University Of Washington

NIH Research Projects · FY 2026 · 2021-09

SUMMARY / ABSTRACT A major fraction of heritability for common diseases, as well as for the penetrance and expressivity of rare diseases, partitions to distal regulatory elements in the human genome, overwhelmingly cell type-specific enhancers. However, a rate-limiting challenge for the field has been how to identify the specific variants, elements and regulated genes that mediate these effects on disease liability. Towards the overall goals of the Impact of Genomic Variation on Function (IGVF) Consortium, we propose to test over one million human regulatory elements or variants for their functional effects on transcriptional regulation, as well as to query over 100,000 distal regulatory elements for the gene(s) that they regulate. A first theme of our proposal is the diversity of multiplex technologies that we will employ to these ends, including massively parallel reporter assays (MPRAs), crisprQTL, saturation genome editing, multiplex prime editing and single cell combinatorial indexing, many of which we pioneered. A second theme is a focus on dynamic cellular systems that enable a given library of variants and/or elements to be tested across a broad range of cell types and states within a single experiment; these will include ESC-derived neuronal progenitors, cardiomyocytes, embryoid bodies, gastruloids and organoids, and in select cases, mice. A third theme involves leveraging our experience (e.g. CADD, a widely used, genome-wide catalog of variant effect predictions) to support the overarching goals of IGVF. Specifically, we envision using functional measurements generated by us and others to produce well-calibrated predictions of enhancer activity and variant effects that are continuous along the branching trajectories that comprise human development. Our specific aims are as follows: (1) To perform massively parallel validation and functional characterization of candidate human enhancers in a broad range of cell type contexts. (2) To perform massively parallel characterization of human genetic variants with potential roles in human disease. (3) To contribute to a comprehensive variant-element-phenotype catalog while taking a leadership role in synergistic interactions within IGVF, in the dissemination of methods, data and predictions, and in the overarching goals of the consortium.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Organ development and function requires that neurons establish precise cellular interactions with tissue­resident support cells. For example, touch­sensing somatosensory neurons project peripheral axons to the skin, where they interact with specialized skin cell types. These specialized skin cells regulate axon development and modu­ late neuronal responses to cutaneous stimuli. Reciprocally, somatosensory neurons influence skin homeostasis. Although the anatomy of vertebrate skin is well described, little is known about the dynamic process of sensory specialization of the skin, partly because most studies have focused on mammalian embryos, which has limited access to live­imaging. The external development and the availability of unique transgenic tools make zebrafish an ideal model for study­ ing the dynamics of neuron and tissue maturation. This proposal investigates the development of a novel popula­ tion of specialized sensory cells that we identified in the zebrafish epidermis. Preliminary cellular, molecular and developmental analyses suggest that these zebrafish epidermal cells are the equivalent of mammalian Merkel cells, specialized mechanosensory cells that detect touch. The experiments proposed here investigate how de­ velopment of these specialized epidermal cells is coordinated with skin and nervous system maturation. In Aim 1, we will use live­imaging and genetic manipulation to characterize how sensory cell addition occurs during skin growth. Aim 2 investigates the establishment of interactions between axons and epidermal cells and how neurons promote skin specialization. Finally, in Aim 3, we will use in vivo photoconversion, lineage tracing and molecular techniques to track the trajectory of skin­resident stem cells as they differentiate into sensory cells. Collectively, these studies will provide mechanistic insights into organ specialization during development, interactions between peripheral axons and their target tissues, and potentially point to the origins of touch­sensing disorders.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY / ABSTRACT Background. Chronic neck pain is one of leading determinants of disability worldwide, a major contributor to the current opioid epidemic, and the cause of enormous health care expenditures. We currently do not understand which molecular mechanisms in the diseased tissues of the neck generate the painful signals that cause this disease burden. This gap in knowledge impedes development of effective, mechanism-specific therapeutics. Aim. We plan to use RNA sequencing on tissues taken from chronic neck pain patients undergoing surgery and then use computational biology and patient phenotyping techniques to gain better insight into molecular drivers that cause chronic neck pain. Approach. We will recruit patients undergoing surgery for chronic neck pain stemming from the atlanto-axial joint. This joint is located between the 1st and 2nd cervical vertebrae on both sides of the spine and is a common site of arthritis. We will also recruit patients undergoing the same surgery