Washington State University

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Molecular mechanisms of ferroptosis induction throughout germline development and aging Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation. Ferroptotic cell death is a component of human diseases, such as neurodegeneration, lung, and kidney disease. Promotion of ferroptosis may be a useful therapeutic to target cancerous tumors. Many unanswered questions surround this relatively newly discovered form of cell death, including the nature of lipid-mediator instigators of ferroptosis, how dietary components affect ferroptosis, and how metabolic processes involving cellular organelles such as mitochondria, ER, and cellular vesicles influence ferroptosis sensitivity. Furthermore, there is a dearth of studies examining whether ferroptosis affects germ cells and influences reproduction. We have developed a highly novel ferroptosis model using the small roundworm Caenorhabditis elegans. Our studies center on the induction of ferroptosis by dietary polyunsaturated fatty acids, which we have shown to specifically induce cell death in germ cells in young C. elegans. The powerful genetic tools in this model have allowed us to make rapid progress in the identification of modulators of diet-induced ferroptosis, especially in relation to lipid metabolism pathways, iron, and antioxidant protection. Importantly, our discoveries have been confirmed in mammalian models, including the important finding that ferroptotic cell death can be induced in cancer cell lines by addition of the polyunsaturated fatty acid dihomo-γ-linolenic acid (DGLA). We propose a multifaceted approach to discover and characterize molecular mechanisms of ferroptosis induction using genetic, dietary, and biochemical approaches. Furthermore, we will expand our studies of ferroptosis in germ cells to older worms to determine the role of ferroptosis in reproductive aging. Our experience and expertise, together with the flexible MIRA mechanism to pursue new research opportunities as they arise, will propel sustained progress. These studies will provide insights into the complex regulation of ferroptosis throughout development in a multicellular, living animal. Our findings may also contribute to novel cancer treatments or provide potential therapeutic targets for treatments of diseases promoted by ferroptosis.

NIH Research Projects · FY 2025 · 2023-12

Project Summary/Abstract Expanded cannabis legalization nationwide and increased use among reproductive-aged women have exposed gaps in our scientific knowledge on whether and/or how cannabis use during lactation affects human milk composition. Human milk is considered optimal nutrition for infants for the first 6 months postpartum. Whereas recent evidence has shown that cannabinoids transfer into human milk, there is only one preliminary study that indicates that cannabis use may be associated with changes in milk factors and no studies that assess how cannabis use patterns or cannabinoid concentrations in milk impact holistic milk composition. This lack of knowledge is particularly concerning as 1) human milk is frequently the sole source of infant nutrition in the first months of life; 2) cannabinoids are lipophilic and, given the high lipid content of the mammary gland and human milk, both may be repositories for these compounds, and 3) delta-9-tetrahydrocannabinol (∆9-THC) levels in cannabis products have increased in the past several decades, suggesting that the milk of mothers who use cannabis and their recipient infants may be exposed to higher doses of cannabinoids than ever before. The overall objectives of this project are to 1) characterize and compare holistic human milk composition (metabolome, lipidome, and proteome profiles) among mothers who use cannabis and those who do not use cannabis and 2) investigate the relationships between and among human milk cannabinoid concentrations, maternal characteristics, cannabis use patterns, and milk metabolome, lipidome, and proteome profiles among breastfeeding women who use cannabis. Our central hypotheses are that 1) cannabis use alters human milk metabolome, lipidome, and proteome profiles, 2) cannabinoid concentrations in milk are related to milk metabolome, lipidome, and proteome profiles, and 3) differences in pre- and postnatal cannabis use patterns and selected maternal characteristics [e.g., body mass index (BMI), time postpartum] are related to variation in milk metabolome, lipidome, and proteome profiles, even when controlling for cannabinoid concentrations in milk. To test these hypotheses, we will compare bioarchived milk samples from 20 healthy women (≤ 6 months postpartum) who use cannabis (≥ 1x weekly) and 20 healthy women who do not use cannabis. We will employ a multi-omics approach to characterize and understand holistic milk composition (i.e., metabolome, lipidome, and proteome profiles). Results from this work are expected to provide data on whether cannabis use during lactation impacts human holistic milk composition and, importantly, constitute the foundation for a NIH R01 proposal. It also takes the initial steps toward providing mothers and healthcare providers data to enable more informed advice and evidence-based decisions during lactation.

NIH Research Projects · FY 2025 · 2023-11

Herpes simplex virus (HSV) remains a major pathogen worldwide causing lifelong latent infections in humans. It is responsible for significant disease, ranging from cold sores and genital infections to blindness and fatal encephalitis. Herpesviral entry is a complex process involving multiple viral and cellular factors. The long-term goal of this project is to understand the molecular mechanisms that govern HSV entry into host cells. HSV particles are enveloped in a host cell-derived lipid bilayer membrane. To accomplish entry, viruses must surmount the barrier posed by the host membrane. Thus, the HSV entry process is intimately associated with membrane interactions. The roles of lipid components of virus and cell membranes in HSV entry are greatly understudied relative to membrane-associated proteins. Sphingomyelin (SM) is the most common sphingolipid component of biological membranes and is a signature constituent of lipid rafts. The specific role of SM in herpesviral entry is not well-understood. Based on our published and preliminary data, two complementary specific aims are proposed. A battery of assays will be employed to assess the importance of SM for specific steps in the entry process including cell attachment, endocytic uptake, intracellular vesicular transport, and membrane fusion. In Specific Aim # 1, we will elucidate the function of HSV envelope sphingomyelin in viral entry. Experiments in Specific Aim #2 follow from preliminary data indicating the selective importance of host cell SM in HSV entry by an endocytosis pathway. The precise step(s) in the entry cascade that require cellular SM will be defined. A combination of molecular, biochemical, and cell biological approaches will be used to fill key gaps in our understanding of HSV entry.

