C6 (C62) that fully inhibits open hHv1 chan- nels, benefit from advanced biophysical and in vivo methods, and two expert collaborators. Three specific aims. (1) Alb activation of hHv1 seeks the structural and mechanistic basis for the action of Alb and a more potent natural metabolite. (2) Alb regulation of the PMN inflammatory re- sponse seeks to delineate the role of hHv1 in PMN using C6, C62 and L* and the basis for peptide action. (3) Inhibiting acute lung injury with Hv1 inhibitors studies an ALI model in WT and Hv1 KO mice. Significance. This work addresses an unmet medical need, recently made more apparent by the ad- vent of COVID-19-related ALI/ARDS and has broader influence because Hv1 in PMN and other phago- cytes is complicit in additional acute and chronic inflammatory disorders. We propose to apply unique hHv1 inhibitors and innovative methods to understand and suppress this pathophysiology.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY    Heavy  metals are  natural  components  in  the earth  but  become  concentrated  and  toxic  in environment  as  a  result  of  human-­caused  activities.  The  term  heavy  metals  comprise  a  number  of  essential  metals  (e.g.  zinc, copper, iron) and non-­essential metals (e.g. cadmium, mercury, lead) for human, whose excess results in  toxicity even under trace amount. Accumulated heavy metals directly bind to and interfere with various cellular  components  (e.g.  proteins,  nucleic  acids),  and  lead  to  severe  cellular  dysfunction  and  even  death.  The  most  commonly  affected  organs  include  liver,  kidney,  intestine,  heart,  blood  and  nervous  systems.  Children  with  their  developing  nervous  systems  are  particularly  vulnerable  to  heavy  metal  intoxication.  Heavy  metal  homeostasis also  plays an  essential  role  in human normal physiology,  whose  defect  results  in  various  human  diseases. Therefore, understanding the fine-­tuned cellular response to overdosed heavy metals will lead to the  development of effective therapeutic methods against heavy metal-­associated intoxication and diseases.   In this application, we propose to elucidate the molecular mechanism involved in heavy metal response  by establishing the Hippo pathway as a key player in this process. Over the past decades, the Hippo pathway  has  been  recognized  as  a  crucial  signaling  pathway  that  controls  tissue/organ  size  by  restricting  cell  proliferation and stimulating cell apoptosis. Interestingly, our preliminary studies have revealed an unexpected  role  of  the  Hippo  pathway  in  heavy  metal  response  by  regulating  metal-­responsive  transcription  factor  1  (MTF1). Moreover, we have revealed a novel regulation of the Hippo pathway by heavy meals. Based on these  findings,  we  hypothesize  that  the  Hippo  pathway  plays  a  critical  role  in  heavy  metal  response.  In  this  application, we propose 1) to characterize the Hippo pathway-­mediated heavy metal response in vivo (Aim 1);;  2)  to  elucidate  the  regulation  of  MTF1  by  the  Hippo  pathway  (Aim  2);;  and  3)  to  study  the  regulation  of  the  Hippo pathway by heavy metals (Aim 3).

