University Of Washington

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Type 1 diabetes affects more than 1.25 million people in the United States and the annual incidence is increasing at an alarming rate of 3-4%. The emotional and financial burden of the disease is overwhelming and we currently have no way to predict or prevent new cases. As we gain a better understanding of the pathophysiological processes in the pancreas and the downstream effects of hyperglycemia (and periodic hypoglycemia during treatment), more robust biomarker assays are needed to improve the reproducibility of research findings and to translate those findings to clinical care. One technology that can provide robust, transferable assays for the measurement of proteins is liquid chromatography-tandem mass spectrometry. By directly detecting the analyte of interest, assays that use mass spectrometry detection can have better specificity than immunoassays and when paired with enrichment strategies, they can also be very sensitive. As we have demonstrated previously, it is straightforward to harmonize the results of mass spectrometric assays, which is significantly more difficult for immunoassays in general. This proposal aims to generate and validate novel transferable protein assays that harness the power of mass spectrometry. We aim to leverage a new method for the enrichment of extracellular vesicles and new de novo proteins for affinity enrichment, called minibinders, to help with sensitivity of the methods. Whenever possible, assays will be multiplexed and if antibodies are required for enrichment, they will be widely distributed through the Iowa Hybridoma Bank. Plasmids encoding minibinders will be deposited at Addgene. Chromatographic data from method development (particularly peptide selection, which will use narrow-window data-independent acquisition rather than relying on algorithms or data- dependent acquisition methods) as well as chromatographic data from method validation will be distributed via Panorama, along with detailed standard operating procedures. As requested in RFA DK-21-031, a portion of the assays produced will target glucagon, other fragments of proglucagon, proinsulin and its fragments, glycated soluble CD59, amylin, and the chromogranins. Our Target Prioritization Committee will help identify the most important proteins to add to this list and focus our development efforts.

NIH Research Projects · FY 2025 · 2023-07

Project summary In hematopoietic stem cells (HSCs), the epigenome confers self-renewal and differentiation functions wherein inheritance of HSC chromatin states is persistent across cell cycles. My postdoctoral studies focused on a fundamental epigenetic feature involving the local recycling of pre-existing, parental nucleosomes, which showed that repressed, but not active, chromatin domains are inherited across DNA replication. While identifying the histone chaperone(s) that facilitate the inheritance of repressed chromatin domains in mouse embryonic stem cells, I discovered that nucleophosmin, NPM1, plays an essential role in this process. NPM1 is a histone chaperone whose genetic mutation and rearrangement are found in ~30% of all adult AML, however its function in normal hematopoiesis remains unknown. Furthermore, key questions persist on the inheritance of H3K27me chromatin domains, such as what brings NPM1 and the polycomb repressive complex 2 (PRC2) to the DNA replication fork during S-phase, how are polycomb chromatin domains inherited in the developing immune system, and whether parental nucleosome segregation has a role in the precise balancing between self-renewal and differentiation capacities that shape a hematopoietic cascade. In this proposal, my group will identify the molecular basis for constructing the heritable human epigenome of HSCs and discover the chromatin dynamics that provide HSCs the ability to both self-renew and differentiate. We will use in vitro human induced pluripotent stem cells and differentiate them to derive a hematopoietic progenitor cell fate. Using this system, we will conduct cutting age genome engineering, proteomics, and imaging technologies to discover the function of histone chaperones and polycomb in constructing the heritable epigenome of HSCs.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT The primary goal of this proposal is to understand how conformational properties of the major pathogenesis factor of E. coli - most common, type 1 fimbrial adhesin of E. coli - defines its ability to elicit a protective antibody response as a vaccine candidate against urinary tract infections caused by E. coli. FimH is an adhesive subunit of the type 1 fimbriae and is one of the major factors in the ability of E. coli to bing human urothelium and cause urinary tract infection. We determined that the mannose-binding lectin domain of FimH can assume two conformational states - with a high- and low-affinity towards terminally-exposed mannosyl residues. The conformational shift in FimH is highly dynamic in nature and is the basis of the ability of FimH to mediate shear-enhanced bacterial adhesion, bind fast and strongly human cell receptors and shed bound antibodies. We intend to perform a comprehensive functional analysis of antibodies against both conformational states of FimH to analyze the conformational switch in FimH in the context of the immune response against the adhesive protein.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The oral streptococci are a major cause of infective endocarditis (IE). Several of these species express Siglec- like, serine-rich repeat (SRR) adhesins that bind a variety of O-linked sialoglycans on human glycoproteins and cells. Expression of the SRR adhesin GspB of Streptococcus gordonii M99 results in increased virulence in animal models of IE. This enhanced pathogenicity is thought to be due the interaction of GspB with the trisaccharide sialyl-T antigen (sTa, a core 1 sialoglycan) on the platelet receptor GPIb, leading to increased attachment of bacteria in the blood stream to cardiac valve surfaces and vegetation formation. Isogenic mutants of M99 expressing GspB variants that preferentially bind core 2 sialoglycans,(e.g., sialyl-lactosamine) are less virulent, as compared with the WT strain. These findings indicate that differences in the type of sialoglycan bound ("selectivity") affect the ability of organisms to both initiate and propagate endocardial infection, with some interactions enhancing pathogenesis, while other interactions reducing disease. We hypothesize that in vivo, the binding of streptococci in the bloodstream to sTa on platelets or cardiac surfaces helps initiate infection, and that this binding is enhanced by hemodynamic effects created by high shear flow. In contrast, binding to core 2 sialoglycans on blood cells may lead to increased bacterial clearance, thereby attenuating virulence. Subsequent interactions of bacteria on valves with core 1 versus core 2 sialoglycans may affect disease progression via different effects on platelet activation, clotting, or endothelial healing. To further address the roles of sialoglycan binding selectivity in the pathogenesis of IE, we will compare by flow the above isogenic strains for their binding to platelets, RBCs and WBCs in vitro. We will also examine whether these strains bind platelets that differ in GPIb sialoglycan content, and whether binding to sTa versus core 2 structures affects platelet activation. In addition, we will assess how selectivity impacts the attachment of bacteria to damaged cardiac surfaces, as modeled by microfluidic chambers lined with immobilized platelets, von Willebrand Factor, collagen or activated human vascular endothelial cells, and the effect of hemodynamic forces on binding. We will also explore the impact of selectivity on vegetation formation by assessing strain differences in triggering platelet contractility and effects on platelet-dependent endothelial healing. We will also compare vegetations produced in vivo by these strains for key features linked to virulence, including co- localization with platelet and biofilm production. These studies will provide novel insights as to how sialoglycan selectivity and hemodynamic forces affect the initiation and propagation of endocardial infection. By defining the mechanisms for these events, this research will redefine our understanding of the key steps in the pathogenesis of streptococcal IE. These concepts and findings will be broadly applicable to other endovascular pathogens, and will provide novel insights into microbial interactions with host glycans.