NIH Research Projects · FY 2025 · 2023-11

PROJECT SUMMARY Antibodies are critical mediators of immune protection against pathogens, simultaneously acting to neutralize entry and activate innate immune cells through interaction between the antibody Fc domain and Fc-receptors (FcRs) on innate immune cells. Leveraging Fc-mediated activation in monoclonal antibody-based therapies is increasingly being investigated for the treatment of infectious diseases but the precise mechanisms and breadth of functions that can be induced by range of FcR-bearing immune cells is still unclear. However, these efforts are limited by the fact that we do not fully understand the spectrum of effector functions and effector programs that are induced via different FcRs, which could greatly impact efficacy and safety of mAb-based therapeutics. Moreover, as some innate immune cells such as monocytes express multiple FcRs, understanding how these pathways interact will help in the development of rational antibody design strategies to effectively harness innate immune cells against a variety of pathogens. There are four activating FcRs that bind IgG and are expressed on innate immune cells (FcγR1, FcγR2A, FcγR3A, and FcγR3B) and one inhibitory receptor (FcγR2B). The interplay between these receptors can impact Fc-effector functions, especially in monocytes that express multiple FcRs. Moreover, antibody-mediated activation of non-classical monocytes that express 3 activating FcγRs must balance protection and pathology, as antibody-dependent enhancement of infection/disease has been observed in some contexts. Thus, defining the transcriptional profiles of FcR-mediated activation of will be critical to better predict in vivo activity of immunotherapies and antibodies induced by vaccination or infection. To that end, we propose to begin to generate an ‘FcR Effector Atlas’ that defines the anti-pathogen pathways that are induced in distinct innate immune cell types mediated by different FcRs. The Atlas can be used to map the pathways induced across all innate immune cells to help guide development of optimally efficacious and safe antibody therapeutics against infectious diseases. By combining Fc-engineered antibodies that facilitate individual or combinatorial activation of specific FcRs and nascent transcriptomics, the goal of this proposal is pioneer an approach to generate the Atlas in nonclassical monocytes. We will use SARS-CoV-2-specfic Fc-engineered monoclonal antibodies as a model system to specifically engage different FcRs on monocytes to define the FcR-mediated transcriptional network using capped small RNA-sequencing (csRNA-seq) that enables transcriptional profiling of nascent RNA transcriptional start sites. Further, we will functionally link induction of specific pathways to restriction of SARS- CoV-2 infection. Together, the approaches and results generated from this proposal will contribute to our fundamental understanding of how antibodies leverage innate immunity and can be used to guide development of highly effective and safe immunotherapeutics for infectious diseases and beyond.

NIH Research Projects · FY 2026 · 2023-11

PROJECT SUMMARY/ABSTRACT After the severe acute respiratory coronavirus (SARS-CoV) emerged in China in 2002, the virus was traced back to animal markets and several genetically related viruses were identified in bats. This early work into coronavirus zoonosis and the concomitant rise of next generation sequencing technologies in the early 2000’s helped initiate global research efforts to identify viruses circulating in wildlife. The genomes for tens of thousands of novel animal viruses have now been sequenced and deposited in online repositories. Coronaviruses are abundant in mammals and birds and comprise approximately 25% of all bat viruses discovered to date. The highly pathogenic human coronaviruses, SARS-CoV, and middle east respiratory syndrome coronavirus (MERS-CoV) are only representative members of their respective sarbeco- and merbeco- subgenera, which encompass hundreds of related viruses found in bats and other wildlife, worldwide. Unfortunately, because there are few tools available for researchers to study uncharacterized animal viruses, virus discovery studies rarely isolate viruses under laboratory conditions or perform experiments beyond genetic sequencing, leaving some of the most essential questions about these viruses – including if they have the potential to infect humans – unanswered. An improved understanding for what species these viruses can infect and how they invade the cells of their hosts is essential for future pandemic preparedness. The most significant species barrier for the coronaviruses that have transmitted to humans is at the level of cell entry and studies have shown that overcoming this barrier allows for coronaviruses to replicate in cells from diverse species. To invade cells, the “spike” glycoprotein on the surface of viral particles binds to host cell receptor molecules. The receptor binding domain (RBD) is a small region on the distal tip of the spike protein, capable of folding independently of spike and contains all amino acids that contact the host receptor. We previously developed “SarbecoType” – a BSL2-compatible, viral pseudotype-based platform to functionally screen the cell entry properties of the RBD from any sarbecovirus. This approach is highly cost- efficient and scalable, requiring synthesis of only a small portion of the spike gene, and has allowed us to characterize the cell entry phenotypes of approximately 95% of all published sarbecoviruses. This dataset identified multiple clades of sarbecovirus RBDs that vary in their zoonotic properties for humans, and has formed a foundational basis for ongoing universal sarbecovirus design. Therefore, we hypothesize uncharacterized coronaviruses pose a threat to humans. Here we propose to functionally screen the much larger and diverse group of merbecoviruses with similar methods (I.e., “MerbecoType”) and use this entry data to predict the entry capabilities of novel sarbeco- and merbeco-virus sequences.