NIH Research Projects · FY 2025 · 2022-08

Project Summary Despite longstanding efforts to improve recruitment of minorities into Alzheimer’s Disease and Related Dementias (ADRD) research, US Hispanics/Latinos continue to be severely underrepresented in ADRD randomized clinical trials. Greater representation of Hispanics/Latinos is critical to ensure the safety and efficacy of interventions and the generalizability of results across an increasingly diverse US population. Preclinical AD trials will be essential in the pursuit of improved ADRD therapies and are unique in requiring asymptomatic individuals to undergo biomarker testing and to enroll with a study partner. It is unclear, however, whether these unique requirements pose a specific challenge to the recruitment of Hispanics/Latinos into preclinical AD trials. It is imperative that we discover and test novel interventions to improve recruitment of Hispanics/Latinos to these trials. Given my background as an epidemiologist with a solid foundation in health disparities research, I have the dedication for undertaking this research. Through this K01 Mentored Research Scientist Career Development Award, I propose to receive the necessary training and research experience to address several gaps and become a leader in ADRD recruitment science. My overarching hypothesis is that older Hispanics/Latinos are less willing than their Non-Hispanic (NHW) counterparts to participate in preclinical AD trials, due in part to modifiable behavioral factors amenable to intervention. To test this hypothesis, I propose three specific research aims: (1) First, I will examine differences in willingness to participate in preclinical AD trials between diverse Hispanics/Latinos and Non-Hispanic whites (NHW) from a national population-based sample of 1,800 older adults who respond to a web panel survey. (2) Next, through in-person cognitive interviews, I will identify key behavioral determinants of intention to participation in a sample of 100 local community dwelling older Hispanics/Latinos, with a particular focus on contextual factors, cultural beliefs/values, research literacy, previous experiences with the healthcare system, research attitudes and perceived need/benefit. (3) Lastly, I will use an Intervention Mapping planning approach to systematically develop and pilot test a behavioral intervention that is culturally and linguistically appropriate and designed to improve Hispanic/Latino participation in AD preclinical trials. Findings will provide preliminary data for a large multicenter trial to test the intervention using a randomized approach. To accomplish these aims and my goal of becoming an independent investigator, I propose to engage in extensive ADRD training through didactic instruction, experiential learning and mentorship by the leaders in the field. I will leverage resources from the University of California, Irvine Alzheimer’s Disease Research Center to (a) acquire foundational knowledge of core principles in ADRD research, (b) gain experience in mixed- methods approaches in recruitment science, (c) become proficient in designing behavioral interventions, and (d) develop the leadership skills necessary to conduct multicenter studies and propel my research forward.

NIH Research Projects · FY 2025 · 2022-08

The main theme of the research is to develop new methodologies for resolving statistical issues emerging from our team's collaborations in cohort studies of underrepresented and overall US aging populations with a particular interest in Alzheimer's disease (AD). We focus on making appropriate statistical inference for censored survival data with partially known risk sets, developing methods for assessing prediction precision for recurrent events survival data including time-dependent ROC and extension of the C-index, and developing new conditional modeling strategies that best model life history processes during the lifespan. We also plan to develop publicly available statistical software with the goal of dissemination and generalization. The proposed methods of estimating time-dependent ROC and C-index for recurrent events extend those for a single survival time by taking into account different models for recurrent events, producing novel predictive models for recurrent events when there are possible missing events. Such predictive models are useful in estimating the partially observed risk sets which would lead to appropriate parameter estimation in regression analysis for censored survival data, in particular, time of AD onset, using the proposed weighted estimating approaches. In AD research, death as a terminal event occurs frequently in aging cohorts. The proposed conditional modeling strategy allows us to investigate AD events during the entire lifespan, which provides a clearer picture of the relationships among AD onset, death, other life evens, and risk factors, thus a better understanding of AD etiology and prevention. The method also provides a straightforward thus more precise estimation of AD prevalence that is potentially impactful to health care policy making. These methods are motivated from and will be applied to a wide range of datasets, including the Indian Health Service – a unique source for studying the disproportionate burden of AD among Native American populations, the Aging, Demographics and Memory Study – a subset of the Health and Retirement Study representing an ideal observation cohort study for studying the epidemiology of AD in the Black and Latinx communities, the National Alzheimer's Coordinating Center uniform data sets collected by more than ADRCs in the US from their longitudinal cohorts, the 90+ study that focuses on aging and dementia of the oldest old population, and the CMS Limited Data Set that is a random sample of Medicare/Medicaid claims data accessible for research purposes – the most representative data of the US aging population. The methods will be widely applicable to problems in many other fields of biomedical research.

