University Of Washington

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT With the surge of ARDS cases associated with SARS-CoV-2 infection, there is an urgent need to understand novel pathways involved in resolution of lung inflammation and injury to provide the basis for new therapeutic approaches. Studies comparing rodent and human lung injury gene expression signatures reveal conserved pathways, including MAPK/ERK activation during injury. More recently, we found a genetic polymorphism in MAP2K2 associates with death in ARDS, suggesting a biological role in ALI recovery. We demonstrated that MAP2K1/MAP2K2 (MEK1/MEK2) activation in macrophages promotes pro-inflammatory pathways and inhibits reparative ones, making it a potential target to manipulate macrophage phenotypes in ALI. In preclinical acute lung injury (ALI) models, inhibition of MEK1/MEK2 improves measures of ALI, including faster recovery of body weight, reduced pulmonary neutrophilia, and greater reparative macrophage activation. These results suggest that MEK pathways could be effective targets in ALI. To address the isoform and cell source driving this improvement in outcome, we generated mice deficient in MEK1 in myeloid cells (LysmCre+Mek1fl). These mice have no phenotype in naïve conditions, but experience 100% mortality with LPS-induced ALI using a moderate LPS dose from which all wild-type mice recover. These mice have a similar early inflammatory response to LPS but fail to turn off inflammation at later time-points. This phenotype can be completely rescued with IFNAR1 blockade, suggesting MEK1 suppression of type I interferon responses is critical for ALI resolution. We also found sustained MEK2/p-ERK activation in the absence of MEK1, suggesting that MEK1 is critical for MEK2 deactivation to promote ALI resolution. In support of this hypothesis, we found that mice lacking MEK2 globally or in the leukocytes compartment have faster ALI resolution. The proposed aims below outline our approach to identify how MEK isoforms work in concert to regulate myeloid cell responses to better define and target a novel regulatory pathway in ALI. In aim 1, we will determine MEK1 and MEK2 cell-specific roles and signaling pathways in ALI and test our will test our hypothesis that MEK1 is required to deactivate MEK2 in alveolar macrophages to promote resolution of lung inflammation. In aim 2, we plan to evaluate MEK1 and MEK2 interactions and mechanisms of MEK2 deactivation. We will use MEK1 mutants to test our hypothesis that MEK2 is deactivated by binding to pT292 MEK1, and we will determine how these mutants alter macrophage inflammatory (LPS) and reparative (IL-4) responses. In aim 3, we plan to test MEK1 and MEK2 degraders as therapeutics in murine models of ALI. We will test our hypothesis that MEK2-specific degradation will result in faster ALI resolution. These studies will advance our understanding of how the immune system stops inflammation and promotes ALI resolution, revealing new therapeutic targets and approaches that could be brought to the bedside.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY More than one million HIV exposed uninfected (HEU) children are born annually, with the majority in sub-Saharan Africa. HEU infants are at an increased risk of poor linear growth, infectious morbidity, and mortality compared to their HIV unexposed uninfected peers. Breastfeeding reduces but does not eliminate poor health outcomes in HEU infants, despite improving maternal health with antiretroviral treatment. The biological mechanisms for vulnerabilities in breastfed HEU infants remain unclear. There are significant gaps in our understanding of how HIV infection affects breastmilk composition and the role of breastmilk composition in the development of poor growth among HEU infants. It is biologically plausible that systemic metabolic dysfunction associated with maternal HIV infection may alter metabolite levels in breastmilk and thereby affect infant outcomes. We hypothesize that HIV will alter the maternal plasma and breastmilk metabolome and that these alterations will be associated with the infant gut microbiome and infant growth. We propose to leverage the Tunza Mwana Kenyan birth cohort (NICHD 5R01HD096999) including lactating women living with and without HIV and their infants. Utilizing existing maternal plasma and breastmilk samples from 60 cohort participants (30 with HIV and 30 without HIV) and prospective infant growth monitoring, we will expand this unique cohort performing targeted metabolomics and machine learning to characterize metabolic relationships between the mother-breastmilk-infant triad and to identify metabolomic profiles in plasma and breastmilk associated with the infant gut microbiome composition and growth. Specific Aims are to 1) determine how maternal plasma metabolites correlate with breastmilk metabolites, and how plasma and breastmilk metabolites are associated with HIV in early (3 weeks) and later (6 months) lactation, 2) evaluate how breastmilk metabolites influence infant gut microbiome composition and diversity at 3 weeks and 6 months, 3) identify maternal plasma and breastmilk metabolites and infant gut microbiome compositions associated with growth to 24 months of life. Career and Learning Objectives are to 1) acquire foundational and advanced skills in epidemiology and biostatistics, 2) develop core competency in the conduct and interpretation of multi-omics studies, and 3) strengthen skills in longitudinal cohort studies and clinical-translational trials. By accomplishing these learning objectives and through the achievement of the specific aims in the research plan, the applicant will be well- positioned to attain competitive external funding to implement future studies assessing the maternal- breastmilk-infant triad and nutrition and growth outcomes. Study Impact: This work will identify key elements in breastmilk that are disrupted and lead to profound effects on infant health. New data generated on maternal metabolomic and infant gut microbiome profiles associated with infant growth will inform potential targets for intervention.