NIH Research Projects · FY 2025 · 2023-09

Virus infections remain major threats to human health worldwide as demonstrated by COVID-19 pandemic caused by SARS-CoV-2 and its variants. All enveloped viruses must fuse with host membranes to initiate the infection process. Membrane fusion is a critical, but poorly understood biological process that is driven by protein conformational changes. Membrane fusion is a highly complex, multistage and multiscale process, which is difficult to investigate through scale-specific techniques (both experimentally and numerically). In this project, we propose to investigate the structural changes of fusion proteins and virus fusion through a combination of multiscale modeling, machine learning, and complementary experimentation. Because of our decades long experience in working with the herpes simplex virus (HSV), we will utilize the herpesvirus fusogen, gB, a class Ill fusion protein, as a model protein, to elucidate protein conformational changes during virus fusion. The specific research aims are: (1) To delineate the conformational changes of viral fusion proteins, through development of the machine learning facilitated enhanced sampling scheme for fusion proteins and delineation of the sequential conformation changes of gB protein for HSV fusion by a combination of machine learning and experiments; (2) To elucidate membrane fusion driven by viral fusion protein conformational changes, through development of a multiscale model for membrane fusion, assessment of the role of membrane fluidity in fusion and elucidation of the importance of the gB membrane proximal region on gB conformational changes and membrane fusion through combined simulations and experiments. This research will establish experimentally validated, powerful modeling platforms for exploration of the protein conformational changes and will bridge the multiple spatial and temporal scales involved in the fusion process. The machine learning method for enhanced sampling is highly innovative and crucial to capture the large-scale structural (conformational) changes and associated energy profiles of fusion proteins, and to identify the appropriate pathways during the fusion process. The integration of the gradient-based optimization method to the machine learning algorithm is novel to identify the most appropriate reaction coordinates.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Bias in how clinicians form relationships with and treat patients exists based on patient race, ethnicity, gender, socio-economic status, LGBTQ+ status, disability, addiction and other factors. Implicit or unconscious biases (the ways in which our beliefs, attitudes, and values influence how we see the world and the people in it) are widespread and affect patients’ health outcomes. Our team has developed Counter Bias Training Simulation (CBTsim), an innovative and unique training program that uses simulation to reduce the impact of implicit bias on how people interact and make decisions that affect others. Versions have been developed for policing and military professionals to enhance their ability to interact with diverse groups of citizens in unbiased ways. CBTsim has great relevance to healthcare, for which existing bias trainings are typically video or lecture based, and may be ineffective at reducing the impact of bias on patient-clinician relationships (PCR) and consequent healthcare delivery. The purpose of this study is to develop “CBTsim Healthcare” and evaluate its effectiveness at reducing bias in how nurses treat their patients. The proposed study will be jointly conducted in the Washington State University College of Nursing Simulation Lab and Providence Medical Center in Spokane. First, we will develop CBTsim Healthcare scenarios based on an extensive review of the literature on healthcare disparities. Then, we will conduct a randomized control trial with 100 nurses to test the effectiveness of CBTsim Healthcare. Nurses will receive 2 hours of baseline testing, then 50 (treatment group) will receive a 4-hour CBTsim Healthcare training and the other 50 (control group) will watch a 1-hour video on implicit bias in healthcare, typical of current standard practice. Then, all 100 nurses will receive 2 hours of post-intervention testing. Testing will include the implicit association test (IAT) to measure implicit bias, questionnaires to measure prejudice, and patient care scenarios using simulation mannequins to test for bias in PCR and other aspects of healthcare delivery. Finally, we will track treatment and control group nurses in the hospital for 6 months following the intervention to assess disparities in healthcare, measured using patient satisfaction with nursing care scales (quantitative measure) and narratives to document experience of PCR (qualitative measure). All major health agencies have identified reduction of implicit bias in healthcare and resulting minority health disparities as a matter of extreme importance and urgency. The economic impact of health disparities is an estimated $230 billion a year, and the social justice impact is immeasurable. Our current focus on nurses is due to our existing relationship (e.g., on AHRQ R01 HS025965-01), however we anticipate that CBTsim Healthcare could be modified for other healthcare professional groups and could have a revolutionary impact on reducing bias in healthcare delivery nationwide.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Poor sleep is common in neurodevelopment disorders such as autism spectrum disorder (ASD), with up to 93% of ASD individuals reporting sleep problems. These problems worsen quality of life and core symptoms of ASD and likely precede an ASD diagnosis, suggesting they start early in life. The potential adverse impact of early life sleep disruption is supported by animal model studies showing long term functional consequences on behavior. However, little is known about the underlying molecular consequences of sleep deprivation (SD) early in life. Our recent work in wildtype mice demonstrates that the cortical transcriptional response to SD is more prominent at postnatal day 24 (P24) with a preferential downregulation on the Wnt-singling pathway compared to adulthood. Nevertheless, the transcriptional response and molecular mechanisms underlying the adverse effects of SD early in life are poorly understood in the context of ASD. My overall goal is to understand the detrimental consequences of sleep loss in neurodevelopmental disorders. Previous work in the lab showed that mice carrying a mutation in the ASD gene Shank3 (Shank3∆C mice) are the only ASD mouse model that replicates the clinical features of insomnia in ASD and show an abnormal transcriptional response to SD in adults. In my graduate work, I used polysomnography in young Shank3∆C mice to show that sleep problems are present and detectable at P24. In the F99 phase, I will investigate how this ASD mutation alters the impact of SD on transcription at P24 using both bulk and single nuclear RNA sequencing. In addition, I will use targeted quantitative proteomic for SHANK3 and -catenin to investigate the effects of Shank3∆C and SD on canonical Wnt-signaling. Nuclear -catenin levels are a well-established marker of canonical Wnt-signaling activation and SHANK3 has been shown to bind -catenin in the nucleus and inhibit its transcriptional activity. My sponsor, Dr. Peixoto has an established track record analyzing transcriptomic response to SD in mouse models and has establishes the Shank3∆C model for the study of sleep in ASD. My co-sponsor, Dr. Frank has extensive experience in developmental neuroscience and has pioneered many of the studies characterizing the role of sleep-in developmental synaptic plasticity. Additional members of my advisory committee, Dr Hicks and Dr. Prasad are world experts on single-cell transcriptomics and proteomics. In the pre-doctoral F99 phase, I will learn to use proteomic and transcriptomics approaches and advanced transcriptomic data analysis. In the postdoctoral K00 phase, I will build upon these skills and use genetic mouse models to integrate circuit level manipulation and multi-omics to better understand the consequences of sleep loss on behavior in neurodevelopmental disorders. Overall, the proposed training will optimally position me to start an independent research career at a leading neuroscience research institute and advance our understanding on the molecular consequences of sleep loss across development.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT In 2020, 28.3 million Americans experienced an alcohol use disorder (AUD); yet only 25% of these people received treatment. During the pandemic, 60% of patients with a substance use disorder reported receiving telehealth-based care for their addiction. The pivot to telehealth provides an opportunity to overcome the barriers to access and retention that affect in-person care (e.g., inconvenience, stigma). In contingency management (CM), people receive tangible reinforcers in exchange for submitting biological samples to verify abstinence. CM is ideal for telehealth delivery and initial studies of telehealth models demonstrated reductions in alcohol use. At the same time, these models have limitations. They require wearing a transdermal monitor or submitting multiple breath samples daily. As a result, these approaches are costly, burdensome, and stigmatizing which limits their feasibility. In part due to limitations of these biomarkers, current CM models are brief, averaging about 12 weeks, despite evidence suggesting that longer CM interventions result in better post-treatment outcomes. Therefore, the overall objective of our program of research is to utilize phosphatidylethanol (PEth), a blood-based biomarker that can detect alcohol use for up to 28 days to deliver a feasible telehealth-based 26-week CM intervention. In a pilot trial, we developed a telehealth-based PEth CM intervention where participants used a medical device, the TASSO-M20 to self-collect blood for PEth testing under the observation of research staff over Zoom. This intervention used a two-phase approach where the frequency of PEth testing and reinforcement was decreased from once a week, to as infrequently as every four weeks once participants achieved a PEth level consistent with two to four weeks of abstinence (< 20 ng/mL). Seventy-one percent of CM participants achieved >4 weeks of abstinence versus 21% of the treatment as usual (TAU) group, and 43% of CM participants achieved >24 weeks of abstinence compared to 0% of the TAU group (p < 0.05). Based on these promising results, we now propose to test our telehealth PEth-based CM model in a sample of adults with AUD (n=200), recruited via online platforms by randomizing individuals to six months of 1) an online cognitive behavioral therapy for AUD (CBT4CBT) and telehealth PEth-based CM (CM condition) or 2) CBT4CBT and reinforcers for submitting blood samples (no abstinence required) (control condition). We will assess group differences in PEth-defined abstinence and regular excessive drinking (PEth ³ 200 ng/mL), and alcohol-related harms (e.g., smoking, drug use). We will address important gaps in CM research by assessing outcomes during a 12-month follow-up, which is much longer than most previous CM studies; using a conceptual model to identify predictors of post-treatment abstinence. The primary barrier to the dissemination of our model is the cost of PEth testing and CM reinforcers. We will conduct an economic analysis to place these costs in the context of downstream CM-associated cost- offsets and improvements in personal and public health. If our model increases alcohol abstinence and is cost- effective it could reach millions of Americans with AUD that cannot or do not seek in-person care.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract This research project directly addresses objective one of the NORA for the Public Safety Sector which calls for research to “Identify exposures experienced by fire service and wildland firefighters” and to “develop improved and cost-efficient technologies for the detection of contaminants that may be present during firefighting” in order to reduce the burden of cardiovascular disease, cancer, and other chronic diseases amongst firefighters, including wildland firefighters. The project also addresses priorities to reduce work-related illness by better describing and characterizing exposures to hazardous chemicals, including those from wildfires in the Agriculture (Objective AG-02) and Forestry (Objective FO-02) Sectors, and the project addresses Cross- Sector priorities to “Develop new field equipment/strategies for the assessment of occupational exposures for the study of respiratory health” and to “develop and evaluate industrial hygiene sampling and analytical methods (including biomonitoring and other methods) for the determination of carcinogen exposures” (Objective 2; CRC Cross-Sector). The objective of this project is to develop a low cost and highly sensitive multiplex biosensor system integrated with a smartphone readout that can rapidly and accurately analyze a panel of woodsmoke-derived biomarkers amongst wildland firefighters at anticipated low concentrations. Of particular importance to this approach is the ability to couple a simple sample separation strategy with a parallel sensor platform and smartphone readout for simultaneous quantification of multiple targets, and to increase sensitivity using single-atom nanozymes (SANs) amplification. This is significant to identify multiple analytes at low concentrations in biological matrices, such as those will be developed in this project. Further integration with a smartphone reader, the proposed biosensor will provide quantitative results, and allow real-time data collection and sharing. In this project, we will collect blood and urine samples from field firefighters to investigate the woodsmoke-associated biomarkers and evaluate the proposed biosensor system. Short-term outcomes of this research will include publication and presentation of research results; whereas, intermediate outcomes will focus on the development/validation of a sensor platform using samples collected from field firefighters with an eventual end outcome (beyond scope of current project) of using the sensor system for measuring and subsequently reducing firefighters’ exposure to wood smoke. The development, validation and subsequent deployment of a multiplex sensor platform as a quantitative tool to measure mixed firefighters’ occupational exposures is fully consistent with the goals of the NIOSH Research to Practice (r2P) initiative. This approach will provide better analytical performance (e.g., sensitivity, dynamic range, detection limit, reliability, accuracy, and speed) as well as operational performance (user-friendly design) than the current laboratory-based method.