NIH Research Projects · FY 2022 · 2022-08

Contact PD/PI: Khodagholy, Dion PROJECT SUMMARY/ABSTRACT A major obstacle to understanding how the dynamic activity of brain circuits permits emergence of cortical func- tion is insufficient capability to acquire and manipulate this activity across the course of brain maturation. Neu- romodulators and neural activity patterns are intimately linked in mediating this maturation. There is urgent need to develop technology to acquire and manipulate neurophysiological signals from small, fragile, immature brains and address this gap in knowledge. Our long-term goal is to causally determine neural correlates of cognitive functions and neuropsychiatric disorders in the developing brain. Here, we step toward this goal by pursuing the overall objective of this project: to establish a fully implantable closed-loop neural interface device that can detect neurophysiologic signals and responsively deliver neurochemicals in mouse pups as they grow and develop. Our central hypothesis is that integrating conducting polymer electrodes, conformable ionic circuits, and organic ion pumps will enable creation of an Organic Closed-loop Electrochemical Array for Neurodevelopment (OCEAN) that will help us elucidate the coordination of neural activity and neurochemistry in the developing brain. This hypothesis is supported by preliminary data demonstrating use of: i) conformable high-density surface electrocorticography arrays (NeuroGrids) to record from cortical networks in developing rodents; ii) conformable, biocompatible ionic circuits capable of processing neurophysiological signals; iii) highly conductive, stretchable, flexible materials for transmission of such signals; iv) organic ion pumps to modulate brain signals with millisec- ond precision. The rationale for the proposed research is that integration of these materials and devices will enable us to address the substantial barriers that essentially preclude chronic, high spatiotemporal monitoring and manipulation of neural networks in vivo during development. The specific aims include: (i) establish expand- able, conformable and biocompatible integrated components for high spatiotemporal resolution electrophysio- logic monitoring; (ii) establish expandable, conformable and biocompatible integrated components for high spa- tiotemporal resolution neurochemical modulation; (iii) integrate neurophysiologic recording and neurochemical delivery to perform proof-of-concept closed-loop modulation. The proposed research is innovative, in our opinion, because it uses organic electronic approaches at all stages – signal acquisition, processing/detection, data trans- mission, device powering, and neurochemical delivery to create for the first time a fully implantable responsive neural interface device compatible with in vivo use in naturally behaving rodents across development. This work is expected to be significant because it will provide the groundwork for interacting with neural networks across time periods associated with brain maturation and emergence of complex brain functions. It will have positive impact on development of previous unattainable experimental paradigms and contribute more broadly to im- provement in design of safe, long-term, minimally invasive bioelectronic devices. Project Summary/Abstract Page 6

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY / ABSTRACT Each year, millions of children sustain injuries and undergo invasive medical procedures in the emergency department (ED). Current guidelines emphasize the need to address the significant pain and distress children experience in the ED, but inadequate pain and distress management remain prevalent. Under-treated pain and distress can result in negative future pain experiences and maladaptive psycho-behavioral outcomes. Recent work suggests that Latinx children are at a particularly high risk for experiencing distress and pain surrounding surgery as well as pain treatment disparities in the ED. This application proposes a large-scale observational, longitudinal cohort study to identify both predictors and outcomes of procedural distress and pain in an ethnically diverse sample of children 2-9 years old undergoing invasive ED procedures. Methods will be based on the NIMHD Minority Health Disparities Research Framework and Triple Aim and will include empirically informed assessments of psychological, sociocultural, environmental, healthcare system, clinical recovery, patient experience, and resource utilization variables. The K23 candidate is an Assistant Professor at the University of California-Irvine School of Medicine and aims to establish an independent interdisciplinary research program that improves pediatric pain care and prevents adverse outcomes and healthcare disparities surrounding injury and painful procedures in the pediatric ED. The candidate’s training plan capitalizes on the expertise of a highly experienced multidisciplinary mentorship team, integrating key training in pain surrounding invasive ED procedures, Triple Aim value-based care framework, sociocultural factors in pediatric pain and pediatric healthcare disparities, advanced statistical modeling, and professional development. This application will be executed within the UCI Center on Stress & Health, a highly productive, well-established research environment that incorporates a unique multidisciplinary approach to training and clinical research. The training plan will incorporate didactic coursework, one-on-one mentoring, and UC Irvine Institute for Clinical and Translational Science (NIH CTSA) resources and seminars focused on career development, training evaluation and research ethics. With enthusiastic and material support from the Children’s Hospital of Orange County senior leadership, the project will be conducted in a high-volume pediatric ED where a large proportion of patients (66%) are Latinx and are part of an innovative population health program. Collectively, this will provide an exceptional training environment to characterize multidimensional contributors to procedural pain and distress in a population at-risk for experiencing care disparities and launch a clinically impactful, independent research program that promotes effective and equitable pediatric pain and injury-related care in the ED.