NIH Research Projects · FY 2025 · 2023-08

PROJECT ABSTRACT Opioid addiction is pervasive and widespread, affecting roughly three million U.S. adults. Currently available opioid addiction treatments, such as opioid replacement therapy, fail to slow the growing opioid pandemic and maintain the risk of addiction and overdose. Moreover, available opioid addiction treatments require long-term commitment to treatment with little evidence of long-lasting abstinence. Lastly, opioid replacement therapy is unable to alleviate addiction-induced cognitive impairments. A deeper understanding of the neural and cognitive systems that underlie addiction is necessary for the development of better targeted treatments for opioid addiction. The rodent model of opioid addiction exhibits behavioral markers analogous to those induced in human opioid addiction. Hence, this is a reliable and feasible model for studies of the neural correlates of addiction-related maladaptive behaviors. An emerging body of research suggests that the evolutionarily conserved lateral habenula in rodents is highly implicated in addiction. The lateral habenula is unique in that it directly regulates dopaminergic and serotonergic structures, both of which exhibit dysfunction in addiction. However, with traditional electrophysiological methods of recording lateral habenula neural activity, it has been difficult to clearly assess responses of large populations of neurons. More recent advances in imaging technology have allowed for week-long monitoring of individual neuron calcium dynamics, easing the feasibility of studying the lateral habenula neural responses. Serotonin agonists, such as psilocybin, have shown promising results in reducing the rates of relapse in alcohol and nicotine addiction and improving cognitive function in unhealthy adults. Importantly, lateral habenula hyperactivity is known to drive aversion and is present in withdrawal. Serotonergic agonists have also been shown to quiet lateral habenula activity, suggesting a potential unexplored treatment avenue. Hence, with the use of calcium imaging, I hypothesize that lateral habenula neuron dynamics will shift to a hyperactive state following morphine withdrawal, and that these neural signatures will correlate with decreased performance on cognitive tasks. Additionally, I hypothesize that psilocybin treatment will reinstate baseline lateral habenula activity and improve cognitive performance. The proposed series of experiments will fill the gap in understanding the neural circuitry that drives maladaptive decisions during opiate withdrawal, as well as the behavioral and neural effect of a novel treatment for opiate addiction.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Major gaps remain in our knowledge of how transcription factors (TFs) interact to bind target cis-regulatory elements (CREs) and dictate gene expression during development. There are ~1600 TFs in vertebrates, and therefore traditional approaches of genetic screens with TF pairwise knockouts would require >2.5 million experiments. Even with high throughput methods, this is not experimentally feasible. I will build novel computational tools and deep neural networks and use multiplexed high-throughput single-cell Assay for Transposase-Accessible Chromatin (scATAC-seq) data from zebrafish throughout development. These deep neural networks will be used for in silico experiments to model CRE interactions to learn the cell-type specific regulatory syntax of T-box proteins during development. These combinations of TF-TF interactions from in silico experiments will then be tested with targeted CRISPR-Cas9 mutagenesis followed by phenotype profiling with in situ hybridization and high-throughput low-cost scATAC and scRNA-seq. In Aim 1, I will make a genome-wide cis-regulatory map of cell-type specific gene regulation of zebrafish to uncover the role of Nodal signaling in zebrafish somitic mesoderm development. In zebrafish, mutations to Nodal, a ligand to TGF-Beta receptor proteins, cause a phenotype of aberrantly undifferentiated trunk somitic mesoderm and correctly differentiated tail somitic mesoderm. The mechanisms driving the differences between these somites are unknown. To resolve this mystery, I will generate single-cell time series wild-type and Nodal deficient embryos across the continuum of zebrafish development using multiplexed high-throughput scATACseq and scRNAseq data. Computationally linking these data will represent a comprehensive reference of zebrafish CRE and transcriptional development and a valuable resource for all zebrafish biologists. By improving the software package, Cicero, to include flexible Poisson lognormal network models, we can achieve the resolution necessary to find novel cell-type specific differences in enhancer-promoter links during development and perturbationc In Aims 2, I will train and validate a deep learning neural network model to predict pairs of transcription factors that interact to activate cell-type specific gene programs. I will use these data and computational tools to perform in silico experiments to learn the cell-type specific regulatory syntax of T-box TFs during development. After performing in silico experiments using this neural network, I will rank candidate TF-TF interactions to test using high-throughput methods for targeted CRISPR-Cas9 mutagenesis to knock out TFs. I will apply this method to uncover the cis-regulatory syntax that allows T-box family transcription factors to exert their DNA loci specificity.

NIH Research Projects · FY 2026 · 2023-08

The scope of the problem is that kidney diseases become more abundant as the US population lives longer. In particular, the risk, incidence, and prevalence of CKD increases with age. As a result, disease severity is higher in elderly patients, the largest group to undergo first-time chronic dialysis. Injury to podocytes remains the primary cause for glomerulosclerosis in both aging and disease. Yet, understanding the intersection of podocyte injury and aging, and the underlying mechanisms responsible are a major unmet need. To close this knowledge gap, our pilot studies showed that podocyte injury in young mice from two models of experimental Focal Segmental Glomerulosclerosis (FSGS) unexpectedly induces short-term replicative- and stress-induced premature senescence. In the long-term this results in premature podocyte aging in early middle-aged mice, which under healthy conditions typically do not exhibit signs of senescence and aging. This phenotype was accompanied by glomerulosclerosis and a reduced kidney function. Finally, the same correlations were also observed in young patients with FSGS. Based on these preliminary data, we propose a novel paradigm that podocyte injury and aging intersect, and because of these overlapping mechanisms, injury amplifies aging. The overall goal of this proposal is to identify novel mechanisms for podocyte injury progression with advancing age. Specific Aim #1 will prove that injury to podocytes causes a premature aging podocyte phenotype. This will be achieved by testing the following hypotheses: (i) Injury to non-aged podocytes causes a p16-dependent replicative senescence and an aged phenotype; (ii) Injury to young podocytes causes a p53- p21 axis-dependent stress-induced premature senescence; (iii) Long-term consequences of injury-induced senescence is a premature podocyte aging phenotype causing glomerular scarring and reduced kidney function. Specific Aim #2 will prove that the mechanisms of podocyte injury and that of aging intersect/overlap and are amplified when superimposed on one another. We will test the following hypotheses: (i) Distinct subpopulations of podocytes are responsible for the intersection between injury and aging; (ii) A combination of inflammatory cytokines, immune modulators and growth factors secreted as a result of the senescent- associated secretory phenotype triggered by podocyte injury promotes disease progression. Innovative experimental approaches used include two models of podocyte injury, loss-of-function approaches using four new podocyte-specific mouse mutants to limit podocyte senescence/ aging, gain-of- function approaches using two new podocyte-specific transgenic mice, single nuclei transcriptomics of injured podocytes over time and the Design-of-Experiment methodology to holistically explore the podocyte signaling environment. The proposal is significant for its short-term translational impact by integrating our mouse data with large transcriptomic data sets from aged and diseased human kidneys and for its long-term impact in developing new strategies to counter the age-dependent demise of kidney function from disease.