NIH Research Projects · FY 2025 · 2023-09

OVERALL ABSTRACT The Community Health and Aging in Native Groups of Elders (CHANGE) Resource Center for Minority Aging Research (RCMAR) is a new iteration of the Native Elder Research Center (NERC) that has been continually funded since its inception in 1998. CHANGE will relocate the RCMAR to Washington State University’s Institute for Research and Education to Advance Community Health and continue to increase the diversity of investigators conducting research with Native populations in the US, defined here as American Indian, Alaska Native, and Native Hawaiian, and Pacific Islander people. We maintain an 18-month cycle, draw on long- established best practices, offer innovative solutions to improvements identified by former NERC Faculty and Scientists, integrate a community-based approach, and expand our diversity, equity, inclusion, and accessibility platform to create a culture of inclusiveness and support for 9 diverse early-career faculty (3 per cycle). CHANGE is led by MPIs Dedra Buchwald, MD, at Washington State University, and Julie Baldwin, PhD (Cherokee Nation of Oklahoma), at Northern Arizona University; they will co-lead the Leadership and Administrative Core. The Research Education Component will be led by Clemma Muller, PhD, MS, at Washington State University; and the Analysis Core will be led by Richard MacLehose, PhD, at University of Minnesota. Gary Ferguson, ND (Unangax̂/Aleut), at Washington State University, will lead Community Integration activities to embed community engagement and education into the Pilot Studies and all aspects of CHANGE, upholding our commitment to principles of participatory research. We feature subcontract affiliations with Mentors Valarie Jernigan, PhD (Choctaw), a former NERC Faculty and Scientist, at Oklahoma State University; Scott Okamoto, PhD, a current RCMAR Mentor, at University of Hawai’i at Mānoa; and Mandy Fretts, PhD (Eel Ground First Nation), at University of Washington. Our Specific Aims are to: 1) Deliver the 18-month CHANGE curriculum to a diverse cohort of 9 Scientists pursuing careers in aging research with Native people; 2) Provide state-of-the-art support for CHANGE Scientists’ Pilot Studies within a holistic framework of mentoring, professional development, and research excellence; 3) Integrate CHANGE Scientists into a network of investigators, RCMAR alumni, and community partners who conduct Native health research and promote Native health interventions; and 4) Embed principles of community outreach, engagement, and participatory research, as well as diversity, equity, inclusion, and accessibility across all CHANGE activities. The literature on training programs for underrepresented minority investigators highlights the success of personalized, culturally-informed training and access to underrepresented minority role models. CHANGE is highly responsive to RFA-AG-23- 026’s requirements of recruiting Scientists and providing Mentors from the parent or affiliated institutions, while concurrently accommodating the real-world limitations presented by the highly-dispersed and relatively-small community of early-stage investigators and senior faculty conducting aging-related research on Native health.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Individuals with Down syndrome (DS) are at higher risk for developing Alzheimer’s disease (AD) compared to the general population. As such, they are considered an ideal target population for anti-AD therapy trials; however, there is no reliable measure for predicting dementia onset in this population. Intraindividual cognitive variability (IICV), a measure of variability in neuropsychological test performance within a person at a single timepoint, is a novel, low-cost, non-invasive biomarker of neurodegeneration and early dementia for the general population. However, IICV has not been investigated in adults with DS. Therefore, the current proposal will fill this knowledge gap by characterizing the associations between IICV, AD biomarkers, and dementia in adults with DS. Aims 1 and 2 of this proposal use data from the Alzheimer’s Biomarker Consortium-Down Syndrome (ABC-DS) study, which is currently composed of cognitive and biomarker data collected at two different timepoints (baseline and 18 months), to calculate IICV measures for memory, executive function and processing speed, visuospatial construction, and multidomain cognition in 300 adults with DS. Using the longitudinal ABC- DS data, we will first examine whether IICV is associated with AD plasma biomarkers (β-amyloid 42/40, p- tau217, and NfL) and/or AD-related pathology (Aβ-PET and tau-PET) (K99, Aim 1). We will also examine whether IICV is associated with the clinical presentation of dementia and cognitive decline (K99, Aim 2). We expect our analyses to show that IICV is positively associated with AD-related biomarkers and pathology, and that IICV at baseline is associated with a follow-up diagnosis of dementia as well as cognitive decline from baseline to follow-up. These data will be critical for optimizing the design of a new cohort study of adults with DS that will test the outcome measures from Aims 1 and 2 in a new, more diverse, cross-cultural cohort of adults with DS from Washington State and São Paulo, Brazil, and include comparisons with a control group of individuals with autosomal dominant AD, due to its similarity with DS in early striatal amyloid- β deposition (R00, Aim 3). To complete these aims, we have developed a comprehensive, mentored training plan for me to (1) gain expertise in the relationship between neuropsychology, plasma biomarkers and neuroimaging; (2) broaden my knowledge of the similarities and differences between autosomal dominant AD and AD in DS; (3) explore cross-cultural similarities and differences in AD risk; and (4) develop advanced statistical skills. The data and training obtained in the K99 phase will lead to the successful implementation of a high-quality, international research program focused on IICV and AD biomarkers in DS. Findings have great potential to be used with the DS population worldwide, increasing the chances of early interventions and inclusion in anti-AD trials. The intense training in the K99 and the support of mentors with extensive expertise in all areas of the proposal, will provide the foundation for an independent scientific career on cross-cultural AD risk prediction in DS and other high-risk populations.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Survivors of Ebola virus disease (EVD) have reported a wide range of symptoms following recovery from infection. These long-term sequelae are severe enough to interfere with their daily lives and are now collectively referred to as post-Ebola syndrome (PES). Although post-viral symptoms have posed a serious problem in the Ebola outbreaks of 1995 and 2013-2016, little is known about the underlying mechanism of PES pathogenesis. Ebola virus (EBOV) RNA has been found in immune-privileged sites, such as the eye and semen, so it is suggested that the virus may persist in tissues to cause continued antigenic stimulation over time. However, not all cases of PES can be attributed to viral persistence. Most of the symptoms that survivors experience are autoimmune-like, the most common being arthralgias and myalgias. Autoantibodies against common human proteins have also been found in survivor serum, alluding to virus-induced autoimmunity. We hypothesize that both virus-specific and autoimmune antibody responses play a role in the development of PES. Through a collaboration with Dr. John Schieffelin at Tulane University, we propose to analyze an existing cohort of EVD survivors and their household contacts from Sierra Leone that have been clinically characterized for development of PES. Our preliminary findings revealed that antibodies against the immunodominant antigen, the Ebola glycoprotein (EBOV GP), in asymptomatic EVD survivors were qualitatively different from survivors experiencing musculoskeletal manifestations of PES. Specifically, antibodies from asymptomatic individuals