NIH Research Projects · FY 2025 · 2022-08

Protein-protein interactions (PPIs) are key to the formation of protein complexes, the active components responsible for a multitude of cellular functions. Aberrant PPIs can have detrimental effects on essential biological processes and lead to various human diseases. Elucidating PPI networks and their structural features within cells is central to understanding fundamental biology and molecular alterations associated with human pathologies, and ultimately facilitating therapeutic development. However, delineation of proteome networks to define the cell’s functional states at the systems-level is challenging due to limitations in existing approaches. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful technology for PPI studies, owing to its unique capability of preserving and capturing protein interactions in their native environments, as well as uncovering PPI identities with structural details. Although current XL-MS technologies are successful in global PPI analysis, unmet challenges still remain especially for complete illustration and quantitative assessment of cellular networks, and spatial PPI mapping. This necessitates new developments to enhance technical capabilities for advancing systems structural biology. The ubiquitin-proteasome system (UPS) is the major degradation pathway in eukaryotes, whose network is remarkably dynamic and made of a large number of compositionally and structurally dynamic machines that orchestrate protein ubiquitination and degradation. Dysregulation of the UPS has been linked to many human diseases including cancer and neurodegenerative disorders. Given the clinical success of proteasome inhibitors, the UPS has become an effective platform for drug discovery. Although basic functions of the UPS are understood, molecular details underlying its multi-layer regulation and mechanistic action remain elusive. Therefore, elucidating the interaction and structural dynamics of the UPS network in its physiological context is essential not only for advancing the understanding of UPS biology, but also for augmenting their therapeutic potential in human health and medicine. In the next five years, we plan to address several outstanding technological challenges in PPI studies to better decipher the UPS pathways, by pursuing the following two research directions: 1) Advancing XL-MS technologies for interactomics and structural proteomics at the systems-level; 2) Mapping the UPS network to uncover molecular details underlying protein ubiquitination and degradation. Specifically, we will center our efforts on developing novel XL- MS technologies to enable in-depth and quantitative analysis of proteome networks with structural details and enhanced spatial resolution to define cellular functional states. In addition, we will employ the newly established technologies to delineate action mechanisms of proteasome regulators in protein degradation, investigate Cullin- RING Ligase mediated protein ubiquitination and dissect the organization of the UPS network. Together, these studies will result in an exciting technological advancement in proteomics research, and facilitate answering important but unresolved biological questions associated with UPS biology.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract This proposal describes a 4-year career development plan for the PI, Dr. Abraham J. Qavi, MD, PhD, with the goal of preparing him for an independent research career as a physician scientist. This plan includes expand training opportunities and a pathway to an independent career that includes the development and implementation of sensor technologies and its application towards filoviruses, namely Ebola. The PI graduated with degrees in Biochemistry & Molecular Biology and Chemistry from the University of California, Irvine. He then enrolled in the Medical Scholars Program at the University of Illinois at Urbana-Champaign, where he earned his MD and PhD in Chemistry. Dr. Qavi continued his medical training as part of the Clinical Pathology Physician Scientist Training Program at Washington University in St. Louis and the Barnes-Jewish Hospital consortium. During his residency elective time, he began research in the laboratory of Dr. Lan Yang, who will serve as his co-mentor together with Dr. Amarasinghe. Dr. Yang is the Edwin H. and Florence G. Skinner Professor of Electrical & Systems Engineering, and an internationally renowned researcher in photonics. In 2019, Dr. Qavi added an additional training component under the co-mentorship