NIH Research Projects · FY 2026 · 2023-07

Cannabis use during pregnancy has increased substantially, in conjunction with widespread decriminalization/legalization, changing public perceptions about harm, and evidence of cannabis's antiemetic properties. Prior outcomes research on prenatal cannabis exposure is narrow in scope, as these older studies included research participants with polysubstance use (e.g., tobacco, alcohol, illicit drugs). In addition, prior research also likely underestimated potential risks specific to cannabis use during pregnancy because modern strains are 10x more potent than they were 40 years ago. We propose to study neurodevelopment in infants exposed in utero to cannabis using state-of-the-art MRI and behavioral measures. Maternal cannabis use will be measured prospectively using weekly reports validated with labels and urine-based assays. By focusing on infancy, we aim to characterize cannabis-induced brain changes at a time when environmental effects are minimized and are less influential than at older. In addition, we will test the hypothesis that prenatal cannabis exposure is more detrimental to male than females. To test our hypotheses, we will recruit 200 pregnant people (where the mother uses cannabis, but not other drugs, tobacco, or alcohol) and 170 pregnant control participants matched on education level. Infants will receive a neonatal neurobehavioral exam and multi-modal imaging (functional magnetic resonance imaging, diffusion tensor imaging, and structural MRI) under natural sleep at 2-4 weeks-of-age and extensive neuropsychological follow-up assessments at 6 and 18 months. This program of research aims to clarify potential health risks, enabling pregnant people to make better-informed choices surrounding cannabis use during pregnancy.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract The epidermis forms a multi-layered epithelium that serves as a protective shield for the body, preventing dehydration and pathogen invasion. Its principal cellular constituents, keratinocytes, continually regenerate the cutaneous barrier via a specialized form of differentiation as they move outward in the tissue. At the end of their life in the skin’s outermost layers, keratinocytes initiate a cellular remodeling program called cornification in which they eliminate their organelles to form flattened, keratinized cells. The importance of this process is underscored by many disorders of cornification linked to defective keratinocyte maturation. Despite the advent of biologic treatments for inflammatory skin diseases, development of similar targeted therapies for epidermal barrier dysfunction has been limited by an incomplete understanding of the pathways driving keratinocyte differentiation. The proposed aims address this knowledge gap by applying advanced microscopy, gene editing, and optogenetics to define the mechanisms mediating organelle degradation in human epidermis. This proposal builds on the PI’s K08 project, which found that cornifying keratinocytes induce autophagy, a lysosomal degradation pathway, to break down organelles. The K08-funded work showed that differentiating keratinocytes upregulate an autophagy receptor, NIX, which marked mitochondria and instructed cells in the upper tissue layers to break down these organelles, a step that was essential for epidermal maturation. Our planned experiments will expand the K08 project scope to include the endoplasmic reticulum (ER), testing the hypothesis that keratinocytes utilize distinct autophagy receptors to orchestrate breakdown of the ER (called reticulophagy). Aim 1 will determine how reticulophagy drives programmed ER degradation during cornification and Aim 2 will assess if reticulophagy mitigates damage from ER stress in keratinocytes. Preliminary studies identified candidate receptors that initiate reticulophagy in either a constitutive manner during cornification or upon organelle injury due to ER stress. We will use gain- and loss-of-function approaches in organotypic skin to determine the role of these receptors in directing epidermal morphogenesis and mitigating ER stress. As well, we will leverage live biosensor imaging and optogenetic tools to define how reticulophagy alters signaling mediators that control keratinocyte differentiation, including calcium and reactive oxygen species. Results from the planned work promise to identify novel strategies for normalizing epidermal differentiation in disorders of cornification but also for repairing barrier dysfunction in common diseases like atopic dermatitis and psoriasis. Augmenting the investment by the University of Washington to ensure his success as a physician-scientist, R03 funding will advance the PI’s career development and position him to compete for R01-level funding as he transitions to an independent investigator. The proposed project enhances the PI’s expertise at the intersection of cell biology and dermatology, building an innovative research program that complements his clinical specialty and provides a platform for future translational work to find novel treatments for skin barrier disorders.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Commensal and pathogenic bacteria inhabit various oxygen-depleted niches in the human body, such as the gastrointestinal tract, wound tissue, and the lung mucosa. Adaptation to these environments requires distinct anaerobic biochemistry to support colonization and survival. An understanding of these biochemical strategies could present unique opportunities to develop novel therapeutics that overcome challenges of antibiotic resistance and bacterial persistence. However, we lack fundamental knowledge of the diverse chemistry that microbes use in anaerobic and microaerobic environments. The proposed studies outline our approach to elucidate the molecular mechanisms, biochemical reactions, and biological roles of metalloenzymes in host- microbe interactions. Metalloenzymes play central roles in cellular redox chemistry. Whereas classes of metalloenzymes that active oxygen for redox reactions have been studied for decades, metalloenzyme families that function in the absence of oxygen remain poorly characterized. In this project, we interrogate the chemical and biological functions of a newly discovered family of metalloenzyme oxidases that are prevalent in bacterial pathogens and human gut microbes. The few known representatives of this family catalyze oxygen-independent hydroxylation reactions in key cellular processes, including cofactor biosynthesis and RNA modification. We will use these known enzymes to establish the requirements for catalysis and to discern their postulated roles in microoxic conditions. Beyond the members with established functions, emerging metalloenzyme families also represent an untapped source of biochemical diversity. We will leverage genomics and protein bioinformatics to discover new enzymatic chemistry within this poorly characterized superfamily. The proposed work will reveal previously unknown redox chemistry, establish biochemical responses to microaerobic conditions, and set the stage to interrogate the importance of these reactions in host-microbe interactions. The ultimate goal of this research program is to gain a molecular understanding of microbial adaptation to O2 limitation that can be leveraged to treat elusive drug-resistant bacterial pathogens.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Adolescent substance use disorder is a public health crisis with over 1 million youth and young adults affected each year in the United States. County-level policies are well-positioned to reduce adolescent substance use disorder through funding and regulatory policies; however, the extant literature documents multiple failures in attempts to effectively leverage policy mechanisms. The challenges of aligning political will, multisector collaboration, use of research evidence, and resource commitments are well-known problems in the public policy and public administration literature. To date, the field of behavioral health policy science does not have a well-supported method of policy design that overcomes downstream policy implementation challenges. We propose to study a method of policy formation, Policy Codesign, designed to anticipate multiple barriers in the downstream implementation of evidence-based, adolescent SUD services. The participant structure and process of Policy Codesign draws from the public policy model of coproduction as well as engineering-based codesign and is supported by promising, preliminary studies. The process includes well-defined policy formation stages within a multisector and community-engagement framework, including: Values mapping, Information gathering, Information integration, Prototyping, Testing, Implementation. The current single-arm, observational study with two geographically diverse counties in Washington State will inform the research procedures to prepare for a rigorous trial of Policy Codesign in counties in Washington State, New York, and Connecticut. The project’s specific aims are 1) to examine the acceptability of Policy Codesign and perceived feasibility of developed adolescent SUD policies, 2) to measure changes in social network growth, cohesion, and bridging activities among multiple sectors central to behavioral health policy implementation, and 3) to examine the perceived replicability of Policy Codesign among well-established behavioral health policy intermediaries. The project is innovative as the first test of a policy design strategy intended to solve downstream implementation problems in evidence-based, adolescent SUD prevention. If found to be effective, Policy Codesign would significantly advance the field’s ability to translate and sustain policy-based solutions to address adolescent substance use. The core investigative team (Walker, Ahrens, Owens) is well-suited to carry out the project with extensive practical expertise in real world behavioral health policy design and community-engaged research. The team is supported by leading policy (Purtle), implementation (Saldana, Palinkas, Aarons) and adolescent SUD treatment (Hogue) researchers widely regarded as the top experts in their fields, as well as behavioral health policy and health design organizations actively involved with county policymaking in adolescent behavioral health systems.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT This R01 comprises a collaboration between scientists in the US and Belgium that seeks to develop synergistic antiviral drug combinations that keep pace with the evolution of the SARS-CoV-2/COVID-19 pandemic and future outbreaks of related coronaviruses. We will exploit i) the principles of synergistic activity that arise with drug combinations and ii) novel mathematical approaches to optimize human dosing for treating emerging and re-emerging coronaviruses with pandemic potential. We will identify combinations of drugs that i) target conserved cellular factors that support virus infection, and/or ii) target viral enzymes that are essential for viral replication, to develop novel antiviral drug combinations against multiple coronaviruses (i.e., a pan-family drug cocktail). Based on our previous work, mathematical modeling approaches will be used to i) identify drug combinations with synergistic antiviral action, ii) prioritize drug combinations and dosing strategies for testing in rodent models of virus infection and disease, and iii) predict in vivo efficacy in humans with various dosing strategies. Our research plan provides a proactive approach for global pandemic response and preparedness, with deliverables consisting of i) combinations of compounds that confer potent, synergistic suppression of multiple coronaviruses and have sufficiently high and durable plasma and tissue concentrations and ii) publicly available harmonized clinical trial designs that will allow for efficient testing of multiple agents in parallel at the inception of new epidemics. As the approach is applicable to any virus family, the project portends global impact by improving protection of the general population against existing viral threats, enhancing preparedness and response for future viral outbreaks, and offering immediately testable and deployable oral drug combinations that fill the time void between virus identification and development and deployment of viral sequence-specific vaccines, antibodies, and drugs.