induced higher levels of antibody-dependent complement deposition and monocyte-mediated phagocytosis, but not neutrophil- mediated phagocytosis, and differed in NK cell activation profiles compared with individuals with PES. In addition, our data has also revealed that IgG1 levels against dsDNA, HSP-60, citrullinated histone, and IFNα are elevated in EVD survivors and GP-seropositive household contacts (HHC) compared to GP-seronegative HHC, indicating a correlation between autoantibodies and EBOV infection. Thus, the purpose of this proposal is to further investigate the role of both virus-specific and autoimmune antibody-mediated innate immune cell activation in PES, and whether this role is protective or pathologic. To do so, we propose to analyze antibodies for induction of innate effector function against EBOV-specific proteins in Aim 1 and Aim 2 will focus on the identification of potential autoimmune antibody responses that are elevated in individuals with PES. Together, these aims will address the role of qualitatively different antibodies with varying specificities in shaping susceptibility to/protection from the development of PES and may help to identify potential therapeutic targets to provide proper and effective treatment to EVD survivors suffering from PES. In addition to providing expertise in immunology, virology, and Ebola virus disease, my sponsor and co-sponsors, Drs. Bronwyn Gunn, John Schieffelin, and Anthony Nicola have developed a comprehensive training plan aimed at providing me with the necessary skills needed for me to succeed in this proposal and as a research scientist.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Organisms adapt to seasonal changes in environmental conditions to survive. These adaptations rely predominantly on photoperiod (i.e., daylength), but are also influenced by temperature. Recent studies indicate that photoperiodic changes affect the neuronal composition of brain areas involved in circadian (i.e., daily) timekeeping and modulate the number of dopaminergic neurons, in a process known as neurotransmitter switching. Other studies show that the brain also undergoes profound structural changes across seasons. However, the relationship between these functional and structural changes in the brain and seasonal adaptations remains a major gap in knowledge. Moreover, whether other relevant seasonal cues, in particular temperature, contribute to these changes is not known. The overall goal of this project is to understand the nature and role of neuronal plasticity in the integration of seasonal cues to promote seasonal adaptations. My hypothesis is that seasonal adaptations are mediated by functional and structural plasticity in neurons from circadian and aminergic circuits in response to environmental cues. To test this, I propose 3 specific aims: investigate structural and functional plasticity of (1) the circadian clock neuronal network and of (2) aminergic circuits in response to seasonal cues and its impact on social and locomotor behavior, and (3) determine how the plastic changes in the circadian clock and aminergic circuits regulate brain connectivity and encode the behavioral output of these circuits. I will accomplish this project in the genetically tractable Drosophila model and will leverage a combination of versatile neurogenetics, high-resolution microscopy, and well-established behavioral analysis. Thus far in my postdoctoral career in the Chiu lab at UC Davis, I obtained training in molecular genetics and biochemistry, which I used to explore the role of circadian peptides in modulating seasonal adaptations in Drosophila. Moving forward, I will build on my current research to study the neuronal mechanisms of seasonal plasticity and behavior. During the K99 training period, I will use available tools in Drosophila to assess the functional and structural changes in the circadian clock neurons and aminergic circuits in response to seasonal cues. Moreover, I will test the functional consequences of these changes by using available genetically encoded sensors and by generating new, more sensitive, sensors to assess aminergic function in vivo under the guidance of Dr. Lin Tian. I will expand the use of these tools in the R00 stage to determine how the interaction between these two circuit systems modulate their functions and how they affect seasonal behavior concertedly. I believe that the mentorship of Drs. Chiu and Tian, together with the support provided by the K99/R00 award, will allow me to build a strong foundation that will enable my success as an independent investigator. The results of the proposed studies will elucidate the neuronal basis underlying sensory integrations in the context of seasonal adaptations, shedding light on the mechanisms behind seasonal modulation of health physiology and disorders.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Pathologically strong or intrusive cocaine memories elicit uncontrollable drug craving and can trigger drug relapse in cocaine-predictive environments even after protracted abstinence, yet the contributions of long-term memory maintenance mechanisms to substance use disorders have been understudied. Cocaine memories are not necessarily retained over time; they become destabilized upon retrieval (memory reactivation) and need to be reconsolidated into long-term memory stores to be maintained, updated, and potentially strengthened. Thus, interference with memory reconsolidation weakens cocaine memories in animal models of drug relapse and reduces craving in individuals suffering from substance use disorders. The long-term goal of this proposal is to advance our understanding of the neural basis of cocaine-memory reconsolidation so that cellular/molecular processes and neural circuits suitable for effective therapeutic targeting can be identified in the future. We have discovered that the CA3 region of the dorsal hippocampus (dCA3) plays a critical role in the reconsolidation of cocaine memories. Building on this finding and strong preliminary data, Specific Aim 1 will be to identify cellular and synaptic-plasticity mechanisms of cocaine-memory reconsolidation within the dCA3. We will test the hypothesis that reconsolidation requires glutamatergic pyramidal neuronal activity in the dCA3 stratum pyramidale (SP) cell layer and GABAergic interneuronal activity in the dCA3 SP and stratum lucidum (SL) cell layers. We will identify and phenotype engram cells in the SP and SL and, also, evaluate the hypothesis that the maintenance of cocaine-memory strength via reconsolidation is associated with lasting plasticity in dentate gyrus mossy fiber (MF)dCA3 engram cell synapses, but not in dCA3 non-engram cell synapses. Specific Aim 2 will be to map dCA3 efferent circuits that regulate cocaine-memory strength during reconsolidation and determine related synaptic plasticity mechanisms. Based on extant literature and strong preliminary data, we will test the hypothesis that direct dCA3dCA1 intra-hippocampal and dCA3dorsolateral septum (dlS) extra-hippocampal circuits regulate cocaine-memory strength in an opposite manner during reconsolidation. Further, we will examine the hypothesis that cocaine-memory maintenance after reconsolidation is associated with increased polysynaptic transmission (MFdCA3dCA1/dlS) and excitatory synaptic plasticity in dCA3dCA1/dlS engram cell synapses. The proposed studies will utilize an instrumental model of drug-memory reconsolidation and relapse, engram cell tagging using Targeted Recombination in Active Population protocols, cell type- and pathway-specific in vivo and ex vivo optogenetics, pharmacology, multi-label immunohistochemistry, and whole cell patch-clamp electrophysiology to increase our understanding of how hippocampal-memory reconsolidation processes regulate cocaine memory strength and drug relapse. The findings are expected to provide an essential conceptual framework for future research and insights for the development of effective treatments for substance- use disorders and other psychiatric disorders characterized by intrusive maladaptive memories.