for acute pain due to fractures. During surgery, we will sample tissues from the joint and the dorsal root ganglion (DRG), in which the nerves supplying the joint are located. These tissues would be removed during surgery in chronic pain patients, independent of the study. For acute pain patients we will use our existing dorsal root ganglion database of people with no pain. We will perform RNA sequencing on samples from both sides, in order to compare tissues from painful and non- painful side of the same individual. We will record clinical pain characteristics, measure pain sensitivity and assess nerve function using pinprick, pressure and cold stimuli. We will also use computational biology methods to assess how diseased tissue interacts with the nervous system to drive pain. These methods will allow us to identify targets that are related to pain outcomes. Aim 1: To determine the RNA expression profile of the joint tissues and DRG from acute and chronic pain patients to determine genes that are differentially expressed in chronic neck pain tissues. Aim 2: To use computational biology techniques to determine how pathological molecules originating in the joint tissues interact with specific receptors on neurons of the DRG to generate the pain signal. Aim 3: To determine the association of RNA sequencing findings in the joint and nerves with clinical manifestations of neck pain, pain sensitivity and nerve function. This will provide information on which molecular changes at the joint and nerves are of specific importance to pain. Impact. The project will enable unprecedented identification of molecular pathways specifically associated with chronic neck pain. Our work will create a unique map of molecular targets for the future treatment of chronic neck pain based entirely on human molecular neuroscience.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Individuals with Down syndrome (DS) (I-DS) are at a heightened risk of obstructive sleep apnea (OSA). Despite the well-established association of OSA with cardiovascular (CV) risks in the general population, whether such an association exists in I-DS is unknown. Despite the high rates of risk factors such as OSA and obesity, there is a suggestion of a lower risk of hypertension and atherosclerotic CV disease (CVD) in I-DS. In this regard, whether increased CV risks are present in I-DS with a high burden of OSA is uncertain. Clarifying the CV impact of OSA may unveil one mechanism by which I-DS are protected from these CV conditions. To address this, we propose a time-sensitive and cost-efficient supplementary study to the NHLBI- sponsored “Sleep Apnea-Specific Nocturnal BP Surge to Determine CV Risks and Therapeutic Benefits in Patients with OSA study R01HL158765” (SASBP study, Project period: 2021-2026). The scope of this project is within the scope of the parent study, which aims to identify blood pressure and other physiological responses to OSA as a way to explain the mediating mechanism of OSA and subclinical CV disease. This study would allow us to assemble a cohort of I-DS across the lifespan to perform deep phenotyping and study co-existing conditions (INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndromE [INCLUDE] component 2) and to conduct clinical trials research inclusive of I-DS (INCLUDE component 3). We will recruit I-DS and control groups with OSA to achieve the following Aims. Aim 1 is to test the hypothesis that immediate CV consequences of OSA events are attenuated in I-DS compared to those without DS. We will compare immediate CV consequences (changes in heart rate and estimated blood pressure) with those of age-, sex-, BMI- and OSA severity- matched (n=15 each group). We will also compare the severity of OSA-related hypoxemia (hypoxic burden) and arousal tendency. Aim 2 is to test the hypothesis that in the presence of OSA, markers of subclinical CV disease are attenuated in I-DS than those without DS. We will compare daytime and nocturnal BP, arterial stiffness, and global longitudinal strain by echocardiography between the two groups. We will also examine the association between the degree of immediate CV consequences and the subclinical CV disease. This represents an inclusive mechanistic study focusing on I-DS with OSA, a major comorbid condition in this special population. The study's findings will facilitate risk stratification of I-DS with OSA and provide pivotal preliminary data to design future clinical trials to test the efficacy of OSA therapy in health outcomes of I-DS.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY An individuals’ biological sex significantly affects their ability to repair and regenerate tissue. A clear example of this is the reduced ability for women to heal and regenerate new, healthy tissue after menopause, which results from a significant loss of sex hormone signaling. This reduction in hormone levels disproportionately enhances the risk for many degenerative diseases including osteoporosis, osteoarthritis, cardiovascular disease, and degenerative brain diseases in which the rate of tissue breakdown exceeds the rate of tissue repair. While it is known that several factors contribute to sex differences in tissue repair including biomechanics, nutrition, physical activity level and sex hormones, the interplay between these parameters is not well understood. Specifically, it is unknown how the native sex differences in tissue structure and the resulting differences in mechanical function dictate cell phenotype and behavior