NIH Research Projects · FY 2025 · 2023-08

One in 60 U.S. children has autism and many are at high risk for poor oral health – a problem that could be addressed in part through improved toothbrushing. Technologies like mobile health apps are an ideal platform to deliver toothbrushing interventions, but few app-based interventions have focused on teens with autism and the work to date has critical limitations. Our team developed an interactive toothbrushing training app called Brush Up, which incorporates concepts from Video Self Modeling. The app helps users develop toothbrushing skills through repeated self-observation and interactions with a cartoon exemplar that reinforces correct brushing. A front-facing (“selfie”) camera on the mobile device displays the child’s face and teeth. Children brush in tandem with the cartoon exemplar. We tested the app with typically developing children ages 5 to 6 years and found significant improvements in toothbrushing duration after only one week of app use. We then piloted the same app in individuals with autism ages 4 to 22 years. Caregivers of teens with autism were particularly enthusiastic about the app and recommended ways to make the app more autism-friendly by including features that account for sensory sensitivities common in autism. We further identified missing app features that if included would increase the odds of long-term behavior change. In this 6-year UG3/UH3 grant, we have 2 Aims: (1) Pilot the app-based intervention; and (2) Conduct a Stage II efficacy trial. During the 2-year UG3 phase, we will use the Discover, Design+Build, and Test Framework to modify our existing app to make it autism-friendly, develop a control app with only a digital mirror and timer, pilot the intervention with 40 adolescents with autism ages 10 to 17 years (Waisman score ≥21), and make necessary adjustments. In the 4-year UH3 phase, we will recruit 270 adolescents with autism, randomize participants to 1 of 2 arms, and compare outcomes. The trial will last 3 months. The primary outcome is toothbrushing distribution, defined as the mean proportion of tooth surfaces brushed during the last month of the 3-month intervention and measured in each arm by the respective app. Secondary outcomes are toothbrushing duration and frequency. We hypothesize adolescents in the experimental arm will exhibit better toothbrushing distribution, duration, and frequency than those in the control arm. App data will be collected continuously during the 3-month trial and up to 3-months post-trial. Survey data will be collected at baseline, 2 weeks, 1 months, 2 months, and 3 months to assess mediators (e.g., self-efficacy) and exploratory outcomes (e.g., habit formation, quality of life). Post-trial surveys will assess long-term change and interviews with 60 participants from the 2 arms will identify ways to further improve the intervention. If found to be efficacious, our findings could address an unresolved problem that affects over 1.2 million U.S. children and teens with autism. Our long-term goal is to develop comprehensive strategies to prevent caries in teens with autism.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract One in 60 U.S. children has autism and many are at high risk for poor oral health – a problem that could be addressed in part through improved toothbrushing. Technologies like mobile health apps are an ideal platform to deliver toothbrushing interventions, but few app-based interventions have focused on teens with autism and the work to date has critical limitations. Our team developed an interactive toothbrushing training app called Brush Up, which incorporates concepts from Video Self Modeling. The app helps users develop toothbrushing skills through repeated self-observation and interactions with a cartoon exemplar that reinforces correct brushing. A front-facing (“selfie”) camera on the mobile device displays the child’s face and teeth. Children brush in tandem with the cartoon exemplar. We tested the app with typically developing children ages 5 to 6 years and found significant improvements in toothbrushing duration after only one week of app use. We then piloted the same app in individuals with autism ages 4 to 22 years. Caregivers of teens with autism were particularly enthusiastic about the app and recommended ways to make the app more autism-friendly by including features that account for sensory sensitivities common in autism. We further identified missing app features that if included would increase the odds of long-term behavior change. In this 6-year UG3/UH3 grant, we have 2 Aims: (1) Pilot the app-based intervention; and (2) Conduct a Stage II efficacy trial. During the 2- year UG3 phase, we will use the Discover, Design+Build, and Test Framework to modify our existing app to make it autism-friendly, develop a control app with only a digital mirror and timer, pilot the intervention with 40 adolescents with autism ages 10 to 17 years (Waisman score ≥21), and make necessary adjustments. In the 4-year UH3 phase, we will recruit 270 adolescents with autism, randomize participants to 1 of 2 arms, and compare outcomes. The trial will last 3 months. The primary outcome is toothbrushing distribution, defined as the mean proportion of tooth surfaces brushed during the last month of the 3-month intervention and measured in each arm by the respective app. Secondary outcomes are toothbrushing duration and frequency. We hypothesize adolescents in the experimental arm will exhibit better toothbrushing distribution, duration, and frequency than those in the control arm. App data will be collected continuously during the 3-month trial and up to 3-months post-trial. Survey data will be collected at baseline, 2 weeks, 1 months, 2 months, and 3 months to assess mediators (e.g., self-efficacy) and exploratory outcomes (e.g., habit formation, quality of life). Post-trial surveys will assess long-term change and interviews with 60 participants from the 2 arms will identify ways to further improve the intervention. If found to be efficacious, our findings could address an unresolved problem that affects over 1.2 million U.S. children and teens with autism. Our long-term goal is to develop comprehensive strategies to prevent caries and reduce oral health inequities that affect teens with autism.