NIH Research Projects · FY 2024 · 2023-07

Project Abstract The sensory receptors for mammalian vestibular organs, called hair cells, encode head movements and relay information along the vestibular nerve to the brainstem and cerebellum. Mammals have two types of hair cell, type I and type II, which different morphological and physiological properties. Type I and II hair cells also have distinct forms of afferent innervation that are differentially distributed across the epithelial zones. Vestibular hair cells die during normal aging and after exposure to ototoxins, and this loss can lead to profound vestibular deficits. After vestibular hair cell damage in adult mice, supporting cells regenerate ~50% of type ll hair cells, but no type I hair cells are replaced. This natural hair cell regeneration does not restore the vestibular system to normal function, as measured by the vestibulo-ocular reflex and other behavioral tests. One interpretation of this finding is that type I hair cells must be replaced in mammals for vestibular function to return. While this is tempting to speculate, there is little evidence that type I hair cells are required for specific vestibular functions. Indeed, we understand little about the respective contributions of type I and II hair cells to our vestibular sense. One step toward solving this puzzle is to use molecular biology to better understand the unique features of each hair cell type and to determine if subtypes of type I and II hair cells exist. Working with Drs. Neil Segil and Litao Tao, I found from single cell RNA sequencing that there seem to be four molecularly distinct populations of hair cells in adult mouse utricles: two type l-like populations, one type ll-like population, and one unknown group of hair cells. In Aim 1, I will continue these studies, examining expression of candidate cell-specific genes in all vestibular organs to determine if four distinct hair cell groups are identifiable in each organ and how these groups are distributed across epithelial zones. I will generate novel insights into hair cell features across vestibular organs, classify new markers for each cell type, and identify genes that may be used to drive cell-selective gene misexpression in future studies. Another step toward testing the different functions of type I and II hair cells is to remove each hair cell population and assess impacts on vestibular function. In Aim 2, I will use CreLoxP technology to ablate all type I hair cells or peripheral type I hair cells in all adult vestibular organs. I will determine the impact of these ablations upon animal behaviors and brainstem electrical responses to vestibular stimuli, which to my knowledge has not been done before in adult mice. I expect to gain new insights into the general function of type I hair cells and the specific functions of type I hair cells in each epithelial zone. Overall, this research provides new information about properties and functions of type I and II hair cells and inform on new therapies to treat balance disorders. Further, this project enables me to receive top-notch research training in a collaborative setting using cutting-edge scientific approaches.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Quorum sensing (QS) is a cell-cell communication system used by many bacteria to regulate gene expression in a coordinated manner. Our work focuses on the QS system of Pseudomonas aeruginosa, a saprophyte and opportunistic pathogen of humans. P. aeruginosa uses QS to regulate dozens of genes, including several important virulence factors. P. aeruginosa QS is mediated by the transcription factors LasR and RhlR, which respond to acyl-homoserine lactone signals, and PqsR, which responds to alkylquniolone signals. Initial characterizations of QS in P. aeruginosa described a hierarchy: LasR regulates RhlR and PqsR activity. Over the past several years we, and others, have shown that this hierarchy is malleable. RhlR and PqsR can be active in the absence of functional LasR in isolates from many settings, particularly those isolated from the chronic lung infections of people with cystic fibrosis (CF). These results suggest a “rewiring” of QS that is critical to the maintenance of virulence functions in chronic infections. This proposal builds on our previous observations to ask about the biological significance of this alternate hierarchy in bacterial isolates from people with CF. We make use of CF isolates and a laboratory model strain to ask i) how is the architecture of QS of P. aeruginosa altered?; ii) in this altered hierarchy, how do RhlR and PqsR QS interact to effect virulence functions in isolates from chronic infection?; and iii) what is the role of a highly-conserved, uniformly QS regulated, non-ribosomal peptide synthetase? The answers to these questions will define the central importance of RhlR and PqsR in P. aeruginosa from chronic infections and inform future efforts to design therapeutics targeting RhlR or PQS QS in this bacterium.