and how this effect interacts with estrogen signaling to overall control tissue repair. Thus, a fundamental, mechanistic understanding of how a cell responds to the spatial and mechanical cues of its environment while mediating estrogen signaling is critical to understand why sex differences occur in tissue repair and homeostasis and for future patient-centered repair and regeneration strategies. The overall goal of our research program aims to develop biomaterial tools to interrogate sex differences in tissue repair and homeostasis. Theme 1: Do male and female MSCs respond to spatial and mechanical properties of the cell microenvironment differently? There is evidence in many tissues that extracellular matrix structure, organization, and resulting function differs between age-matched males and females. However, there are no studies showing how this affects cell response. Biomaterials engineered to mimic both the fibrous properties of structural collagens and the viscoelastic properties of proteoglycans in the native extracellular matrix will be used to assess sex differences in cell response to controlled changes in matrix properties. Theme 2: How does estrogen presentation to the cell affect downstream transcription and behavior? While estrogen is known to play a role on cell processes, these results are dependent on the concentration and the temporal presentation of estrogen to the cell. To address this limitation, we will use concentration gradient generator microchips to quickly and accurately determine the effect of estrogen concentration and timing on cell transcriptional activity. Theme 3: Can we engineer biomaterial systems to control release and presentation of estrogen to the cells? Release rates in a range of hours to months will be controlled by modulating diffusion out of the biomaterials via material chemistry and architecture. The ability to control the rate of release and localize to a specific tissue in the body is critical to promote the estrogen effects at the site while reducing the negative and potentially deadly off-target effects. Results from these studies will provide future avenues of study to understand how estrogen and the cell microenvironment drive sex differences in stem cell behavior which is critical for tissue repair and homeostasis in both women and men.

NIH Research Projects · FY 2024 · 2021-09

IMPACT CENTER OVERALL SUMMARY: Optimizing Evidence-Based Practice Implementation for Clinical Impact The goal of the IMPACT Center is to accelerate the impact of evidence-based practices (EBPs) for youth receiving mental healthcare in low-resourced community-based settings. These settings, which include commu- nity mental health centers and schools, provide mental healthcare to most low-income, ethnically diverse youth who receive services. In these settings, EBPs are not implemented widely or with fidelity, and youth have high unmet mental health needs, leading to adverse outcomes. In IMPACT, we focus on optimizing EBP implemen- tation in low-resourced community settings for four of the most common youth mental health conditions: depres- sion, anxiety including posttraumatic stress, and behavioral conditions. To provide high-quality treatment for youth, community settings need support to optimize EBP implementation. The field of implementation science has tried to address this research-practice gap; however, a recent editorial call for more “practical implementation science” that generates solutions in partnership with the practice community leading to tools they can use inde- pendently for EBP implementation. In IMPACT, we will partner with stakeholders in developing, refining, employ- ing, and disseminating user-friendly methods and tools to accelerate the impact of EBPs for youth. IMPACT targets 3 implementation challenges: (I) Identify and prioritize determinants to EBP implementation; (II) Match implementation strategies to prioritized determinants; and (III) Test and optimize strategies according to practice partner goals (e.g., reach, preference, impact, efficiency, affordability). Our Methods Core is a trans- disciplinary team from clinical, community, and organizational psychology; information, computer, and imple- mentation sciences; anthropology; medicine; public health; and user-centered design. We will develop methods and toolkits for each challenge, co-designed and refined with practice partners for acceptability, feasibility, and appropriateness. Our partnership with the WA EBP Initiative is an ideal laboratory for developing new methods. The IMPACT Center, a Kaiser Permanente Washington Health Research Institute-University of Washington partnership, is codirected by Lewis and Dorsey. The Administrative Core will support stakeholder engagement, maximize Center learning, and foster synergy and integration across the Cores, exploratory projects, and pilot grant program. This Core will lead dissemination to the practice and scientific communities, training, and evalu- ation of IMPACT. The Methods Core will collaboratively develop new methods, support investigators and train- ees, and actively participate in outreach. The exploratory projects will employ and refine methods from IMPACT's three challenges, developing and testing practical implementation strategies to improve treatment quality and client outcomes via EBP implementation. Project 1 optimizes measurement-based care, a foundational frame- work to guide EBP delivery. Project 2 optimizes and tests a peer-led, frontline leader-focused strategy to optimize cognitive behavioral therapy (CBT) for four most common youth conditions. Project 3 increases reach and impact of an effective, multicomponent engagement strategy for clinicians in schools to use trauma-focused CBT.