with Dr. Gaya Amarasinghe, Professor of Pathology & Immunology, to expand the application of his previously developed sensor technology to infectious disease reach. The goal of this study is the development of a rapid, multiplexed optofluidic sensor platform for the rapid detection of pathogens, with an initial focus on Ebola. Filoviruses, such as Ebola, are among the most lethal human pathogens, with high case fatality rates during outbreaks. Critical in the identification and management of outbreaks are robust detection methods that can be implemented rapidly and sensitively. To address this critical need, Whispering Gallery Mode (WGM) sensors will be leveraged. WGM devices are a class of optical sensors in which light is confined within a micron-scale volume. These devices have incredibly high sensitivity, small sensor footprint, ease of integration with conventional electronics, and low fabrication costs. Microbubble resonators (MBRs), a subclass of WGM sensors, offer the advantages of conventional WGM devices while enabling coupling of optical and the fluidic components into a single component. The goal of this proposal is the use of an optofluidic sensor platform based on MBRs for the rapid, multiplexed detection of Ebola. The workflow and advancements, including MBR development, engineered antibodies, biophysical validation and multiplexing, will provide the framework to extend the work beyond the initial goals and promote facile transition to an independent career for Dr. Qavi.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Critical periods are restricted windows of heightened plasticity during which developing neural circuits are particularly sensitive to sensory input. Unlike the transient alterations that come with adult plasticity, changes occurring during developmental critical periods are lasting and cemented after closure. The transplantation of inhibitory progenitors into adult amblyopic mouse visual cortex has been shown to reactivate critical period plasticity and rescue visual deficits. Recent findings suggest that this reactivation stems from the transplant- induced developmental rejuvenation of host inhibitory neurons. The preliminary transcriptional profile of rejuvenated host inhibitory neurons identifies Calbindin (Calb1), an understudied plasticizing molecule, as a key upregulated factor. The long-term goal of this work is to investigate the role of Calb1 in the transplant- induced rejuvenation of host inhibitory neurons and subsequent reactivation of critical period plasticity. Aim 1a tests whether Calb1 is necessary for transplant-reactivated plasticity by virally-inactivating Calb1 function in recipient adults and using intrinsic signal optical imaging to assess for a shift in ocular dominance following monocular deprivation. To test whether Calb1 is necessary for developmental critical period plasticity, the same approach will be applied to non-transplanted juveniles. Aim 1b tests whether Calb1 is sufficient to reactivate plasticity, non-transplanted adults with virally-overexpressed Calb1 show shifts in ocular dominance post- monocular deprivation. Aim 2a utilizes weighted gene co-expression network analyses of the previously generated transcriptional data to identify the biological pathways underlying transplant-induced rejuvenation. Expression of identified pathway components will be validated and characterized in Aim 2b using immunohistochemistry. This work will yield novel insight into the cell and molecular mechanisms underlying inhibitory neuron transplantation, visual circuit development, and reveal new translatable targets for the development of neurotherapeutics. Brain repair strategies that catalyze endogenous cellular rejuvenation hold great clinical promise for a broad range of neurological and neuropsychiatric disease. The technical aspect of my training plan focuses on the acquisition of viral injection and functional imaging (Aim 1), and bioinformatic and statistical analysis skills (Aim 2). Aim 1 training will be carried out under sponsor and visual neurophysiologist Dr. Gandhi and postdoctoral fellow Dr. Figueroa-Velez, and Aim 2 training will be guided by co-sponsor and transcriptomics expert Dr. Spitale and bioinformatician Dr. England. Technical training will be complemented by a heavy emphasis on scientific communication, professional development, teaching, and outreach. UC Irvine is a rich, well-established training ground for successful graduate students and offers a breadth of resources, structured courses, and networking opportunities.