NIH Research Projects · FY 2026 · 2023-05

Abstract Our long-term goal is to decipher the molecular mechanisms of telomerase regulation and telomere homeostasis during development. Telomerase elongates telomeres to compensate for their loss during cell proliferation. Its regulation is critical for human aging and susceptibilities to cancer and many age-related degenerative diseases. The TERT gene, encoding the human telomerase reverse transcriptase, is regulated primarily at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most somatic cells. Recent progresses on telomerase regulation in cancer cells have greatly improved our understanding of TERT gene activation during cancer development. However, the mechanisms of its repression in most differentiated cells and expression in certain somatic cells remain to be elucidated. Regulation of transcription during development and differentiation often involves distal elements and chromatin reorganization. We previously reported that the endogenous TERT gene was embedded in a condensed chromatin domain and stringently repressed in a histone deacetylase-dependent manner in somatic cells. To identify distal regulatory sequences required for establishing the repressive chromatin of the TERT locus and to understand its regulation in vivo, our laboratory has developed two innovative technical platforms in the past decade. The first is recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. This technique, together with the new CRISPR-mediated gene editing, enables us to study distal regulatory elements of the TERT gene in their genomic contexts. Consequently, we have discovered that a polymorphic tandem DNA repeat in intron 2 (VNTR2-1) functions as an enhancer for TERT transcription. In addition, we have engineered a humanized mTert allele (hmTert) for studying human-specific telomerase regulation in mice. In the next funding period, we plan to use these tools and focus on the following three directions: (1) Identify transcription factors that regulate TERT gene via VNTR2-1; (2) Identify key distal regulatory elements responsible for TERT repression; and (3) Study the roles of these distal elements in regulating telomere homeostasis in vivo using our mouse model with humanized telomeres. In short, using our unique tools, we will address some of the fundamental mechanisms critical to telomerase regulation and telomere homeostasis in humans, and ultimately telomere-associated human diseases.

NIH Research Projects · FY 2024 · 2023-01

Project Summary Monosodium urate-induced inflammation begins with hyperuricemia leading to monosodium urate (MSU) crystal deposition in the joints and periarticular tissues. These deposits can cause inflammation, pain, and tissue destruction through either acute inflammatory flares or chronic disease. In MSU-induced inflammation, activated macrophages produce the pleiotropic cytokine Interleukin-1β (IL-1β) which is recognized as the major driver of pathogenesis and a key inducer of other pro-inflammatory molecules. The inflammatory signaling cascade begins with activation of Toll-like/IL-1β receptors (TIRs) in macrophages, which triggers production of pro-IL-1β and other inflammasome components. Pro-IL-1β is then activated by cleaved caspase from the NOD-Like Receptor Protein 3 (NLRP3) inflammasome before being exported and initiating further inflammation. Several treatment options exist for gout patients, mostly falling into two categories – reduction of circulating levels of soluble urate, or the suppression of pain and inflammation. These therapies, however, leave much to be desired as they have significant adverse effects, deleterious drug-drug interactions, high cost and low/non-responder groups. This project investigates a potential novel therapeutic agent for MSU-induced inflammation – pentagalloyl glucose (PGG) – that is known to have strong antioxidant and anti-inflammatory effects. Additionally, our preliminary data shows that this compound inhibits xanthine oxidase which produces soluble urate. We also demonstrate that PGG inhibits TGFβ-activated kinase which plays a role in the upregulation of proinflammatory mediators critical to MSU-induced inflammation. Finally, we have demonstrated that PGG inhibits both a critical downstream kinase and proinflammatory cytokines. In Aim one we will investigate the mechanism by which PGG reduces MSU-induced inflammation in vitro using THP-1 macrophages, investigate the atomistic interactions of PGG with MSU in-silico, then use phosphoproteomics to identify global changes induced by MSU and PGG. In Aim two we will determine the effects of PGG treatment in vivo using mouse models of gout previously published by our group. Taken together, these findings will provide a more complete knowledge of MSU-induced inflammatory signaling while exploring a potential novel therapeutic and providing a training mechanism for this student.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Unhealthy diet is a key behavioral risk factor for several chronic diseases, particularly type-2 diabetes, cardiovascular disease, and some cancers. Long-term measures of diet are typically self-reported and subject to known sources of error and bias. A recognized population-level determinant of diet is access to and use of food retailing, particularly consuming food away from home (FAFH) from quick-service or “fast food” outlets. Currently, there are few methods to objectively measure long-term patterns of FAFH in health research or to understand how built environments shape these behaviors. However, the pervasive use of smartphones provides an emerging opportunity to address these questions. We will use Google Location History Timeline (GTL) data, which includes high-resolution geographic location data collected passively on smartphones with Google Apps, to develop new measures of long-term patterns of food retailer utilization, especially use of FAFH outlets. We will use data from 500 adults from the Washington State Twin Registry who are contributing their GTL data for another NIH-funded study on built environments and active travel (R21 ES031226-01A1). These data include daily time-activity patterns from 2012 to present. We will evaluate these measures against objective measures of contemporaneous food outlet utilization and detailed dietary intake measures in a subset of twins who have previously completed additional surveys, including food frequency questionnaires and travel diaries. Finally, we will measure how metrics of FAFH use relate to health outcomes, particularly weight status, weight change, and self-rated overall health, both cross-sectionally and longitudinally. New measures of food access behaviors associated with diet and health outcomes can be used in epidemiological research and advance the field of built environment influences on human health.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Meiotic studies of human fetal ovaries we conducted for over a decade have generated the largest available human female meiotic database. This unique resource includes matched maternal/fetal biospecimens for each sample collected from 2008 to 2017, a period of rapid changes in both the spectrum and levels of endocrine disrupting chemical (EDC) contaminants. The proposed studies will test the hypothesis that the high meiotic error rate in the human female is driven by both an innate propensity to error (e.g., due to the protracted nature of female meiosis and differences in cell cycle control) and environmental factors. We postulate that environmentally-induced effects will be discernable because they disturb well-defined meiotic relationships. We will expand our meiotic database for the cohort and characterize the features of a well-known cause of human aneuploidy, recombination failure. Meiotic profiles for each case in conjunction with exposure profiles will be used to determine if and how fetal exposure affects the early stages of female germline development in humans. We also will obtain data on temporal changes in human exposure to common endocrine disrupting chemical (EDC) contaminants and compare the ability of different EDCs to transit from the maternal circulation to the developing fetus.