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Incidence of Alzheimer’s disease and related dementias (ADRD) is 40–100% higher among Black compared with White Americans. A key reason for this disparity may include residence in segregated, socially disadvan- taged, and polluted communities. Extant studies suggest that community socioeconomic deprivation is related to decreased brain volume, Alzheimer’s neuropathology, and poor cognitive function, and that fine particulate matter (PM2.5) may contribute to neurodegeneration. Early-life exposures may be particularly important, as early-life community-level disadvantage and ambient air pollution could disrupt the accumulation of cognitive reserve, reduce cognitive resilience, and dampen social trajectories. Community-level social factors and air pollution often co-occur; thus, comprehensive understanding of how these factors operate independently and synergistically requires rigorous evaluation of both. Moreover, identifying the extent to which these factors modify underlying genetic risk—APOE-ε4 genotype and ADRD polygenic risk scores—would inform under- standing of the etiology of ADRD. The overall objective of this application is to evaluate the effects of early-life community-level social and environmental factors on late-life ADRD and the extent to which these factors con- tribute to racial disparities on ADRD in a nationally representative sample. The central hypothesis is that early- life community-level social factors and ambient air pollution have independent and synergistic effects on late- life brain health and ADRD disparities. This project leverages the recently completed data linkage between the 1940 census and the national Health and Retirement Study (HRS) (n≈8,700). Participants in the HRS were an average age of 69 years at first memory assessment between 1995–1998, so HRS includes up to 23 years of longitudinal data on memory scores and dementia, and a subsample also has genotype information. The central hypothesis will be tested in four specific aims among Black and White HRS participants: (1) Investigate the effects of early-life community- level social factors on late-life cognitive health; (2) Examine the effects of early-life ambient air pollution expo- sure on late-life cognitive health; (3) Estimate synergistic effects of early-life community-level social factors and air pollution exposure on late-life cognitive health; and (4) Evaluate the extent to which early-life community- level social factors and exposure to ambient air pollution modify effects of ADRD genetic risk on late-life cogni- tive health. The proposed research is innovative because it assesses joint early-life social and environmental community-level exposures, including via novel air pollution metrics and late-1930s redlining, for ADRD. The proposed work is expected to advance the field by providing new policy-relevant evidence on potential strate- gies to prevent ADRD and eliminate ADRD disparities.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract: Macrophage polarization is involved in the progression of normal physiological and pathological processes. The near universal importance of macrophage has led to widescale assessment of the genomic and proteomic to classify their function. However, it is still a challenge to measure macrophage polarization in real time such as in the context of assessing the status of a biomaterial implant or the efficacy of a nanoparticle drug delivery system in a solid tumor. Our preliminary data show that bioluminescence microscopy is able to deliver the protein expression data valuable for macrophage polarization combined the cell tracking capability of traditional imaging system. This research program will address the current gap in real-time assessment of macrophage polarization by 1) further developing the technology to measure macrophage polarization dynamics using bioluminescence microscopy and 2) assessing the intrinsic and extracellular environmental stimuli that are most important for sensing macrophage polarization in a diseased environment context. To accomplish these goals, we will employ ex-vivo culture systems and live bioluminescence imaging. This work will identify novel strategies for measuring macrophage polarization and validate the importance of analyzing polarization in a disease context. Ultimately, this research will lead to new strategies for synthetic biology development, enhancement of diagnostic testing and development of new therapeutic targets. In addition, the knowledge and tools developed can be applied to other cells where polarization is paramount such as stem cell differentiation, chemoattraction, and cell metabolism.