NIH Research Projects · FY 2025 · 2022-08

Cardiovascular disease (CVD) is the leading global cause of death, accounting for approximately 18.6 million deaths in 2019. Between 2015 and 2018, 126.9 million American adults had CVD, resulting in an annual cost of $363.4 billion in healthcare, lost productivity and mortality. The CVD burden is not distributed equally among racial/ethnic (R/E) groups: ~60% of African American (AA) adults have CVD compared to ~48% of Hispanic/ Latinx (HL) and Non-Hispanic Whites (NHW). R/E disparities in CVD are likely due to an interplay of genetic and sociocultural factors, which are exacerbated by the chronic stress burden that some R/E groups endure. Chronic stress elicits repeated activation of the stress response systems, increasing allostatic load (AL) and compromising health. High AL may increase risk for early vascular aging (EVA): arterial stiffness, subclinical endothelial dysfunction, hypertension and increased left ventricular mass. R/E disparities in EVA emerge by adolescence, before overt signs of CVD, but no studies simultaneously measured vertically integrated CVD markers early in development. Links between chronic stress, AL and CVD have been proposed but not studied comprehensively. Factors that may protect against CVD in some high-risk groups (e.g., HL) have not been explored across R/E groups. Current CVD prevention and treatment guidelines were developed from data obtained primarily in men, contributing to missed or delayed diagnoses, non-optimal treatment and poor outcomes, especially in R/E minority women. Research on the mechanisms of CVD risk in AA was conducted almost exclusively in men despite high CVD prevalence in both sexes. Importantly, R/E disparities in CVD are more marked in women. CVDs explain 33% of the mortality variation between AA and NHW men but 45% for women. Many CVD risk factors have a higher prevalence (e.g., obesity) or a greater impact in women; for example, chronic stress, which disproportionately affects R/E minorities, is a stronger predictor of CVD-related mortality in women. Our current study measures multidomain CVD risk factors including cumulative stress, hypothalamic-pituitary-adrenal activity, inflammatory markers and obesity indices (anthropometry, adiposity, diet, metabolic markers, and adipokines) in 13 to 17-year-old AA, HL and NHW adolescent girls. We propose a follow-up study of this well-characterized cohort 4 to 6 years later in emerging adulthood, a critical period when physical maturity is largely complete but biopsychosocial risk factors that have longterm implications for CVD emerge. We will determine the magnitude of cumulative stress and AL burden over time, incorporate vertically integrated markers of EVA with state-of-the-art techniques, and examine the effects of cumulative stress, AL and adaptive cultural coping practices on EVA. Characterizing R/E differences in modifiable bio- psychosocial risk and protective factors associated with subclinical CVD during a developmental phase when humans can exert more control over their lives (with increased autonomy but few adult responsibilities) offers an opportunity to preempt the transition from health to disease and reduce CVD disparities in at-risk groups.

NIH Research Projects · FY 2024 · 2022-08

Project Summary Cataract is an opacity of the eye lens that can result in blindness. In 2010, cataract affected 15% of the population by age 60 and nearly 70% of the population by age 80. Despite the disease’s prevalence, surgery that replaces the lens with a synthetic implant remains the current standard of care. This strategy prevents access to treatment for people without adequate healthcare. Cataract is caused by protein aggregation in the lens, the majority of which is comprised of a class of proteins called crystallins. Understanding the mechanisms by which crystallins aggregate to form cataract is critical for developing therapeutics that can prevent their formation as an alternative to cataract surgery. Maturation of the lens is accompanied by loss of organelles and protein degradation mechanisms; therefore, the eye lens is metabolically inactive and crystallins by necessity are extremely long- lived proteins. Research on their biophysical properties have proven that crystallins are unusually soluble and stable. However, due to subjection to decades of damaging radiation as well as the loss of lens homeostasis mechanisms, crystallins accumulate post-translational modifications (PTMs) such as oxidation and deamidation. These PTMs are thought to alter their solubility and stability, thereby promoting cataract-related aggregation in the lens. Deamidation, the conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, is the most common PTM. It has been shown that variants with one or two deamidation sites have increased susceptibility to aggregation and oxidation as well as decreased stability. The long-term goal of this project is investigate how deamidation promotes aggregation. This research will be performed on variants with 3, 5, 7, and 9 sites of deamidation and builds on previous insights developed by our lab. I hypothesize that the mechanism of aggregation of these variants will depend on the extent of deamidation. In variants with fewer sites of deamidation, I predict aggregation is formed by increased anion-p interactions. In highly deamidated variants, I propose aggregation increases hydrophobic exposure from altered protein dynamics. Here, I will investigate these possibilities with solution-state NMR and mass spectrometry dynamics experiments. These will be complimented with biophysical techniques that quantify the extent of aggregation, dimerization, and oxidation. I will then look structure of deamidation variant aggregates with solid-state NMR. Finally, preliminary evidence in our lens suggests the HgS could be performing a novel active role in the lens as a last resort redox buffer. I will investigate this with proteomics experiments that monitor the disulfide linkages that have been implicated in this role.