NIH Research Projects · FY 2026 · 2023-07

Project Summary HIV infection is a chronic viral infection that if untreated leads to progressive loss of the CD4 T cell compartment and eventually AIDS. In addition to loss of HIV-susceptible CD4 T cells, chronic HIV infection is characterized by robust systemic immune activation including B and T cell activation and proliferation and elevated levels of pro-inflammatory molecules. Indeed, the level of immune activation is strongly associated with HIV disease progression. Even upon antiretroviral therapy (ART) initiation and viral suppression, chronic HIV infection is associated with dysfunctional circulating immunity rather than a return to immune quiescence. Further, immune activation in mucosal compartments such as the gut can persist in chronically infected individuals, even with long-term ART. This chronic immune activation during HIV infection was first identified largely through study of men with HIV, though more recent studies have suggested that HIV-associated immune activation may manifest differently in women. Given that women are increasingly affected by HIV, with UNAIDS reporting that 53% of people living with HIV are women and girls as of 2020, it's evident that there is a gap in our understanding of immune activation and dysfunction in women, particularly within the female genital tract (FGT) mucosa. A few initial studies have suggested that immune activation is elevated in the FGT of women with HIV, and that ART does not restore FGT immune status to homeostatic levels within the initial month of treatment. Thus, we propose to comprehensively evaluate immune activation and dysfunction in the FGT in settings of HIV infection with or without viral suppression for up to 24 months. In a well-characterized cohort of women with and without HIV infection in Mombasa, Kenya, we will test two primary hypotheses: 1) We hypothesize that HIV infection leads to increased immune activation in the FGT that persists after ART initiation and viral suppression, and 2) We hypothesize that that chronic and persistent HIV infection leads to exhaustion of mucosal tissue T cells within the FGT. We will advance the prior research by including a more thorough investigation of immune activation including a focus on regulatory T cell (Treg)-mediated immunoregulatory mechanisms, and T cell exhaustion through use of high-throughput single-cell analysis, and we will examine the effects of longer-term viral suppression on immune activation and dysfunction in both the circulation and FGT. This will allow us to better understand how HIV infection may lead to negative FGT health outcomes.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Mortality from out-of-hospital sudden cardiac arrest (OHCA) is a large public health burden, accounting for approximately 10% of all deaths in the US. Because OHCA is a leading cause of mortality, advances in resuscitation have the potential to improve public health. Currently resuscitation protocols use a one-size-fits- all approach. However, we now understand that OHCA occurs via heterogeneous mechanisms and manifests a time-dependent pathophysiology, which influences prognosis. The heterogeneity suggests discrete clinical phenotypes and an opportunity for individualized therapy. Distinguishing information about patient physiology can be harnessed from the defibrillator. Continuous bio-measures of ECG, end-tidal carbon dioxide (ETCO2), and transthoracic impedance (TI) can determine physiologic status and potentially guide optimal treatment. However, a real-time continuous approach to characterize a patient’s physiology and prognosis by accurately determining the underlying rhythm and its vitality is not presently feasible without repeatedly interrupting CPR. CPR interruption is required because chest compressions introduce ECG artifact that prevents rhythm identification, prognostic assessment of rhythm morphology, and a patient’s underlying vital status (vitality). However, CPR interruption is harmful since it disrupts perfusion in the otherwise pulseless OHCA victim. Consequently, the current protocol is a compromise: CPR is interrupted every 2 minutes to help inform care decisions though treatment proceeds empirically as CPR resumes and providers are typically “blinded” to the actual underlying rhythm and vital status. Emerging evidence from the proposal team highlight the ability to use signal processing techniques to investigate the ECG, ETCO2, and TI defibrillator signals during CPR to improve OHCA resuscitation. These investigations use artificial intelligence (AI) methods to determine a patient’s instantaneous physiological status and predict downstream resuscitation outcomes. We propose an investigative plan that will: 1. Derive and validate an integrated ventricular fibrillation (VF) OHCA algorithm that incorporates and builds upon previously-validated modular algorithms, using artificial intelligence (AI) methods that process and integrate ECG, TI, and ETCO2 bio-signals during active CPR. 2. Prospectively evaluate the integrated algorithms and their validated building block components in distinct adult and pediatric OHCA populations. 3. Conduct a simulated randomized trial among EMS to compare the described precision strategy to the current-day, fixed protocol to understand how dynamic prompts of a precision strategy affect CPR metrics. The project leverages an unparalleled data resource and a tested, multidisciplinary team with a track-record of impactful resuscitation investigations involving novel approaches to AI and resuscitation. This consequent precision strategy ultimately could transform resuscitation and improve public health.