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY About a quarter of the human cerebral cortex is visual, with dozens of retinotopic maps and category selective regions. Localizing these areas is crucial to much of human visual neuroscience research and to clinical care. Area localization, such as delineating the boundaries of primary visual cortex, is a necessary step in studies that measure neural activity or anatomical properties because knowledge can only be aggregated across labs, groups, studies, and the lifespan if researchers can identify the same visual areas in each person. Moreover, brain area boundaries often serve as dependent measures themselves, for instance in studies that track the size of face or word-selective areas during development, or that link the surface area of a visual map to performance–e.g., V1 and acuity or hV4 and recognition in clutter. However, while dozens of functional visual areas have been identified on cortex, many are technically difficult to localize, and few have been carefully characterized across large populations of individuals. The field lacks validated computational methods to parcellate visual cortex into its component retinotopic maps and category-selective regions at the level of individual subjects, and it lacks a quantitative description of how anatomical map properties (size, tissue thickness, myelination, white matter inputs, etc) vary across large populations. The central aim of this proposal is to quantify how the components of the cortical visual system vary (and covary) in large populations of human subjects. We take two complementary approaches. One is to develop machine learning (ML) algorithms to delineate visual areas using various combinations of MRI inputs. The other is to establish descriptive models of the distribution, heritability, and patterns of co-variation of properties of visual cortical areas and their associated white matter tracts. Currently, accurately identifying visual system components in individuals remains challenging, hampering both scientific and clinical progress. The results of this project will greatly increase the ability of basic scientists and clinicians to accurately identify visual areas in individual humans and to quantify how individual variability impacts vision. This in turn furthers the neuroscientific understanding of vision and empowers translational research into the cortical origins of human visual disorders.

NIH Research Projects · FY 2025 · 2021-08

Summary: Upon cellular death, cells release damaged mitochondria, contributing to local inflammation. The overall aims of the present study are to investigate how mitochondria are cleared to avoid evoking an aberrant immune response, and to determine if autoantibodies targeting mitochondrial proteins have clinical utility in assessing development of thrombosis in patients with systemic lupus erythematosus (SLE). The premise of the study is that SLE patients have impaired clearance of mitochondria, promoting development of mitochondrial antibodies, inflammation and organ damage, including thrombosis. To investigate this we have three main aims. The first aim investigates how mitochondrial extrusion promotes autoimmunity towards mitochondrial protein antigens, attempting to define the exact target of novel main mitochondrial autoantibody, AMA-17, the processes involved in exposing the autoantigen in vitro, as well as potential therapeutic targets, e.g. mitochondrial ROS, involved in generating AMA-17 in vivo. The second aim investigates how anti-mitochondrial antibodies partake in thrombosis development. Using a large longitudinal SLE cohort (n=500), followed over 10 years, we will determine the capacity of AMA-17 to associate with and/or predict development of venous thrombosis. Affinity-purified AMA-17 antibodies will be tested for platelet activation and thrombus formation in vitro using flow cytometry, aggregometry and a cutting-edge model of engineered microvessels, as well as in vivo using a novel model of antibody-mediated venous thrombosis developed by Dr. Knight. Finally, in Aim 3, we will investigate underlying mechanisms involved in clearance of mitochondria, with an emphasis on the role of complement C1q and C3 to facilitate silent clearance. These studies will be done both in vitro using select isolated complement components, as well as in vivo using unique C1q and C3 deficient mice. Outcome measures include phagocytosis, cytokine production, and NET formation. Downstream signaling pathways involved in mitochondrial-mediated inflammation will be identified using mass spectrometry-based phosphoproteomics. In all, our study aims at identifying fundamental mechanisms regulating inflammation, thrombosis and autoimmunity in the context of human disease, with an emphasis on the role of the complement system in silent removal of mitochondria. We expect the proposed research to provide novel therapeutic targets disrupting the inflammatory and immunogenic properties of mitochondria, applicable for many diseases, including SLE and rheumatoid arthritis, as well as identify prognostic mitochondrial- derived biomarkers enabling early and preventive treatment of venous thrombosis.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Nearly all of the ~9 billion possible single nucleotide variants compatible with life exist among the 7.8 billion individuals alive today. Understanding the effects of these variants, especially in disease-associated protein coding genes, is central to understanding human biology and to using genome sequence information to guide the diagnosis and treatment of disease. Unfortunately, most new variants revealed by genetic testing are variants of uncertain significance, meaning insufficient information exists to definitively interpret the variant as either pathogenic or benign. Variants of uncertain significance cannot be used to guide patient care and reflect our incomplete understanding of variant effects. To overcome this challenge, we developed saturation genome editing (SGE) and variant abundance by massively parallel sequencing (VAMP-seq), multiplexed assays of variant effect that can make and measure the functional effect of massive numbers of variants. In SGE, single nucleotide variants are edited directly into the genome, revealing the effect of these variants on cell survival due to effects on splicing or protein function, thereby enabling accurate identification of both pathogenic and benign variants. VAMP-seq measures the effects of missense variants on protein abundance inside cells, and can identify up to 80% of pathogenic variants. Together, SGE and VAMP-seq can be applied to at least 40% of genes to produce high quality, clinically useful functional data at single nucleotide resolution. Already, variant functional data produced by each of these methods are being used by clinicians to interpret genetic variants. Our proposed Center for Actionable Variant Analysis (CAVA) will harness SGE and VAMP-seq to contribute single nucleotide variant functional data for ~200,000 variants in ~32 of the most clinically impactful protein coding genes to the IGVF Variant/Element/Phenotype Catalog. To accomplish this transformative goal we propose four Aims. In Aim 1, we will choose target genes and assays using a framework that maximizes clinical need, clinical impact and practicality. Each target/assay pair will be rigorously validated prior to entering production. We will contribute to the Consortium during the first year and beyond by developing standards, sharing reagents and initiating collaborative projects. In Aim 2, SGE and VAMP-seq will be performed on ~32 genes to high quality standards tracked using well-defined metrics. These include assay dynamic range and reproducibility, individual measurement error, and concordance with existing functional data and gold standard clinical data. A data analysis pipeline, integrated with our LIMS, will ensure reproducibility and enable careful progress tracking. In Aim 3, we will share the multiplexed variant functional data. Rigorously defined data sharing standards and metadata will ensure discoverability, computability and durability. We will work with the Consortium to achieve consensus and we will revise our plans accordingly. In Aim 4, we will enable labs to quickly stand up SGE or VAMP-seq. We will create a predict-evaluate-revise cycle that leverages the data we will generate and work collaboratively to generate data to improve modeling efforts.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The adaptive immune system consists of highly diverse B- and T-cell receptors, which can recognize and neutralize a multitude of diverse pathogens. Immune recognition relies on molecular interactions between immune receptors and pathogens, which in turn is determined by the complementarity of their 3D structures and amino acid compositions, i.e., their shapes. Immune shape space has been previously introduced as an abstraction for such molecular recognition to explain how immune repertoires are organized to counter diverse pathogens. However, the relationships between immune receptor sequence, shape, and specificity are very difficult to quantify in practice. We propose to use recent advances in machine learning and the wealth of molecular data to infer an effective shape space, grounded in biophysics of protein interactions. The key is to find a representation of proteins in general, and of immune receptors, in particular, that reflects the relevant biophysical properties that determine a protein receptor’s stability, function, and interaction with pathogens. Representation learning is a powerful technique in machine learning that uses large amounts of data to infer a reduced representation. Since protein function is closely related to the 3D structure, we will develop novel machine learning methods that use atomic coordinates of a protein structure as input and, through transformations that respect the physical symmetries in the data, learn representations that reflect biophysical properties of proteins and protein-protein interactions. We believe a key innovation in our approach is the analysis of amino acid neighborhoods within 3D protein structures. The distribution of these neighborhoods will reveal how they differ at the surface, in the bulk, and at functionally important regions such as catalytic sites. The learned protein representation will enable us to characterize how specific compositions of amino acid neighborhoods are the building blocks of protein structure and protein function. We will transfer the representation of protein universe to immune receptors to learn the immune shape space. The leaned immune shape space will enable us to address how affinity and specificity are encoded by immune receptors in different cell types. We will study how the modular structure of immune receptors, with separate pathogen engaging and framework regions, enables receptors to diversify and target a multitude of pathogens, without compromising their stability. We will use the complementary aspect of shape recognition to predict the antigenic targets of the immune receptors, and through collaborations, we will experimentally validate our predictions. Our approach opens a new path towards interpretable computational models of proteins and immune receptors that describe how biological properties and biological function emerge from protein subunits. Additionally, the inferred molecular representations can be used as a generative model, where desired properties, such as antigenic targets, are specified and new proteins can be generated.