NIH Research Projects · FY 2025 · 2022-08

Abstract The DECREASE SSI Trial (Decolonization to Reduce After-Surgery Events of Surgical Site Infection) Colon and small bowel surgery are high-volume, high-cost surgeries performed in over 675,000 patients in the United States each year. These surgeries carry one of the highest surgical site infection (SSI) rates, with estimates of 11-45%, or 2- to 3-fold higher than nationally reported rates, when comprehensive surveillance efforts are used, such as in clinical trials. Comprehensive tracking also uncovers a disproportionate amount of post-discharge incisional SSIs. These incisional SSIs are strongly tied to patient anxiety, stress, embarrassment, dissatisfaction, and lower quality of life scores. Mounting evidence shows that social and economic disadvantage can adversely affect the risk of SSI, likely due to the differential ability to protect a fresh incision from infection after hospital discharge. The Decolonization to Reduce After-Surgery Events of Surgical Site Infection (DECREASE SSI) Trial will test the value of adding a post-operative intervention to current pre-operative and intra-operative prevention practices. The DECREASE SSI Trial is a multi-site pragmatic randomized controlled clinical trial comparing routine post- discharge wound care to a post-discharge decolonization regimen of chlorhexidine bathing and nasal iodophor to prevent SSI events in the 30 days following discharge for open colon and small bowel surgery. If successful, this trial will provide a simple prevention measure for the 675,000 persons who undergo non-laparoscopic surgery involving the small and large intestines each year.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract The effective treatment of bacterial infections of skin, deep soft tissues and wounds continues to be a major unmet challenge in healthcare settings, especially among patients with chronic diabetes. Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria that are isolated from chronic, non- healing wounds. Antibiotic resistance has arisen in these particular bacteria, causing these infections to become increasingly difficult to treat and giving rise to multi-drug resistant strains, including Methicillin-resistant Staphylococcus aureus (MRSA). The goal of the proposed work is to develop the next generation of antimicrobials for which the design is inspired by a better mechanistic understanding of mammalian antimicrobial defense pathways. We focus our attention on the antimicrobial activities of neutrophil extracellular traps (NETs), which use histones to kill or suppress microbial proliferation. The antimicrobial mechanism of histones has not been understood. The Siryaporn and Gross labs recently reported that the pairing of histones with an additional component found in NETs – the antimicrobial peptide (AMP) LL-37 (cathelicidin) – produces potent antimicrobial synergy. LL-37 forms pores in the bacterial membrane, which enable histones to enter the bacterium and interfere with gene expression. This has an irreversible bactericidal (killing) effect on bacteria. The work proposed here will exploit this discovery by identifying combinations of human histones and membrane-/cell wall-targeting antimicrobials (MTAs) that produce potent antimicrobial activity and synergy. The overall objective of the project is to better understand the mechanism of antimicrobial synergy between histones and MTAs, and to harness it to establish a class of new therapeutics for the treatment of skin infections and wounds. We will accomplish this objective by identifying combinations of human histones with LL-37 and other MTAs that produce the greatest antimicrobial activities and synergies. We will test these against S. aureus, P. aeruginosa, and communities of skin bacteria in vitro (Aim 1). We will attempt to augment the antimicrobial activity by engineering in factors that impact histone function in NETs, specifically chemical modification through citrullination and tethering histones to DNA fibers (Aim 2). To validate our approach, we will test the combinations of histones and MTAs identified in Aims 1 and 2 in a standardized mouse skin infection model (Aim 3). To additionally address the unmet challenge of treating skin infection and wounds in diabetes patients, we will perform the tests in a diabetic mouse model. The results of this work will provide a mechanistic understanding of antimicrobial synergy and develop a strategy to combat the rise of antibiotic resistance. The results of the study could create a new class of antimicrobial therapeutics for the treatment of skin infections and wounds in diabetic and non-diabetic patients. This would represent a game-changer in the approach to antimicrobial treatments.