NIH Research Projects · FY 2025 · 2023-08

The deleterious health impacts of disasters create long lasting effects across the United States. However, the evidence base guiding our nation’s preparedness and response has been described as “seriously deficient.” Research conducted in the aftermath of a disaster is essential to building this evidence base, but is inherently challenging. Accordingly, an established and trained disaster research workforce is essential. However, we are unaware of any training that provides researchers hands-on, experiential training in environmental and public health disaster research methods and follow-on support. In response, the Investigations in Disasters and Emergencies: Advancing Applied Learning in Disaster Research Response (IDEAAL DR2) program will provide intensive skills-based training in environmental and public health disaster research. IDEAAL DR2 will train up to 100 early career researchers across the U.S.to 1) Increase the knowledge and awareness of environmental and public health disaster research methods and skills among interdisciplinary, early career researchers who are interested in conducting public health-focused hazards and disaster research; and 2) Provide follow-on mentorship, financial support, and access to necessary training, equipment, and technology to support early-career researchers in the development and completion of their own research projects that advance public or environmental health disaster science. The course will include five modules with pre-course, in-person, and post-course components: 1) foundations of disaster research; 2) community engagement in disaster research, 3) disaster research methods; 4) tools and instrumentation for disaster research; and 5) disaster research design and implementation. For 12 months following the in-person course, fellows will design and implement an independent disaster research project, leading to a paper of publishable quality or equivalent career-advancing products, supported by monthly training workshops and peer accountability groups. In addition, five fellows annually, will receive dedicated research advising and financial support for their project. Interdisciplinary program faculty have a strong and long-standing history of collaboration. They will be guided by an advisory committee and results from a rigorous formative and summative evaluation program. Course and evaluation materials will be disseminated broadly through the NIEHS DR2 and NIEHS Partnerships for Environmental Public Health Resources Portal, and evaluation findings will be shared with NIEHS and through conference presentations and publications.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Morbidity and mortality related to opioid use continue to rise, with fentanyl now the most common opioid involved in overdose death. Despite three FDA approved medications for opioid use disorder (MOUD)—methadone, buprenorphine, and naltrexone--as many as 3 out of 4 people with OUD are not on any of these lifesaving medications, which speaks to failures in implementation. The penetration of fentanyl into the opioid supply has made strategies for initiation of buprenorphine even more complex. Growing literature describes increased precipitated withdrawal at the time of buprenorphine initiation, thought to be secondary to the lipophilic nature of fentanyl. There is an urgent need to better understand how we can successfully engage more people in treatment that we know decreases morbidity and mortality related to OUD. The work proposed in this K23 application will investigate the initiation of buprenorphine in the setting of fentanyl use from both the patient and provider perspectives. We will utilize novel methods to develop a buprenorphine initiation toolkit and then will pilot the implementation of this new tool. The specific aims of the research are to: 1. Use qualitative methods to explore the experience of both patients and providers initiating buprenorphine for OUD in the setting of fentanyl use. 2a. Conduct a modified Delphi process to investigate expert consensus regarding buprenorphine initiation in the setting of fentanyl. 2b. Work with a multidisciplinary group to develop a buprenorphine initiation toolkit, and 3: Conduct a pilot study to evaluate the acceptability, feasibility and effectiveness of the buprenorphine initiation toolkit. Along with the multipronged training plan, this research will support my overarching goal of becoming an independent physician researcher focused on improving outcomes for people with substance use disorders through innovative implementation approaches.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY The placenta, often referred to as the “intestine” of the fetus, is an essential organ that controls the exchange of nutrients (including vitamins) and xenobiotics (i.e., dietary supplements and FDA approved drugs) between the mother and her fetus. The fetus can also ingest nutrients and xenobiotics via the digestive tract, so do neonates and infants through breastfeeding after birth. The fetal, neonate, and infant blood-brain barrier (BBB) serves a critical role in protecting the developing brain from xenobiotics and supplying nutrients to the brain. Thus, the placenta, the developing BBB and gut are key organs responsible for nutrient and xenobiotic distribution and absorption impacting early human development and xenobiotic toxicity. Transporters can play an essential role in the absorption, systemic exposure, and tissue distribution of nutrients and xenobiotics in the fetus, neonates, and infants across the placental, intestinal, and blood-brain barriers. Identification and quantification of transporters in these tissue barriers is important for understanding and predicting fetal or neonate/infant uptake of, and exposure to, nutrients and xenobiotics, and hence impacting early development as well as the safe and efficacious use of medications/supplements in these vulnerable populations. While the expression and function of a few ABC transporters in human term placenta, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), have been well-delineated, such data are sorely missing for transporters in early gestation placenta, and in the developing gut and BBB during pregnancy and after birth. In this application, we propose to establish a Transporter Elucidation Center (TEC) at the University of Washington that addresses the goals articulated in RFA-HD-23-003. Using quantitative global and targeted proteomics, we will systematically identify and quantify the ontogeny of transporters in the human placenta (from early gestation to term), the developing gut as well as the developing BBB (from early and mid-gestation and after birth). Then, through in vitro (transporter-transfected cells, immunohistochemistry, immunolocalization) and ex vivo (e.g., placental perfusion, intestinal organoids, and iPSC-derived human fetal BBB models) transport studies, we will determine novel substrates, cellular localization, and transport activity of highly abundant transporters in these tissues. Combined, these studies will address a critical knowledge gap in our understanding of transporters that control essential physiological functions and xenobiotic disposition in the developing fetus and neonate/infant. Consequently, the proposed studies will enhance our ability to predict the toxicity or efficacy of xenobiotics and physiological efficacy of nutrients (or lack thereof) in these vulnerable populations.