NIH Research Projects · FY 2025 · 2021-08

Project summary The brain shows marked plasticity across a variety of learning and memory tasks as well as during recovery after injury. Many have proposed to leverage this innate plasticity using brain stimulation to treat neural disorders. Implementing such treatments requires advanced engineering tools as well as a solid understanding of how stimulation-induced plasticity drives changes in network dynamics and connectivity at a large scale and across multiple brain areas. Here, I propose to use our novel engineering tools to precisely manipulate neural activity in macaque sensorimotor cortex to investigate and induce targeted cortical reorganization. I hypothesize that a closed-loop optogenetic stimulation of somatosensory cortex based on the natural functional connectivity of the sensorimotor system can drive cortical plasticity and induce functional recovery. The functional connectivity maps of the somatosensory and motor cortical areas will be estimated as a guide for targeted stimulation. In Aim 1 the effectiveness of volitional control of activity- dependent stimulation will be investigated to both strengthen the natural existing connections and to induce new connections. In Aim 2 the volitional control of activity-dependent stimulation will be evaluated to induce new connections in the presence of an ischemic lesion. These aims are designed to provide us with both behavioral and electrophysiological measures to assess the targeted cortical reorganization. The combination of these measures can shed light on different aspects of brain plasticity and functional recovery mechanisms. The results of these aims will be a proof of concept for the power of refined stimulation patterns for targeted rehabilitation and rewiring of the brain that not only can be used for neurorehabilitation but also can help understand the circuits and connectivity in these cortical areas.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Older adults with Alzheimer's Disease and Related Dementias and limited-English proficiency (LEP) are at high risk of receiving suboptimal, goal-discordant care because of language and cultural barriers to effective communication about goals of care. Palliative care provides a way to improve quality of life and facilitate goal- concordant care by supporting effective person-family-clinician communication, but is under-utilized by older adults with Dementia and LEP and their families. No studies have sought to identify targets for intervention to address barriers to palliative care at multiple levels (i.e., patient/family, clinician, system, and community- levels) for this vulnerable population. Addressing this gap will be essential for the development of effective interventions to improve the quality of care for older adults with Dementia and LEP. The long-term goal of this work is to develop, evaluate, and disseminate interventions to facilitate culturally-sensitive palliative care for older adults with Dementia and LEP and their families. The rationale for this study is that before effective culturally- and linguistically-appropriate interventions to improve palliative care for older adults with Dementia and LEP can be developed, quantitative and qualitative data is needed to evaluate the scope of existing inequities and identify modifiable multi-level barriers to the delivery of high-quality palliative care for this population. Aim 1 utilizes quantitative methods to compare the quality of care received by decedents with advanced Dementia and LEP to those with English proficiency in four key palliative care domains (utilization of care, documentation of patient goals and preferences, symptom assessment, and circumstances of death) using EHR-based quality metrics and novel machine learning methods. Aim 2 utilizes qualitative interviews with key stakeholders (older adults with Dementia and LEP and their family members, caseworker-cultural mediators and interpreters, and clinicians and administrators) to identify modifiable targets for intervention across multiple levels. Aim 3 utilizes qualitative interviews with leaders of community-based organizations to assess community-level resources and capacity to support high quality palliative care for older adults with Dementia and LEP. Our interdisciplinary team is experienced in Alzheimer's Disease and Related Dementia research, community engagement with LEP populations, and quantitative and qualitative methods. The proposal is innovative because it evaluates