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY The last decade has seen a devastating epidemic of opioid addiction in the U.S with the fatal opioid overdose incidence doubling in 10 years. Genetic factors clearly contribute to addiction vulnerability, but they alone cannot account for this rise. Therefore, environmental factors must also play role, and their enduring effects would be maximal during sensitive/critical periods early in life. Indeed, early life adversity (ELA) is associated with opioid addiction vulnerability, and in experimental models, it promotes such vulnerability. Although the responsible mechanisms have yet to be elucidated, the enduring nature of changes set in motion by ELA strongly suggest that epigenetic mechanisms may underlie these stable changes. The epigenome is a signal integration platform superimposed on the genome, which is modified by both ELA and drugs of abuse, and which also encodes aspects of sex differences. Epigenetic mechanisms are known to establish stable changes in cell function (via coordinating gene regulation) that give rise to persistently altered behaviors, including drug use and drug seeking behaviors. Thus, understanding epigenetic and transcriptional changes associated with addiction propensity following ELA may answer several key questions about the causes and mechanisms of opioid use disorder (OUD). For example, how is ELA epigenetically encoded, thereby increasing risk for opioid abuse and relapse- associated behaviors? Does heroin differentially affect the epigenome and transcriptome in those with ELA history, promoting this vulnerability? These questions will be addressed here using robust rat models of ELA and of heroin self-administration and relapse. Capitalizing on our findings that ELA augments heroin vulnerability and relapse in a sex-divergent manner4, consistent with human data20-23, we will test the driving hypothesis that both ELA and initial heroin use regulate epigenomic processes, and these, in turn, coordinate gene regulation that ultimately affects cellular signaling, and OUD-like behaviors. The epigenomic and transcriptomic changes induced by ELA and heroin will be determined by employing both bulk- and single-cell multi-omics in selected brain regions at different points in the OUD trajectory. They will be complemented by sampling of CSF and blood extracellular vesicle (EV) miRNA profiles to enable potential development of predictive markers of OUD risk and/or progression. Using state of the art sequencing and analysis methods, we will uncover gene regulatory networks associated with ELA, initial heroin use, and heroin abuse/relapse in females and males. This approach will allow us to identify novel targets for OUD prevention and intervention in vulnerable individuals.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY / ABSTRACT Complex behaviors rely on the combination of sensory clues and internal factors like goals, expectations, memories, and attention. A breakdown of these interactions is thought to be at the core of many neuropsychiatric disorders. Sensory information is carried by feedforward inputs while internal factors are conveyed via feedback afferents. Robust evidence from humans, primates, and rodents indicates that sensory and feedback neuronal pathways converge in the posterior parietal cortex (PPC). Indeed, the PPC is thought to be fundamental to an array of cognitive processes including working memory and decision-making. These data suggest that PPC neurons play a key role in integrating sensory and cognitive information streams. Yet, we have a critical gap in our knowledge regarding the synaptic mechanisms integrating feedforward inputs with feedback signals in the PPC. This lack of insight limits our ability to understand how neuronal circuit interactions drive behavior and how impaired integration leads to neuropsychiatric disorders. In preliminary experiments, we used two-color optogenetics to independently control cortical afferent pathways. We discovered that PPC neurons receive direct, monosynaptic innervation from both feedforward and feedback sources. Furthermore, we found marked differences in how functionally distinct subclasses of layer 5 pyramidal neurons integrate inputs from discrete long-range afferents. Specifically, intratelencephalically projecting (IT) cells exhibited nonlinear response enhancement while subcortically projecting extratelencephalic (ET) neurons summed inputs linearly. These data motivate our central hypothesis that cell- type specific integration of feedforward and feedback synapses drives input / output transformations in the PPC. To test this hypothesis, we propose a complementary use of opto- and chemogenetic circuit manipulation, brain slice electrophysiology and computational modelling. First, we will determine the temporal rules governing the interaction of feedback and feedforward afferents in distinct layer 5 projection neurons (Aim 1). Then we will combine two-color optogenetics with circuit specific chemogenetic silencing (DREADDs) to determine how cell-type specific synaptic integration drives the functional output of the PPC network (Aim 2). Finally, we will take advantage of computational methods to determine what ionic conductances underlie the cell-type specificity of feedforward-feedback integration (Aim 3). Completion of this research will provide novel insights into the cellular mechanisms underpinning the interaction of sensory and feedback information streams. This knowledge will further our understanding of the principles that guide information processing in the neocortex and provide the foundation for future basic and translational research.