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Heart failure (HF) is a major barrier to healthy aging among people with HIV (PWH) in sub-Saharan Africa (SSA). Women with HIV (WWH) may be most vulnerable, with a nearly two-fold increased risk for HIV- attributable HF among women vs. men. Why HIV-attributable HF risk is higher in women is incompletely understood, but among WWH, chronic inflammation, metabolic factors such as obesity, and other hormonal factors such as accelerated reproductive aging are hypothesized to play key roles. Once HF is established among PWH, the 1-year mortality rate is 31% and sudden cardiac death (SCD) from ventricular arrhythmias is common. In this context, strong imperatives exist to identify strategies to prevent the development of HF and SCD among WWH. The most important pathologic processes upstream of HF and SCD are myocardial fibrosis and myocardial steatosis. Cardiovascular magnetic resonance (CMR) and spectroscopy (MRS) are considered gold-standard techniques for identifying myocardial tissue characteristics, including diffuse interstitial fibrosis, focal scar, and steatosis. Among PWH, myocardial fibrosis and steatosis correlate with diastolic dysfunction; in addition, myocardial fibrosis predicts adverse cardiovascular outcomes and SCD. To date, no studies have characterized the extent of myocardial fibrosis and steatosis among ART-treated WWH in SSA or examined predictors of fibrosis/steatosis progression specific to this group. Through this innovative proposal focus on WWH in SSA, we will: 1) characterize myocardial fibrosis burden and identify novel infectious/immunologic predictors of progression; and 2) quantify myocardial steatosis burden and identify hormonal/metabolic predictors of progression. We hypothesize that among WWH in SSA, predictors of myocardial fibrosis progression will include endemic co-infections (e.g. cytomegalovirus and latent tuberculosis), immune activation/inflammation indices (e.g. osteopontin and circulating immune cell subsets), and novel metabolomic signatures. We further hypothesize that among this group, predictors of myocardial steatosis progression will include reduced ovarian reserve (anteceding overt menopause; characterized by menstrual history and levels of anti-Mullerian hormone), obesity and/or increased fat in ectopic depots (visceral and epicardial fat by MRI), longer cumulative exposure to select ART subtypes including integrase inhibitors, and novel metabolomic and lipidomic signatures (some overlapping with and some distinct from the signatures associated with fibrosis). This work will inform the design of HF prevention strategies targeting: select immune/inflammatory pathways (e.g. dual CCR2/CCR5 antagonism); vs. viral co-infections (e.g. letermovir for treatment of cytomegalovirus); vs. early/abrupt decrement in endogenous estrogen production (e.g. transdermal estrogen); vs. ART-associated weight gain (e.g. culturally-specific diet/exercise intervention timed to initiation of or switch to culprit antiretroviral therapeutics). Overall, this work will have high-impact to preserve healthy cardiometabolic aging among 13 million WWH in SSA, representing 2/3 of all WWH globally.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The presence of aneuploidy (chromosomal abnormalities) in embryos is considered one of the major limitations to successful human reproduction and a significant cause of gestation failure, accounting for approximately 50% of early miscarriages. Aneuploidy rates are strikingly high in in vitro fertilized human embryos, and around 60% of these embryos are mosaic, containing both aneuploid and normal euploid cells. The frequent occurrence of mosaicism exists in both naturally conceived and IVF pregnancies. However, despite the high incidence of aneuploidy in human embryos, our knowledge of the molecular mechanisms and developmental fate of these cells is restricted due to the considerable ethical limitations associated with human embryo and fetal research. My previous work demonstrated lineage-specific behavior of aneuploidy in early differentiation using an in vitro human embryonic stem cell (hESC) model. To further characterize the cellular physiology of aneuploidy after implantation, an in vivo animal model is required. Common marmosets exhibit naturally occurring aneuploidy, making them a more representative model for humans than rodents are. Therefore, I propose a marmoset model to further dissect aneuploidy cell fate and its molecular and cellular consequences during early development. My preliminary data uncovered that aneuploid marmoset embryonic stem cells (cj-ESCs) preferentially differentiate into trophectoderm lineages in response to BMP4 stimulation, similar to the behavior I observed with hESCs in my previous work, suggesting a conserved role of aneuploidy in restricting stem cells to extraembryonic fates. During the training period, I will use a unique marmoset stem cell model (gastruloid) that recapitulates early lineage specification and gastrulation to investigate the role of BMP4 signaling in the phenotypic manifestation of aneuploidy (Aim 1). To further investigate the elimination and allocation of aneuploidy, I will construct mosaic marmoset embryos to probe aneuploidy cell fate and behaviors during pre-and post-implantation embryonic development in vitro (Aim 2). Since my preliminary data indicates a higher tolerance of aneuploidy in the extraembryonic lineages, during the independent phase of the award period, I propose to analyze the gene expression profile of aneuploidy in the marmoset placenta to understand the effects of aneuploidy on the cellular physiology of extraembryonic tissue. In addition, during this phase, I will construct a placental/trophoblast organoid from cj-ESCs to further dissect the behaviors of aneuploidy in different placental lineages (Aim 3). Together, the proposed research will present a comprehensive model for studying a previously uncharacterized mechanism underlying the elimination of aneuploidy during embryogenesis, paving the way for translational applications to assisted reproductive technologies. The proposed project will also serve as a platform for me to obtain training and scientific expertise in molecular and developmental biology, animal reproductive sciences, and computational genomics which will contribute significantly to my career development as an independent investigator in the field of reproductive biology.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The goal of this innovative project is to bring together multidisciplinary expertise to dissect the functional, structural, and dynamic nature of interactions between the HIV-1 envelope (Env) cytoplasmic tail (CT), the underlying Gag matrix (MA) lattice, and the lipid containing membrane in the context of native virions. We will utilize state-of-the-art cryo-electron tomography (cryo-ET), molecular dynamics (MD) simulations, modeling, and in vitro studies to produce a comprehensive rendition of how interactions between these key determinants of assembly and infectivity are orchestrated. Our studies are based on recent cryo-ET data in which the positioning of Env CT and MA interactions differed from previous models. In Aim 1, we will harness the power of cryo-ET to advance the resolution of Env CT-MA interactions to facilitate further interrogation of the mechanics of Env CT- MA-membrane interactions. In Aim 2, we will generate atomistic models of Env CT-MA in the context of the viral membrane using high resolution structures and cryo-ET density, and strategic mutations and truncations of Env CT and MA. In Aim 3, we will exploit conserved structural features to design novel antiviral agents comprised of membrane-permeable cyclic peptide inhibitors. These Aims are supported by comprehensive biochemical and virologic approaches and will use a well characterized clade C transmitted/founder (T/F) HIV-1 infectious molecular clone that is fully representative of global isolates and presents fully matched Env and Gag components, which distinguishes our work. Our studies will reveal in unprecedented detail how Env CT, MA, and membrane lipids engage one another, with all components in near native context. These powerful approaches will produce an integrated model, including flexible regions that are difficult to resolve, to bring the complete Env- MA-membrane machinery to life.