NIH Research Projects · FY 2025 · 2022-09

A key aspect of survival is an organism’ ability to adapt their behavior based on experience. At the cellular level, a well-established view of how experience can stably change behavior is that new mRNA and protein synthesis occurs, and that this new state becomes stably encoded in the chromatin, the complex of DNA and proteins that determines transcriptional state. Sleep is an extremely conserved drive found across the animal kingdom that may facilitate these processes. We hypothesize that one of the evolutionary conserved functions of sleep is to influence gene expression and chromatin regulation. There are multiple lines of evidence that support the idea that sleep is important for transcriptional regulation and chromatin stability, but to date no one has directly compared transcriptional responses to sleep loss across vertebrate species or between brain and other tissues within the same species. This is important because to determine conserved mechanisms it is necessary to compare across evolutionary time. Another key limitation of all omics studies of sleep is the lack of resolution at the single-cell level. This is important because, as show in our preliminary studies, different cell types respond differently to sleep loss. In addition, the lack of resolution at the single cell-level can make mapping correspondence between changes in gene expression and changes in chromatin inaccurate. The application of single-cell transcriptomic and epigenomic analysis is a promising avenue to understand transcriptional regulation. However, single-cell approaches have not yet been applied to understand how sleep influences transcription and chromatin regulation. In this Maximizing Investigators Research Award (MIRA) we will utilize state of the art single-cell genomic technology to define, for the first time, common sleep-dependent transcriptional and epigenetic programs across different cell-types between two distantly related vertebrate species: Mouse and Zebrafish. This proposal leverages expertise in comparative genomics and evolution, computational biology and the application of transcriptomic and epigenomic technology to study brain and behavior, training I have acquired throughout my PhD and post-doctoral fellowship. The goal is to establish a program of research focused on understanding how sleep across different species can alter transcription and chromatin accessibility in different cell-types. In the short-term this proposal will produce a comprehensive cell atlas of sleep-dependent regulation of gene expression and chromatin states, as well as publicly available software for single-cell data analysis. In the long-term these studies will serve as the basis for functional studies to define evolutionary conserved mechanisms by which sleep can modulate gene expression and chromatin architecture.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Formerly incarcerated people with serious mental illnesses (SMI) experience the criminal legal system unequally and have elevated rates of recidivism, homelessness, general medical problems, and substance use disorders. Permanent supportive housing (PSH) is a housing intervention often used during reentry, but it has limited resources for addressing community integration, a key component of reentry. PSH are often located in high-poverty environments with increased criminogenic risk. The geography of PSH also includes public spaces, which are associated with greater self-esteem, life satisfaction, a positive orientation toward recovery, independent employment, and access to tangible and social resources. The risk environment framework provides a structure for understanding the geography of PSH through its focus on the physical, social, economic, and policy influences on both the micro and macro environments. During reentry, individual, interpersonal, and environmental factors can interact with these environments to produce or reduce risk. If addressed, these factors can contribute to reentry wellbeing, through improved community participation and treatment engagement and reduced psychiatric distress and substance use, ultimately supporting more targeted interventions. The proposed study uses a rigorous nontraditional QUAL + QUAN (spatial) concurrent mixed-methods design to examine how individual, interpersonal, and environmental factors situated in the risk environments of PSH interact with public and private spaces to inform reentry wellbeing. Eighty multi-method interviews (i.e., qualitative, quantitative, and participatory mapping methods) that look at individual, interpersonal, and environmental reentry factors will be conducted with formerly incarcerated clients with SMI. Data collected will be triangulated with go-along interviews with up to 20 of the participants. Participatory mapping will then be geocoded and sites identified as places of importance, frequent participation, and belonging will be evaluated in relation to objective features of spaces like resource and treatment availability or accessibility using GIS methods in order to develop a community resilience index model. A cluster analysis will additionally be conducted to identify and map areas of increased and decreased drug overdose and arrest and overlaid with the community resilience index model to examine the relationship between community resilience and the risk environments. I will then partner with a community advisory board of 2 providers and 6 formerly incarcerated PSH residents to integrate the findings for the purpose of co-designing a targeted multilevel intervention aimed at improving reentry wellbeing through public space engagement. My proposed research plan integrates activities, formal training, and mentorship from experts (Drs. Michael McDonell, Benjamin Henwood, Ofer Amram, Chyrell Bellamy, Susan Collins, and Mark Salzer) in supportive housing, the risk environment framework, spatial analysis, intervention development, community-based participatory research, and the mental health and criminal legal systems. The Mentored Research Scientist Development Award (K01) will build on my previous training and research to allow me to pursue my long-term career goal to become an independent investigator focused on the use and interaction of public and private spaces in supporting reentry from prisons and jails for people with SMI.

NIH Research Projects · FY 2025 · 2022-09

Abstract The goal of this project is to develop a mouse model that acquires human-like telomere homeostasis for the study of human aging, cancer, and other age-related diseases. In humans, most somatic cells lack telomerase expression and cannot replenish their telomeres. Telomeres progressively shorten upon successive cell divisions, functioning as an aging clock. Consequently, telomere attrition is a critical factor of human aging and telomere stabilization, predominantly via telomerase activation, is an essential event in the development of most human cancers. On the other hand, many other organisms, including laboratory mice, do not exhibit telomere-mediated replicative aging. Mice possess long telomeres and ubiquitous telomerase activity in adult tissues. This interspecies difference has become a bottleneck for addressing many fundamental questions in human aging and cancer biology using mouse models. To tackle this challenge, we have started to create a mouse strain with humanized telomere homeostasis. We have engineered a humanized mouse Tert allele (hmTert) by using regulatory sequences from the human TERT gene (hTERT) to replace their mouse counterparts. We found that the hmTert gene regulation recapitulated that of the hTERT gene during mouse development and in mouse adult tissues. Remarkably, the hmTert allele imposed a much shorter telomere length setpoint than the wildtype mTert gene in mice. In this application, we propose three specific aims: 1) Create a mouse strain with hmTert alleles and human-like short telomeres; 2) Study replicative aging in mice with humanized telomere homeostasis; and 3) Determine lifespan and health-span of mice with humanized telomeres.