NIH Research Projects · FY 2025 · 2023-07

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that is activated by nutrients and energy, phosphorylating substrates to promote and coordinate anabolic metabolism. mTOR was identified about thirty years ago in a yeast screen for mutants resistant to growth inhibition by the drug rapamycin; shortly thereafter the highly conserved mammalian ortholog was discovered independently by biochemical methods. Over the past thirty years, biochemical and genetic approaches have identified numerous evolutionarily conserved components of the mTOR signaling pathway as well as the proteins forming the two distinct mTOR complexes, mTORC1 and mTORC2. mTORC1 is composed of three core components, mTOR, Raptor, and mLST8 (mammalian lethal with SEC13 protein 8); Rictor replaces Raptor in mTORC2. mTORC1 is inhibited by rapamycin whereas mTORC2 is relatively insensitive. mTORC1 has been intensely studied because it functions as a central regulator of the cell’s response to nutrients and energy and thereby impacts clinically important conditions such as cancer, aging, and hypoxic injury. Through an unbiased mutant screen in C. elegans for hypoxia resistant mutants, we have identified a missense partial-loss-of-function mutation in daf- 15, which encodes the sole C. elegans ortholog of Raptor (Ce-Raptor). A CRISPR/CAS9-generated mutant with the identical lesion confirmed the mutation as conferring hypoxia resistance. Our discovery that reduction of function of Ce-Raptor imparts resistance to hypoxic injury is consistent with published data using mTORC1 inhibitors in mammalian models. Our Ce-Raptor mutant called daf-15(gc67) is unique among published metazoan Raptor mutants in that it is conditional; Raptor pathway functions can be turned off and on again by simply varying temperature. daf-15(gc67) is essentially wild type at the standard culture temperature of 20°C, hypoxia resistant at 22°C, and developmentally arrested at 25°C, the phenotype of daf-15 null mutants. The graded temperature-conditional nature of the daf-15(gc67) phenotypes overcomes a critical barrier in the field by providing a genetic reagent that allows temporal and tunable genetic control of Raptor and mTORC1 function for the first time in metazoans. This project will use this unique reagent to answer key questions about how and when Raptor regulates hypoxic injury. In Aim 1, we will combine genetic and proteomic methods to test specific hypotheses and discover the Raptor-regulated proteins that determine how and when Raptor controls hypoxic injury. Using our conditional mutant, we have completed a screen for suppressors of Raptor loss of function. In Aim 2, we will identify mutated genes suppressing Raptor loss-of-function and thereby discover regulators of Raptor-mediated hypoxic sensitivity. Through these two aims, we will define how Raptor regulates hypoxic sensitivity. Given the unbiased nature of the suppressor screen and proteomic studies, our project has potential to identify novel components of the mTORC1 pathway and thereby more broadly advance our fundamental understanding of mTORC1 signaling pathways, including those regulating cancer and aging.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Pyroptosis is a programmed process of lytic, pro-inflammatory cell death that is involved in the pathogenesis of leading global causes of mortality. Inflammasome-mediated innate immune signaling activates caspase-1 family proteases to initiate pyroptosis by cleaving the pore-forming protein, gasdermin D. Recent data demonstrate that the protein ninjurin-1 oligomerizes during pyroptosis and is required for plasma membrane rupture, or cell lysis, downstream of gasdermin D pore formation. Cellular factors released during pyroptotic lysis cause local and systemic inflammation and pathology, but processes that regulate plasma membrane rupture and whether these can be therapeutically targeted, are not well-understood. We recently identified muscimol as a novel inhibitor of pyroptotic lysis, but its mechanism of action is not yet known. Muscimol is well-studied as an agonist of neuronal GABA receptors, but our preliminary data suggest that inhibition of pyroptotic lysis is not mediated by these receptors. This proposal aims to understand how muscimol prevents pyroptotic lysis and identify muscimol analogs with potent and specific activity. The experiments outlined in this proposal will systematically examine steps in the process of pyroptosis for inhibition by muscimol. Based on preliminary data, we will focus experiments on the hypothesis that muscimol interferes with ninjurin-1 oligomerization, while also testing other possibilities. We will use complementary models of pyroptosis induced by inflammasome-dependent and -independent stimuli, and further employ reductionist systems based on our findings. Our preliminary data suggest that there are specific molecular determinants for muscimol inhibition of pyroptotic lysis, as analogs demonstrate varied potency from the parent molecule, not correlating with GABA receptor activity. We will systematically test a panel of rationally-selected, already synthesized, muscimol analogs for inhibition of pyroptotic lysis. We hypothesize that our results will reveal a novel structure-activity relationship for muscimol inhibition of plasma membrane rupture compared to its canonical activity at neuronal receptors. In addition, these experiments may yield analogs with increased potency and / or specific activity to prevent pyroptotic lysis, without activity at GABA receptors. Finally, we will utilize the unique chemical properties of muscimol, coupled with advances in proteomics and the expertise of our collaborators, to identify novel muscimol-binding proteins. Together, the results of these experiments will inform a precise molecular understanding of the mechanism of action to disrupt pyroptotic lysis and provide the foundation for a novel therapeutic strategy for the many diseases in which pyroptosis has been implicated.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY / ABSTRACT Well controlled antiviral innate immunity is essential for restricting viral pathogens while preventing aberrant inflammation. A key antiviral signaling pathway is initiated by RIG-I-like receptors (RLRs) which sense viral RNA in the cytosol to induce the production of interferons through a higher order signaling platform centered on the adaptor protein MAVS. Although the components of the MAVS signalosome are basally expressed, RLR signaling only proceeds when they coalesce around MAVS at ER-mitochondrial contact sites. Many of the protein-protein interactions and post-translational modifications required for the proper function of the MAVS signalosome are known. While, functional interactions in protein complexes can be mediated by RNA molecules, whether RNA molecules play a role in signaling through the MAVS signalosome remains unexplored. I have found that 1) MAVS is associated with non-viral RNA, that 2) RNA promotes signaling through the MAVS signalosome, and that 3) canonical RNA-binding proteins (RBPs) interact with MAVS. This proposal aims to define the functional roles for RNA and RBPs in regulating antiviral signaling through the MAVS signalosome. During the mentored phase, I will gain new training in the characterization of RNA-protein interactions through biochemical, sequencing-based, and quantitative proteomic approaches, as well as the virological techniques required to study RNA viruses and innate immunity. In Aim 1, I will define the RNA-dependent interactions between components of the MAVS signalosome during the K99 phase, and identify novel proteins that associate with RNA during RLR signaling at the R00 phase. Through Aim 2, I will pinpoint the sequences in MAVS required for RNA-association, and identify a high-confidence set of MAVS-bound RNAs during the K99 phase. During the R00 stage, I will functionally dissect the roles for these RNAs at the MAVS signalosome. In Aim 3, I will screen a shortlist of MAVS-associated RBPs to identify those that influence MAVS signaling during the K99 stage, and determine the molecular functions of three RBPs (STAU1, HNRNPL, and RBM10) in antiviral signaling at the R00 stage. The overall outcome of these experiments will be to define new RNA-centric principles by which the MAVS signalosome is organized. Understanding how RNA molecules influence antiviral signaling could unlock new host-directed therapeutic strategies against viral diseases as well as autoimmune disorders. In addition to my advisor Dr. Savan, I have assembled an Advisory Committee with expertise in the different facets of my research. Together, this excellent training environment at the University of Washington will augment my research during the mentored phase, and equip me with the skills required to transition to an independent academic researcher studying the RNA regulation of innate immune processes.