NIH Research Projects · FY 2024 · 2023-08

Abstract This proposal is in response to NIH FOA (PAR-22-126), which “supports exploratory research leading to the development of innovative technologies for biomedical research...” This R21 project aims to harness the high sensitivity and resolving power of optical microscopy and asymmetric bipolar microelectrode arrays to develop a low-cost, highly sensitive bipolar electrochemical array platform for detecting and counting individual biological target species. Many biologically or clinically relevant species, such as certain cancer biomarkers, present at very low concentrations, sometimes down to a few copies. These species have been difficult to detect and quantify with existing bioanalytical methods due to their insufficient sensitivity, selectivity, and response speed. To address this challenge, we propose to develop a bipolar electrochemical single entity bioanalyzer, which will allow us to analyze individual biological species, such as single viruses and circulating tumor exosomes. The success of this project builds upon the strong expertise of the PI’s group in bipolar electrochemical arrays, microfabricated sensors, and single entity electroanalysis and has three specific aims. Aim 1 builds the asymmetric bipolar microelectrode arrays containing 250,000 electrodes and characterizes and optimizes them for single entity analysis. Aim 2 will synthesize and characterize Janus Pt/silica nanoparticles (NPs) and use them to label analyte species pre-concentrated onto magnetic microbeads. Aim 3 will build an integrated analytical platform by combining the bipolar microelectrode array with optical microscopy, characterize and optimize its analytical performance for single entity bioanalysis of pseudovirus particles and exosomes. The proposed analytical platform is innovative and powerful for single entity bioanalysis due to the use of an optical signal to amplify and read a small electrochemical signal. The use of a large array of 250,000 bipolar microelectrodes and magnetic beads for pre-concentration further enhances the sensitivity, selectivity, and throughput. Future work will develop a standalone benchtop analytical instrument, which will be useful for their general use in research and diagnosis involving single entity bioanalysis.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Melioidosis is an often-fatal infection caused by inhalation, inoculation, or ingestion of the Gram-negative facultative intracellular pathogen and Tier 1 select agent Burkholderia pseudomallei (Bps). Bps has recently been isolated from the soil in the southern United States. Worldwide, 165,000 cases of melioidosis are estimated to occur each year; 85,000 (52%) of these patients die. Pneumonia is present in over 50% of melioidosis cases and more than doubles the risk of death. Yet, to develop novel, targeted therapeutics necessitates a deeper understanding of pulmonary and systemic mechanisms of host defense. Our team combines expertise in human and experimental melioidosis, pulmonary host defense, sepsis, and bioinformatics. We have developed a robust murine model of Bps pneumonia displaying mild and severe disease phenotypes. In parallel we have performed unbiased multi-omics analyses on a large prospective cohort of hospitalized patients with infection in NE Thailand with the goal of classifying melioidosis cases and understanding the host response to Bps. We identified distinct transcriptional and metabolomic profiles associated with melioidosis compared to other infected patients, and have built robust classifiers in each omics domain to predict death in human melioidosis. However, to comprehensively investigate mechanistic underpinnings requires a tractable experimental model with sufficient comparability to human disease. Moreover, to study the lethality of respiratory melioidosis requires sampling of lung tissue. We hypothesize that applying a comparative multi-omic approach to mice and humans with respiratory melioidosis will both a) yield critical insights into the pulmonary host defense mechanisms that fail to contain the infection and contribute to severe outcomes and b) establish comparability of the experimental murine model with human infection. We submit the following specific aims: 1). Define the temporal trajectory of multi-omic features of systemic host defense in murine respiratory melioidosis and identify perturbations representing success or failure of host defense. 2) Define lung cell-specific transcriptomic changes in murine respiratory melioidosis and identify signals that are associated with success or failure of pulmonary host defense. 3) Identify shared multi-omic signatures between murine and human respiratory melioidosis. The results of these studies will generate a rich compendium of data about the systemic and pulmonary host response to murine respiratory melioidosis and provide novel and comprehensive insights into the heterogeneity and key biological pathways underlying failed host response phenotypes of melioidosis pneumonia. Intersecting these findings with existing human melioidosis data will help to define clinically relevant targets for further investigation while simultaneously providing essential information about advantages and limitations of the animal model in recapitulating human infection at the multi-omic level.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Apical surface interactions (ASIs) arising between cells in opposing three dimensional architectures are relatively common in tissue structures, including vessels and tubes in a variety of different organs and stages, but little is known about the function or mechanisms of these interactions. The goal of this proposal is to illuminate the role of ASIs in tissue architecture and responses as an under-explored dimension of cell-cell interactions. Based on our data and the literature, we hypothesize that close-range ASIs (< 1 µm) are governed by electrostatic charge interactions between membrane glycoproteins, while long-range ASIs (1-20 µm) function through primary cilia which extend up from the cell surface to organize signaling pathways. To develop accurate methodologies to measure and characterize the forces arising between whole sheets of cells with geometrical separation, we have designed a novel method called Bilayer Intermolecular Force Microscopy (BIFM) to induce and measure ASIs between two opposite surfaces. BIFM will be applied to measure the force generated between two cell layers as they approach each other from opposite sides. For close-range ASIs, we predict that chemicals affecting electrostatic charge interactions will modulate force-response. Cell sheets with knockout mutations in PODXL, encoding an apical sialomucin (podocalyxin) with proposed anti-adhesive properties, will exhibit lower resistance force in proportion to reduced electrostatic charge repulsion. Antibodies targeting podocalyxin, in therapeutic development, will also be assessed. For long-range ASIs, we will determine the role of primary cilia, antenna-like organelles with sensory and signaling functions. Using our BIFM device, we will induce ciliary ASIs and assess their effects on signaling. As a negative control, we will employ cell lines that we have genetically engineered to ablate primary cilia (KIF3A-/- or KIF3B-/-). We will furthermore modify our device to enable microfluidic flow to perfuse between two sheets of cells within the BIFM at adjustable speed, to assess flow start/stop in a physiological context, and monitored for changes in signaling activity. These studies will reveal how cilia serve as ASI sensors. To validate findings in vivo, we will analyze physiological tissue structures exhibiting a range of apical surface interactions, focusing on arborized networks such as ductal trees and blood vessel plexi. Expression of podocalyxin or cilia will be correlated with geometric properties and differential gene expression patterns. In summary, our project will provide novel conceptual and technical advances for understanding ASIs as a novel dimension in tissue architecture and physiology. Cross-cutting impact includes (1) revealing functional roles for both close-range and long-range ASIs; (2) establishing a novel biophysical device to measure interactions between cell sheets; and (3) testing mechanisms of cell adhesion and signaling. Our prior experience in modeling and developing biophysical tools positions us well to succeed. Collectively these activities will establish an innovative new area for future investigation, with fundamental importance.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Immune systems across kingdoms of life recognize pathogen-associated molecules through germline-encoded innate immune receptors. Receptor repertoires in plants have evolved to detect an especially diverse set of ligands due to massive expansion of the receptor kinase gene family with specialized ligand recognition functions. Pairing receptor sequence diversity with specific recognition functions across 100 million RK genes (350,000 plant species * 500 receptors per genome) is a grand challenge in plant molecular biology. It also presents the opportunity to develop a new class of protein-based sensors for biotechnology. The Steinbrenner lab aims to characterize and deploy this vast plant immunodiversity for ligand-induced modulation of engineered signaling pathways. First, we will define the full ligand space that is monitored by plant receptors by focusing on the large subfamily of leucine-rich repeat receptor kinases (termed receptors here) which bind small peptide epitopes to initiate immune signaling. We will combine evolution- and structure-guided approaches to decode the basis of receptor:ligand specificity, including an extensive phylogenomic analysis, peptide variant libraries, and ancestral sequence reconstruction. We hypothesize that transitions in ligand specificity are marked by amino acid substitutions in predicted ligand binding sites among ancestral receptor genes. For “orphan” receptors lacking defined functions, we will conduct a genomic screen using synthetic DNA libraries encoding candidate pathogen epitopes using both plant and yeast models as reporters for receptor activation. We hypothesize that most receptors involved in plant innate immunity will be activated by specific pathogen-derived peptide sequences. Combined, these approaches will provide basic insights into receptor:ligand specificity as well as a toolkit of extracellular sensor domains responsive to specific peptide agonists. Second, we will leverage the unique network architecture of plant immune networks to engineer synthetic signaling pathways that do not interfere with endogenous animal signaling pathways. The plant receptors studied here signal through heterodimerization with a common co-receptor called BAK1. Co-receptor activation culminates in phosphorylation of substrates based on defined phosphocode motifs. We are currently engineering the human inflammation signaling pathway to accept orthogonal input from plant receptors by incorporating plant kinase substrates into specific, phosphoregulated signaling factors. In parallel, we will use plant receptor:co-receptor heterodimerization as a platform to scaffold endogenous human immune signaling domains from Toll-like receptors. We hypothesize that engineered pathways will allow modular tuning by diverse peptide ligands, providing an alternative to current immunoglobulin or GPCR-based synthetic tools. In summary our lab is poised to deploy tools for receptor de-orphanization and signaling pathway engineering to leverage the immense diversity driven by plant-pathogen co-evolution. (30 lines)

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Autism spectrum disorder (ASD) is a complex disorder of brain development characterized by difficulties in social interaction and communication and the presence of restricted and repetitive behaviors and is often accompanied by disruptions of sensory and perceptual processing. Altered sensory and perceptual processing leads to a range of individual experiences including heightened and dampened sensory reactions that can profoundly affect quality of life. Despite the prominence of sensory and perceptual symptoms in ASD there is no unifying explanation for their etiology. The central premise of this proposal is that the sensory and perceptual processing differences that occur in ASD derive from differences in attention. Specifically, we will test the hypothesis that spatial- and feature-based attention is more narrowly focused and that rapid oscillations of attention occur at a slower rate in people with ASD. Our proposal employs computational models of visual cortical responses in combination with psychophysical and brain imaging measures of neural responses. Overall, our results will significantly advance understanding of the neural and computational basis of sensory and perceptual changes in ASD.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT COVID-19 vaccination is safe, immunogenic, and durable in individuals with treated and virally suppressed HIV infection but is less immunogenic in immunosuppressed individuals and those with unsuppressed HIV infection. The rollout of COVID-19 vaccines is still limited in countries with high HIV prevalence and low access to antiretroviral therapy (ART), necessary to suppress HIV viral replication and reduce HIV-associated comorbidities. Thus, studying COVID-19 vaccination in immunosuppressed and untreated HIV populations is needed. We generated an Alphavirus-derived replicon RNA (repRNA) SARS-CoV-2 vaccine candidate, repRNA- CoV2S, encoding the SARS-CoV-2 spike protein and delivered by a novel Lipid InOrganic Nanoparticle (LION), a cationic nanoemulsion (CNE). This vaccine platform generates robust and durable protective immunity against SARS-CoV-2 infection in mice and nonhuman primates. Preliminary studies indicate this vaccine is immunogenic in non-human primates with HIV-induced immunosuppression and those with B-cell deficiencies, demonstrating that a repRNA-CoV2S vaccine could be employed to induce strong immunity against COVID-19 in immunosuppressed individuals living with HIV. Here, in a highly relevant pre-clinical SIV macaque model for HIV infection, we will test a 2nd generation COVID-19 vaccine, repRNA- Omicron, that 1) encodes the SARS-CoV-2 Omicron S protein and 2) is comprised of a novel chimeric immunogen (SHARP) which focuses immune responses to the receptor binding domain (RBD) and promotes neutralizing antibodies. We will evaluate the immunogenicity and durability of repRNA-Omicron during untreated SIV-associated immunosuppression and examine the role of SIV-induced immune activation and exhaustion on vaccine immune memory. Furthermore, our studies will aim to understand the mechanisms driving humoral memory by the novel repRNA/LION vaccine platform. If successful, this work will further contribute to understanding the mechanisms driving SARS-CoV-2 vaccine breakthrough infections and reinfections in people living with HIV and will inform improved treatment and vaccine strategies for people living with HIV and other immunocompromised individuals.