NIH Research Projects · FY 2025 · 2022-09

The effect of alcohol consumption on drug metabolism and interactions of alcohol with drugs constitute a significant health problem. The mechanisms of these effects are tightly related to the influence of alcohol on drug- metabolizing system, and on cytochrome P450 ensemble in particular. Functional versatility of the cytochrome P450 system is achieved through the presence of over a dozen of P450 species differing in their substrate specificity. The composition of this ensemble is known to be largely affected by alcohol consumption. It is generally presumed that the integral properties of this ensemble represent a simple aggregate of the properties of the constituting P450 enzymes. Although this premise remains to be a foundation of rational analysis of the routes of drug metabolism, its validity became essentially compromised. The body of evidence of system-wide integration in the properties of the P450 ensemble continues to grow. The current project is prompted by recognition of a far-reaching physiological significance and pharmacological importance of inter-species crosstalk in the P450 ensemble and its thorough relevance to the effects of alcohol on drug metabolism and P450- dependent cellular signaling. Our central hypothesis is that the results of the alcohol-induced changes in the composition of the cytochrome P450 pool are not limited to direct consequences of the activity of the alcohol-induced enzymes. They also involve alterations of the metabolism of a broad range of exogenous and endogenous substrates of various P450 enzymes due to their interactions with alcohol-inducible P450 species. These indirect effects are deeply involved in clinically-important cases of alcohol interactions with drugs and alcohol-induced alteration of cellular signaling through eicosanoids, retinoic acid and other P450-dependent messengers. This proposal is aimed to explore the alcohol-induced changes in P450 expression and their effects of on the network of protein-protein interaction in the ER membranes and, consequently, on the system-wide properties of the P450 ensemble. Our long-term goal is to obtain a complete picture of functional interactions between human cytochromes P450, and the alcohol-inducible P450 species in particular, and characterize their role in dictating the integral properties of the drug-metabolizing ensemble and their changes by alcohol consumption. Specific Aims of the project are: (1) To refine our knowledge on alcohol-induced changes in the composition of the cytochrome P450 ensemble in liver cells; (2) To identify the principal intermolecular interactions of alcohol- induced P450 species in HLM; (3) To identify the major functional interactions of alcohol-inducible P450 enzymes with other P450 species and explore their effect on the metabolism of drugs and endogenous substrates by the P450 ensemble. Fulfilling these aims will allow in-depth characterization of alcohol-induced changes in drug-metabolism and P450-dependent cellular signaling and understand their role in deleterious alcohol-drug interactions and other adverse effects of alcohol consumption.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Dr. Eric Lofgren, the PI of this proposal, runs the Lofgren Lab at Washington State University. The goal of this research group is to strengthen the methodological foundation of modeling disease dynamics in small, structured populations, with a focus on how organizations or groups structure themselves. Contact patterns between individuals in small populations arise not only from individual-level decisions, but also from structural constraints such as the staffing levels, work assignments, or the built environment. When aggregated over an entire state or country, the dynamics of these small groups and the structural influences on them can be extremely important to public health. In healthcare environments, these contact patterns can greatly affect patients’ risk of acquiring a healthcare-associated infection. However, the methods and tools to study these populations using mathematical modeling are underdeveloped as compared to those that consider much larger populations. Borrowing from epidemiology and disease ecology, the Lofgren Lab’s research uses stochastic models of disease transmission that incorporate both individual-level variation in how people interact and higher-level contact patterns that are externally imposed, for example, examining how workplace policies or a building’s architecture influences who comes into contact with whom. The proposed five-year research program seeks to strengthen the field’s methods for modeling small, structured populations. The goals of the program are to produce a number of important research outputs. These include not only modeling papers exploring disease dynamics in this population, but also development of computational tools necessary for others to advance this area, including novel synthetic data for methods development. Additionally, the program seeks to develop web-based software platforms that allow clinicians and policymakers to use the methods produced in this program without needing to understand the underlying computational machinery. The proposed research program will also extend the Lab’s work geographically, extending it to rural populations in the United States, Argentina and Eastern Africa. The overall vision of this research program is to employ a four-pronged approach: (i) developing the theoretical basis for particular modeling approaches; (ii) examining phenomena that arise in these populations (e.g., small but intensive outbreaks within healthcare facilities as an early mark of emerging epidemics); (iii) leveraging computational tools to address unique challenges in small populations (e.g., utilizing machine learning and information theory approaches to detect an effect within the extremely noisy data that emerges from these populations); and (iv) producing policy-relevant and actionable information for clinicians and public health decision-makers.

NIH Research Projects · FY 2024 · 2022-08

Abstract Opioids are commonly utilized in pain management therapies. Opioids are mu-opioid receptor (MOR) agonists that suppress excitatory neurotransmitter release, decrease overall excitability, and hyperpolarize neuronal cells, resulting in the desired analgesic effect. One of the most common opioids used is hydrocodone. Hydrocodone is primarily metabolized by cytochrome P450 (CYP) 3A4 and CYP2D6 through N-demethylation and O- demethylation to form its primary inactive metabolite, norhydrocodone, and an active metabolite, hydromorphone. Hydromorphone is further metabolized by UDP-glucuronosyltransferase (UGT) 2B7 through O- glucuronidation to its inactive metabolite hydromorphone-3-glucuronide. A growing concern over the past decade has been the opioid epidemic, where individuals are misusing prescription opioids including hydrocodone that are often used in combination with other illicit drugs, including cannabis. Our lab and previous studies have shown that major cannabinoids including Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), and the THC metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-Δ9- tetrahydrocannabinol-carboxylic acid glucuronide (THC-COO-Gluc) inhibit several major drug metabolizing enzymes. Specifically, THC, 11-OH-THC, THC-COO-Gluc, and CBD inhibit CYP2D6, while THC, 11-OH-THC, and CBD inhibits CYP3A4. Our lab has also shown that THC, 11-OH-THC, THC-COO-Gluc, and CBD inhibit UGT2B7. We hypothesize that major cannabinoids and their metabolites will inhibit hydrocodone metabolism potentially leading to clinically relevant adverse drug-drug interactions (DDI) in individuals who use them concomitantly. The proposed study will examine the inhibitory effect of THC, CBD, CBN, 11-OH-THC, 11-COOH- THC, and THC-COO-Gluc against both wildtype (wt) and prevalent polymorphic variants involved in hydrocodone metabolism in vitro and examine the potential for DDI in vivo. The proposed research will identify the cannabinoids/cannabinoid metabolites that inhibit wt drug metabolizing enzymes (Aim 1) and their major polymorphic variants (Aim 2) that are involved in hydrocodone metabolism. The proposed research will also include a clinical trial investigating the potential DDI between cannabis and hydrocodone, with the pharmacokinetic data used to develop DDI models between hydrocodone and cannabis utilizing physiologically based pharmacokinetic (PBPK) modeling (Aim 3). These in vitro and in vivo studies will aid in evaluating the clinical relevance of the potential DDI between cannabis and hydrocodone in humans.