NIH Research Projects · FY 2024 · 2023-07

Project Summary: The primary goal of this training proposal is to understand how dynorphinergic regulation of circuits in the dorsomedial striatum (DMS) enhances seeking behaviors for natural (sucrose) and drug (fentanyl) rewards. Enhanced dynorphin-kappa opioid receptor (dyn-KOR) signaling and aberrant striatal activity is associated with the transition from recreational to persistent opioid-seeking. Yet how dyn neuron activity and subsequent -KOR modulation of the striatum regulates sucrose or fentanyl-seeking is unknown. During the first phase of my postdoctoral research, using a combination of pharmacology, genetics, in vivo photometry and optogenetics, I identified that retrograde dyn transmission at BLA inputs to the DMS enhances BLA-DMS activity, and promotes natural reward-seeking behaviors. However, when exactly dyn neuron activity and release are engaged in reward-seeking is unknown. Hence, I will obtain training in, and use in vivo two-photon imaging to understand how DMS dyn ensembles encode reward-seeking for sucrose, (Aim 1A, K99). I will also extensively characterize and use a novel dyn biosensor using in vivo photometry to determine if the pattern of ensemble activity is reflected in subsequent DMS dyn release, (Aim 1B, K99). Furthermore, because aberrant dyn-KOR signaling is linked to maladaptive opioid-seeking, and my preliminary data suggests a role for dyn-KOR activity to enhance sucrose-seeking, I will develop and use an oral fentanyl self-administration paradigm in conjunction with in vivo photometry to dissect when BLA-DMS terminals are engaged during fentanyl-seeking (Aim 2A, K99). I will also multiplex conditional deletions of dyn in the DMS, or KOR in the BLA, and stimulate dyn release in the DMS, with in vivo photometry during fentanyl-seeking to determine whether enhanced dyn-KOR signaling negatively modulates fentanyl-seeking (Aim 2B, K99). For the R00 “independent” phase of my proposal, I propose to extend the findings from Aims 1 and 2 in my own lab. I will delineate how DMS dyn ensemble activity encodes fentanyl- seeking, manipulate specific ensembles to control behavior via spatial light modulation, and dissect the necessity for BLA inputs in the DMS to induce dyn release during fentanyl-seeking (Aim 3, R00). The proposed studies specifically address how elevated endogenous dynorphin-KOR signaling, via its control of activity in the DMS, regulates natural and fentanyl reward-seeking. During the proposed K99 training period, I will be trained in in vivo two-photon calcium imaging approaches, opioid biosensor imaging and oral self-administration. Additionally, I will actively participate in scientific society conferences, obtain career development training (budgeting and administrative tasks), and continue to further my scholarly knowledge base (planned interactions with my mentor and committee members). Altogether, this award will greatly facilitate the development of my own research program, thereby preparing me for an independent neuroscience career as a principal investigator.

NIH Research Projects · FY 2026 · 2023-07

We propose to test the malleability of a promising, cognitive factor that predicts alcohol misuse—drinking identity (a facet of the self-concept linked to alcohol and drinking)—and its subsequent effect on drinking during key transitions that occur during emerging adulthood (the developmental period from 18-25). This period is associated with changes in risk for alcohol misuse (e.g., heavy alcohol consumption and experiencing negative alcohol-related consequences), which has substantial individual and public health costs. Changes in risk for alcohol misuse coincide with two major life transitions experienced by a substantial proportion of the population: graduating high school and graduating college. On average, the risk for alcohol misuse increases after high school and decreases after college, though there is considerable heterogeneity in these trends. When considering novel cognitive targets for intervention efforts, emerging adulthood is also a time of self- exploration and self-concept and identity changes, suggesting that identity-based factors may be particularly relevant. Our labs have been at the forefront of research evaluating identity and drinking. We have demonstrated that drinking identity is a unique cognitive predictor of alcohol misuse and that changes in drinking identity are associated with changes in drinking in the early college years and during the transition out of college. Key next steps involve the validation of strategies for shifting drinking identity to prevent the typical escalation of alcohol misuse associated with the transition out of high school and expedite reductions in alcohol misuse associated with the transition out of college. An extensive psychology literature on the self- concept, identity motivation, possible selves, and self-regulation provides a strong theoretical foundation for adapting a future possible selves task to shift drinking identity and, in turn, reduce alcohol misuse. Our overarching aims are to evaluate changes in drinking identity in response to the future possible selves task, to demonstrate that those changes are, in turn, followed by changes in alcohol misuse and key risk factors, and to establish boundary conditions for these changes—e.g., their durability, the most effective task version, and the need for multiple (vs. single) task doses. We propose to conduct two experimental studies (Study 1: high school transition; Study 2: college transition) that will be implemented at two demographically diverse sites (N = 528 per study; n = 264 per study site). We will use a 2 (imagine and write about a future possible desired self vs. not) x 2 (imagine and write about a future possible feared self vs. not) x 2 (1 vs. 3 writing session[s]) design. The effects of the future possible selves task on drinking identity, risk factors, and alcohol misuse will be evaluated out to 12 months. Our approach provides a robust test of the task’s efficacy as an alcohol misuse prevention [Study 1] and intervention [Study 2] strategy. If successful, this application will support drinking identity as a malleable cognitive factor underlying alcohol misuse and present a novel, scalable, theory-based strategy to address alcohol misuse during key developmental transitions in emerging adulthood.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY: Opioid use disorder and opioid overdose death rates in the United States reached unprecedented levels during the COVID-19 pandemic. Understanding how endogenous opioid activity affects natural reward seeking is crucial to understanding the neuropharmacological basis of opioid use disorder. Previous research implicates the mesolimbic dopamine pathway, which refers to dopamine neurons projecting from the Ventral Tegmental Area (VTA) to the Nucleus Accumbens (NAc), in reward and addiction. Recent studies show that gamma-aminobutyric acid (GABA) containing neurons in the VTA (VTAGABA) provide local inhibition of dopamine neurons that synapse onto the NAc, thus playing a role in regulating reward behaviors. Importantly, these VTAGABA neurons contain a variety of G-protein coupled receptors, specifically the µ-opioid receptor (MOR). However, the exact role of these receptors and their signaling play in VTAGABA neurons and consequent regulation of natural reward is unknown. The source of endogenous opioid neuropeptide onto these MORs, and how these impacts signaling and activity has not been described. The central hypothesis of this proposal is that µ-opioid receptor signaling on VTAGABA neurons is regulated by afferent endogenous opioid peptides, resulting in disinhibition of VTAGABA neuron excitability. This results in control of dopamine neuron activity, and ultimately the expression of natural reward-seeking. This proposal directly addresses NIDA's Priority Scientific Area 1 that aims to further understand the molecular, neuropharmacological and circuit changes induced by drug use. Aim 1 will isolate the role of endogenous µ-opioid peptides in the VTA on dopamine signaling and natural reward- seeking. Aim1A uses ex vivo two-photon imaging and neuropharmacology approaches to visualize VTAGABA and VTA DA dynamics. Aim1B will investigate the effects of MOR ablation in the VTA on dopamine activity and behavior. Aim 2 will isolate the source and dynamics of endogenous µ-opioids in the VTA during natural reward behavior. In Aim 2A, I will use viral tracing techniques to anatomically visualize inputs from the lateral hypothalamus (LH) to the VTA. Aim 2B will test the effects of endogenous µ-opioid signaling on VTAGABA and DA activity in the NAc during reward seeking behaviors. In Aim 2C, I will use molecular approaches including CRISPR gene-editing and a recently developed µ-opioid biosensor (µMASS1) to understand the spatiotemporal aspects of MOR signaling in VTAGABA neurons during reward seeking. This career development training and series of experiments will provide insight into the role of endogenous MOR signaling on dopaminergic activity and natural reward-seeking. For this proposal, I will train in slice electrophysiology, two-photon slice imaging, gene-editing, molecular neuropharmacology, and behavioral approaches to understand how specific neuropeptides regulate reward circuits. This F31 proposal will greatly advance my career development plan and prepare me for a career as an independent, academic neuropharmacologist.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT The microbiome of the eye and adnexal structures has not been well-characterized to date. The periocular eyelid margin has a substantial microbiome, and may be involved in disease states including blepharitis, Meibomian gland disease, dry eye syndrome, hordeolum, and chalazion. Preliminary data from our group suggest that, while the lid margin microbiome is relatively consistent between eyes and across time in individuals, there is substantial variation between individuals. The full extent of variability and the causes for these differences are not known. We propose studying the well-characterized, healthy TwinsUK cohort. This long-standing registry includes over 11,000 mono- and di-zygotic twins in the United Kingdom. Subjects will be recruited from this group, with all subjects completing questionnaires, having an ocular surface examination, and allowing a lid-margin swab to be obtained. DNA and RNA will be extracted and subjected to state-of-the-art metagenomic sequencing, with rigorous positive and negative controls. In the first aim, we will characterize the lid margin microbiome in this cohort. A subset of subjects will be sampled bilaterally and a subset longitudinally to determine the consistency and stability of findings. In the second aim, we will take advantage of the pre-existing large genomics database on these subjects to determine the heritability of the periocular microbiome and attempt to identify the genetic determinants of specific profiles. In the third aim, we will utilize both sampling of unrelated cohabitants as well as extensive pre-existing demographic, nutritional, and environmental data on this cohort to attempt to identify the environmental factors influencing the periocular microbiome. At the conclusion of this study we anticipate having generated the definitive catalogue of lid margin flora (including viruses and phage); and having established the genetic and environmental factors giving rise to specific microbial communities. All data will be made publicly available. These data will inform future studies on the relationship between ocular adnexal microbiome, eye disease, and specific interventions.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Prolonged elevated levels of stress are comorbid with many neuropsychiatric illnesses such as major depressive disorder (MDD), and can have devastating effects on individuals, their caretakers, and healthcare professionals. MDD is typically demarcated as exhibiting emotions of sadness, significant loss of motivation, and can be defined by social deficits. A growing literature indicates that major depressive disorder is influenced by dysregulation in limbic brain regions including stress and reward circuitry. Despite a significant clinical awareness, direct preclinical characterization of affective disorder circuitry and associated neuronal mechanisms inclusive of sex as a biological variable are currently absent, and notably do not include social decision making and motivation as behavioral metrics. Therefore, the aim of my proposal is to obtain the necessary computational, behavioral and anatomical training to identify, interrogate, and manipulate neural populations that modulate these complex social behaviors. This project will focus on identifying nucleus accumbens (NAc) circuit and cell type specific mechanisms that regulate resiliency to operant social stress (OSS), and then further interrogate the afferent projections driving these populations. OSS incorporates social decision making and social reward as metrics for social stress resiliency. The choice of this brain region is based on converging preliminary data, implicating that the NAc dopamine receptor (Drd) 1 and Drd2 medium spiny neurons (MSNs) confer opposing roles in regulating resiliency to social stress. NAc circuit and cell-type specific activation will be identified using a combination of whole brain clearing, Fos (a marker of neuronal activity) immunohistochemistry, and retrograde viral tracing. Because of the limited temporal resolution of Fos during these behaviors, awake-behaving fiber photometry recordings will be used to observe how NAc MSNs encode varying levels of susceptibility and resiliency to OSS in real-time. Next, the causal significance of the NAc and its afferent projections will be examined using region, circuit, and cell type specific optogenetic manipulations. Understanding the neural mechanisms driving operant social stress in a sex-dependent manner will allow for the development of more specific and effective treatments for affective mood disorders such as major depressive disorder (MDD).