NIH Research Projects · FY 2025 · 2023-08

Behavioral strategies for obesity treatment reliably result in weight loss and health benefits. However, weight loss typically plateaus at ~7–10% of total body weight (a plateau phase) after which weight regain is common, limiting the ability of most intervention strategies to modify obesity-related disease risk in a sustained manner. Overcoming these limitations requires advancing understanding of metabolic, appetitive, and neurological factors that counteract—and eventually halt—weight loss. Existing literature shows that weight loss evokes appetitive adaptations that include increased hunger and decreased satiety. The investigative team’s preliminary data add evidence that an impaired satiety response in brain regions controlling reward and motivation follows weight loss. Documented metabolic responses to weight loss include declines in total body and resting energy expenditure as well as increased metabolic efficiency in muscle and adipose tissue. We now present preliminary data using novel measures of mitochondrial metabolism indicating that a weight loss plateau is characterized by reductions in mitochondrial metabolic capacity in peripheral blood mononuclear cells. The proposed research aims to 1) test if an involuntary weight loss plateau is distinguished from other phases of weight loss by alterations in cellular bioenergetics across multiple tissue types including circulating blood cells, muscle, and adipose tissue; 2) test for neurophysiologic changes consistent with reduced satiety at an involuntary weight loss plateau and assess their relation to changes in metabolism; and 3) investigate predictors of a weight loss plateau using data collected at baseline and in real time during behavioral weight loss. To address these three Specific Aims, the multidisciplinary study team proposes a basic experimental study in humans using a remotely delivered group behavioral weight loss intervention to evoke and study plateau physiology. Real-time remote monitoring of weight loss trajectories will be used to individualize assessments for 130 adults with overweight or obesity to occur at baseline, during active weight loss, and at a weight loss plateau. Intensive phenotyping will include serial behavioral assessments and tasks, fasting and postprandial blood sampling, functional and structural brain magnetic resonance imaging, indirect calorimetry, and adipose and muscle biopsies to investigate the contribution of mitochondrial function and other pathways to this weight loss plateau. Future work will probe underlying mechanisms that trigger plateau physiology in a tissue-specific manner, using identified predictors to target individuals and timepoints. In sum, the proposed research uses an innovative, individualized approach to improve understanding of the physiological factors that make additional weight loss difficult once a weight loss plateau is reached. The long-term goal is to develop new approaches for extending weight loss and improving weight maintenance.

NIH Research Projects · FY 2026 · 2023-08

Malformations of the oral cavity, which include dental anomalies (hypodontia, hyperdontia), cleft lip and or cleft plate (orofacial cleft, OFC), and salivary gland anomalies (ectopic or aplasia), are among the most common birth defects in the US. The design of preventative therapies for these disorders will require a precise understanding of the transcriptional regulatory networks (TRNs) governing development of the relevant tissues. Studies in model organisms have been invaluable, for instance revealing that mesenchyme in these structures derives from neural crest and epithelia in them derives largely from oral ectoderm. However, it is unclear how these TRNs are deployed over developmental time and within spatial domains of the mouth. Moreover, aspects of these TRNs are likely to be human specific, for instance those regulating the development of secondary dentition, which does not occur in rodents. Finally, all of the disorders mentioned above have a genetic basis, in none has all of the heritable risk been fully explained. Knowledge of the TRNs in human tissue is the surest way to find candidate genes to harbor such risk. Recent advances in our spatial transcriptomics (sciSpace), and access to donated human fetal tissue, permit these important questions to be addressed in a precise spatio-temporal manner. Here we propose, in Aim 1, to conduct sciSpace over the entire human face at four critical developmental timepoints (7-9, 10-12, 13-15, and 16-18 weeks post conception). We will then focus on the secondary palate and the genetic underpinnings of OFC. We will use computational algorithms to deduce the membership and regulatory hierarchy of TRNs regulating differentiation of distinct domains of palate epithelium and palate mesenchyme; top ranking members of these TRNs are strong candidates to harbor the missing heritability for OFC. In Aim 2, we will use the results of the first aim to develop protocols for converting induced pluripotent stem cells (iPSC) into palate epithelium and mesenchymal cells. We will engineer iPSC with 2 coding and 2 non-coding variants associated with OFC, differentiate the engineered iPSC into palate cell types, and subject the differentiated cells to single cell RNA-seq. This will reveal the specific cell types, and the step in their development, that is affected by the variants, illuminating the pathogenic mechanisms of OFC. These experiments will identify strong candidates for the missing heritability for orofacial cleft, improve functional tests of DNA variants associated with it, and provide the datasets to similarly analyze other inherited craniofacial disorders.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT The components of chronic kidney disease-mineral and bone disorder (CKD-MBD) include calcitriol deficiency, hyperphosphatemia, and excesses of fibroblast growth factor-23 (FGF-23) and parathyroid hormone (PTH), all of which consistently and strongly associate with cardiovascular disease (CVD) in patients with kidney failure on dialysis. Vascular calcifications are present in >80% of these patients and may be a mechanism through which CKD-MBD increases CVD risk. Intravenously-administered (IV) vitamin D receptor agonists (VDRA) are routinely used as first-line therapy for CKD-MBD in kidney failure, supported by experimental and epidemiological evidence that suggest VDRAs improve CVD and survival. However, the long-term risks and benefits of the current treatment approach, which encourages escalating VDRA doses to reduce PTH concentrations towards the normal range, have never been tested by clinical outcomes trials. This approach often leads to high VDRA doses which incur several risks: (1) a dose-dependent rise in FGF-23, a major risk factor for CVD that is associated with monotonically increasing risk of death in kidney failure; (2) hypercalcemia, a key promoter of vascular calcification; (3) low turnover bone disease, which drive calcium and phosphate into the vasculature because they cannot deposit into bone; (4) held doses of VDRA as a result of (2) and (3), which interrupts therapy and deprives patients of its likely health benefits. The goal of this project is to evaluate the safety, efficacy, and feasibility of a low fixed-dose vitamin D strategy to treat CKD-MBD in kidney failure to move the field towards identifying an optimal strategy that balances the risks and benefits of VDRA use. The two Aims proposed will leverage a unique partnership with Northwest Kidney Centers (NKC), the largest provider of dialysis in Seattle, WA. Aim 1 is a 12-month pragmatic randomized clinical trial of 90 NKC patients on hemodialysis that will compare the effects of a low fixed-dose oral calcitriol strategy (intervention) with the current usual care of PTH-titrated IV VDRAs (control) on a comprehensive panel of biomarkers assessing mineral metabolism, bone turnover, and serum calcification propensity, as well as measures of feasibility and acceptability. Aim 2 is a complementary retrospective cohort study using the NKC electronic health records database that will test associations between mean VDRA dose and variability in VDRA doses with CVD event and hospitalization rates. These Aims test whether a low fixed- dose vitamin D strategy has causal effects that improve intermediate outcomes (Aim 1) and associate with improved clinical outcomes (Aim 2), building a foundation for future large-scale clinical outcomes trials. Integrated with this research agenda is a career development plan designed to position me to become a successful independent investigator in the field of disordered mineral metabolism in CKD. This mentored approach to training includes regular meetings with co-mentors who are established leaders in multi- disciplinary fields, structured didactics, attendance at local and national conferences, and experiential training.