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Pulmonary arterial hypertension (PAH) is a rare but life-threatening disease characterized by vascular remodeling and progressive elevation in pulmonary vascular resistance. These events result in right heart failure, significant morbidity, and a high mortality. The timing of right ventricular (RV) dilation and dysfunction varies from patient to patient, even among individuals with otherwise similar RV afterload; however, the biological basis for this heterogeneity in RV adaptation is not yet known. Identifying and understanding molecular mechanisms of RV maladaptation are important steps for discovering novel right heart targeted therapy. Dr. Pi recently explored plasma metabolomic signatures in a deeply phenotyped cohort of PAH participants using a systems biology approach and found distinct metabolic pathways and profiles associated with metrics of right heart failure and mortality. In particular, polyamine and histidine metabolism were consistently associated with these outcomes. It is important to appreciate that while right heart adaptation is related to right heart failure, it is a distinct condition. Specifically, right heart failure may merely represent the severity of pulmonary hypertension, while right heart adaptation reflects the ability or inability of the right heart to respond to any severity of pulmonary vascular disease. When the metabolomics analyses of right heart failure were adjusted to account for differences in pulmonary vascular resistance, an association between sphingomyelin metabolism and RV adaptation emerged in individuals with otherwise similar right heart afterload. Using the same prospective observational PAH cohort from University of Washington, in this proposal Dr. Pi aims to extend her metabolomics work by (i) identifying proteomic profiles associated with RV maladaptation and mortality in PAH; (ii) integrating the multi-omic (metabolomic and proteomic) signals to find dysregulated pathways and features associated with poor outcomes. Importantly, the scientific aims directly support an ongoing and rigorous training program in systems biology and bioinformatics that will specifically enhance the proteomic analysis and integrative omics approaches. Successful completion of this project will generate novel data and methods that will provide a solid foundation for a K23 proposal focused on RV adaptation and ultimately an independently funded career leveraging “big data” to understand complex cardiopulmonary disease.

NIH Research Projects · FY 2025 · 2023-07

Red blood cells, white blood cells, and platelets are important for the clinical diagnosis of intrinsic blood cell and hematopoietic disorders, and also as predictors of various heart, lung, and blood disease outcomes. Moreover, hematologic quantitative traits are highly heritable and serve as a model system for studying the genetic architecture of complex traits. While significant strides in understanding the genetic basis of hematological traits have been made over the past decade, the wealth of whole genome sequencing (WGS) data from emerging resources such as the NHLBI Trans-Omics for Precision Medicine (TOPMed) program provides an unprecedented opportunity to gain further insight in several key areas, including the role of structural variants (SVs). While a few common SVs (e.g., α-globin) are known to be associated with blood cell traits, a more systematic and agnostic genome-wide search for SVs in large samples is required to identify new biology. The centralized availability of deeply sequenced DNA from the NHLBI TOPMed and the NHGRI Centers for Common Disease Genomics (CCDG) programs, along with genome-wide data from UK Biobank and other cohorts, allows for full characterization of SVs genome-wide at population-scale. By improving the accuracy of genome-wide SV calling for WGS data as implemented in our new Genvisis software package and by validating candidate causal SVs using state-of-the-art gene-editing technologies in hematopoietic cells, our interdisciplinary approach will facilitate the translation of genetic association findings into mechanistic insights, discover new biology underlying hematopoiesis, and ultimately identify factors that account for individual differences in pathobiology or response to treatments. In Aim 1, using WGS data from TOPMed and CCDG participants, we will apply novel methodology to generate high-quality and more accurate SV calls than the SV calling algorithms currently available for both WGS and existing array data. In Aim 2, we will use the newly generated SV calls to conduct single-variant and gene-based segmental association analyses of SVs with blood cell traits and related clinical outcomes in up to 570,319 participants. Association findings will be replicated in up to 760,000 participants in populations/studies not used in the discovery phase. SVs that are significantly associated with blood cell traits will subsequently be tested for association with other blood disorders including clonal hematopoiesis of indeterminate potential (CHIP) and VTE. In Aim 3, targeted long-range sequencing will be performed in selected samples to precisely localize newly identified blood trait-associated SVs in complex genomic regions. We will also perform functional genomic annotation of replicated blood cell trait-SV associations followed by state-of-the art gene-editing approaches to understand novel mechanisms underlying genetic regulation of hematopoiesis. This model integrative approach to advancing precision medicine research in heart, lung, and blood diseases will demonstrate for the first time the role of SVs in the genetic architecture of hematologic traits and contribute to a better understanding of hematopoiesis and pave the way for new research into Precision Medicine for blood diseases.

NIH Research Projects · FY 2025 · 2023-07

Intestinal epithelial cells (IECs) are central regulators of intestinal homeostasis and organismal well-being. In response to infection or injury, intestinal epithelial stem cells (ISCs) alter their differentiation to promote host-protective inflammation in the epithelium. However, intestinal epithelial inflammation must be tightly regulated to prevent pathology. Type 2 intestinal inflammation occurs during food allergy and parasitic helminth infection that together cause substantial morbidity worldwide. In this context, bioactive lipids are released locally and lipid receptor expression is dynamically regulated, perfectly positioning lipids to fine tune IEC activities. However, how lipids regulate Type 2 epithelial inflammation is poorly defined, representing a gap in our understanding of how intestinal homeostasis is restored following allergy or worm infection. In intestinal Type 2 inflammation, IECs sense stimuli and release alarmins that activate immune cells to secrete the cytokine IL-13, which acts on ISCs to promote their differentiation to secretory goblet and tuft cells. Studies from our laboratory have recently shown that CRTH2, a receptor for the bioactive lipid prostaglandin D2 (PGD2), is expressed in murine and human IECs and enriched in ISCs. CRTH2-deficient ISCs were more likely to differentiate into goblet cells in vitro. PGD2, dependent on CRTH2, counteracted effects of IL-13 on murine small intestine IECs in vitro and in vivo, suppressing goblet cell differentiation during infection with a parasitic helminth. Finally, CRTH2-deficient mice retained goblet cell hyperplasia and increased barrier permeability after the resolution of Type 2 immune activation. Based on these data, we hypothesize that PGD2 acts on CRTH2+ ISCs to return the intestine to homeostasis after a Type 2 inflammatory event and to shape the IEC response to new stimuli. To test this idea, we propose 2 Aims. Aim 1 includes mechanistic studies that will test if PGD2 suppresses ISC differentiation to secretory lineages in response to Type 2 cytokines. Aim 1 will use a novel CRTH2-reporter mouse to identify PGD2-responsive IECs and will test how the PGD2-CRTH2 pathway affects murine and human ISC biology. Aim 2 includes studies that assess the biological significance of PGD2-CRTH2-mediated suppression of IEC responses. Aim 2 will test how PGD2 and CRTH2 affect barrier function and homeostasis and epigenetically program ISCs after Type 2 inflammation and how IEC CRTH2 deficiency during Type 2 inflammation impacts intestinal repair and pathology during a subsequent, acute or chronic inflammatory event. These studies are essential for understanding how PGD2 and CRTH2 aid in restoring gut homeostasis after Type 2 inflammation and will inform the use of existing CRTH2 modulators and other drugs that target lipids in the treatment of intestinal inflammatory disorders.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT People experience more positive than negative consequences from drinking,1 but negative consequences often have the potential to have a larger impact on a person (e.g., romantic or friendship problems, poor academic performance, and unsafe driving).2,3 According to alcohol expectancy theory4 and social learning theory,5 the experience of consequences after drinking leads to the development or updating of expectancies (e.g., beliefs about what consequence will occur as a result of drinking).4 However, understanding an individual’s expectancy evaluations such as likelihoods and valences likely depend, at least in part, on how individuals perceive recently experienced consequences. Previous research has not consistently evaluated people’s evaluations of consequences,6–9 and if they do they only evaluate the consequence once either cross- sectionally or retrospectively the next day. This prevents us from gaining an understanding of how people view these consequences and how their views change with time. In addition to the changing consequence evaluations, research has typically aggregated across positive and negative expectancies6,8,10–13 which has prevented us from developing an understanding of how expectancy evaluations are altered following the experience of specific consequences and how these may change leading up to a drinking event. To address these gaps, the proposed F31 will use a complex ecological momentary assessment (EMA) design to assess consequence and expectancy evaluations over the short term to examine changes in people’s perceptions based on their experiences. Specific Aims include (1) test whether Fading Affect Bias (FAB) can be observed as changes in people’s evaluations of positive and negative drinking consequences following weekend drinking episodes over the course of a week and (2) test whether likelihoods change over the short term, to what extent these changes depend on prior experience of these consequences, whether FAB can be observed as changes in people’s valences of negative expectancies, and whether increases can be observed as changes in people’s valences of positive expectancies leading up to a drinking episode, and (3) test how the changes in consequence evaluations are associated with changes in likelihoods and valences over the short term. To complement these aims, the applicant will receive training in (1) alcohol expectancy theory and young adult alcohol consequences and expectancies, (2) design and implementation of longitudinal ecological momentary assessment (EMA) studies, (3) quantitative analysis, and (4) research dissemination via manuscript preparation and conference presentations. Study findings will have important implications for future alcohol expectancies prevention research. Specifically, results can be used to inform individuals on a daily-level of when they may be susceptible to experiencing a greater number of negative consequences from drinking on a given occasion and prompt greater reflection of upcoming plans.