NIH Research Projects · FY 2025 · 2023-08

Project Summary / Abstract As scientific practice evolves in response to exponential increases in data volume, availability of rapid computational statistics, and the so-called “reproducibility crisis”, researchers are developing new methods for collecting and analyze data in rigorous and responsible fashion. The aim of this proposal is to develop three training units for researchers in the neurosciences that will bring learners up to speed on these developments, improving the rigor and quality of their scientific research by deepening their understanding of the role of statistics in biomedical research. Each unit, developed iteratively in a cycle of testing, evaluation, and revision will be designed for online or classroom use suitable for diverse learning styles. Units will comprise a series of short video segments and interactive exercises that lead learners in a process of guided discovery and self-reflection as they move toward a set of well-specified learning goals. Our units will teach neuroscientists to avoid common pitfalls in designing and analyzing data. In the first unit, we address a set of easy-to-make mistakes wherein a researcher alters her plans midway through the process of data analysis. The practice of HARKing—hypothesizing after the results are known—involves testing hypotheses that are formulated after viewing research outcomes. Outcome switching occurs when a study yields negative results based on the pre- specified outcome measures, but other measures are reported instead. The Garden of Forking Paths refers to the latitude that researchers have in shaping a statistical analysis as they go along. The second unit addresses the problem of publication bias, which arises when authors and journals prefer to publish positive results in favor of negative one, and can lead researchers to reduplicate efforts or draw mistaken inferences from published data. The aim of this unit is to make students aware of problem, teach them how to adjust when reading the literature, and suggest strategies for avoiding publication bias in their own work. The third unit will train students how to figure out whether when a statistical analysis rigorous and reliable. Students will learn how to ask “Are the data appropriate what we want to learn?” “Is the choice of statistical test reasonable?” “Are the inferences supported by the evidence?” By developing this set of units, to be included in a broader neuroscience curriculum, we can train a new generation of biomedical scientists who are well-equipped to work with the vast datasets that are becoming available thanks to new research tools and technologies. These scientists will be able to work more accurately, make new discoveries more efficiently, and advance our knowledge in the health and life sciences at a faster rate than ever before.

NIH Research Projects · FY 2025 · 2023-08

Through a coordinated program of research, mentoring, coursework, and partnerships, we will train a cohort of students and postdoctoral fellows who will become leaders in interdisciplinary, computational research that advances central questions in the cellular and circuit basis of cognition. We will foster a community of trainees equipped with a skillset that unites fundamentals of neurobiology, mathematics, scientific computing, and equips them to engage in real dialogue and innovation across fields using collaborative and reproducible approaches. The projects undertaken by trainees in this research program will directly address key questions relevant to the NIH’s mission including: the study and advancement of theoretical and computational models that describe how information processing arises from dynamics of neurons and their networks, the study and advancement of the analysis of multi-modal, highdimensional neural data, and the investigation of complex neural circuit dynamics and function in health and disease. Each trainee will receive joint research mentorship between a leading computational and experimental neuroscientist at UW, thus gaining the skills and experience to ensure that their computational work occurs within a virtuous cycle that allows theory to further drive novel experiments. Our program will build bridges across departments and schools, and connect trainees with emerging opportunities within and beyond academia. We will admit up to three students and two new postdoctoral fellows each year for a two-year period, training up to 13 students and 9 new postdocs in total over the course of the program. Trainees will have backgrounds in the computational, biological, and mathematical sciences.

NIH Research Projects · FY 2025 · 2023-08

Project summary Chronic pain is a highly prevalent and impactful condition. It persists past the time expected for normal tissue healing from an acutely painful event, suggesting plasticity is occurring within the central nervous system to perpetuate the pain experience. Furthermore, there are several conditions known to occur in the absence of any tissue injury, such as fibromyalgia and tension-type headaches. Collectively, these conditions are referred to as nociplastic pain syndromes. The broad goal of this proposal is to identify regions within the central nervous system that exhibit plasticity in response to chronic pain. To model chronic pain, I will employ the partial sciatic nerve ligation (pSNL). I will focus my investigation on the parabrachial nucleus (PBN), a hub for the relay of aversive sensory information. My preliminary data suggests that the population of neurons expressing Calca within the PBN exhibits an increase in neuronal activity acutely following chronic pain induction, however this activity does not persist through the entire chronic pain experience. Additionally, silencing the PBN Calca population prevents pSNL driven pain and chronically stimulating this population produces pain that persists beyond stimulus cessation. Together this information informs my central hypothesis that PBN Calca neurons are involved in the initiation and perpetuation of chronic pain. To test this hypothesis, I will both manipulate Calca neurons and observe their activity during chronic pain. The experiments detailed in Aim 1 will probe whether PBN Calca neurons are involved in maintaining the chronic pain experience by assessing whether transient inhibition of PBN Calca neurons will ameliorate pSNL driven pain. Aim 2 will explore how much and what type of stimulation is required for the manifestation of persistent pain driven by PBN Calca neurons. This aim will also determine whether expression of the Calca gene is required for the manifestation of chronic pain. Finally, in Aim 3 I will observe the activity of PBN Calca neurons preceding and following the induction of chronic pain via calcium imaging. This will reveal when PBN Calca neurons exhibit increased activity during a chronic pain experience. Collectively, these experiments will delineate the role Calca neurons play in the manifestation and perpetuation of chronic pain. This work will further our understanding of how chronic pain conditions arise and persist in the absence of a driving injury and may suggest a target for novel pain therapies.