multi-level barriers to palliative care across three diverse LEP populations (Latinx, Chinese, and Vietnamese), and uses machine learning methods to evaluate the quality of care across several palliative care domains in a large health system. The proposed research is significant because these data will enable the identification and prioritization of multi-level targets for the development of culturally and linguistically-appropriate interventions to improve palliative care for older adults with Dementia and LEP. Ultimately, this work will advance a model of care that facilitates goal-concordant, high-quality care for older adults with Dementia and LEP and supports the palliative care needs of their family members.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Severe traumatic injury and sepsis are acute pro-inflammatory insults that trigger a “genomic and cytokine storm” by the host innate immune response that can result in multiple organ failure (MOF). Whereas many patients previously succumbed to early refractory MOF, progressive advancements in resuscitation and organ support have led to an increasing number of patients surviving to enter a state of chronic critical illness (CCI), defined as a patient with an extended intensive care unit (ICU) stay and non-resolving organ dysfunction. Currently, as many as 25% of trauma and 40% of septic patients in the ICU develop CCI. In addition to a prolonged hospital course, these ICU “survivors” have recurrent infections, are unable to physically rehabilitate, and are frequently discharged to high-resource care facilities with dismal long-term outcomes. Two clinical manifestations dominate the course of the CCI phenotype: 1) recurrent nosocomial and post-discharge infections indicative of a state of chronic immunosuppression, and 2) acute muscle wasting, weakness and physical debilitation indicative of persistent inflammation. There is an expanding body of evidence that an underlying syndrome of persistent inflammation, immunosuppression and catabolism (PICS) is a key mechanistic driver of CCI. We hypothesize that PICS is its own unique immunological endotype independent of the index event, and is the shared mechanistic pathway leading from either trauma or sepsis to the clinical phenotype of CCI. This maladaptive host response is sustained by the ongoing release of endogenous alarmins (DAMPs) associated with end-organ injury, as well as microbial products from primary/secondary infections (PAMPs). This failure to return to immunologic homeostasis also drives the persistent organ dysfunction seen in CCI. Muscle, being both clinically relevant and understudied, serves as a novel area of study from the standpoint of inflammation-mediated end- organ injury both as a driver of acute muscle wasting, as well as a potential source of ongoing alarmin production and release. The goals for our laboratory’s research program over the next five years include the following: 1) characterize the heterogeneity of the host response after severe trauma and sepsis by identifying distinct endotypes based on immune trajectory over time, and whether these endotypes are modified by sex, age and ethnicity/race; 2) determine if the PICS endotype is the common mechanistic pathway after trauma or sepsis to the clinical development of CCI; and 3) determine whether muscle inflammation is both a component of chronic end-organ injury, as well as a mediator of systemic inflammation through the systemic release of endogenous alarmins. The proposed work is novel, innovative and vital. There are currently no therapeutic interventions other than supportive therapies for the increasingly common condition of CCI after trauma or sepsis. We believe that only through a complete understanding of the immunological endotype of CCI can effective therapeutic interventions be designed. Focusing on interactions between host immunity and muscle inflammation is a novel, under-explored area of research for the long-term management of severe trauma and sepsis.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The objective of the proposed project is to continue the laboratory’s development of a neuroprosthetic therapy that uses targeted, activity-dependent spinal stimulation to improve arm and hand motor recovery after cervical spinal cord injury (SCI). The project seeks to advance clinical practice through the use of brain-computer-interface technology to harness physiological mechanisms of neural plasticity. Motor deficits severely impact the quality of life of people with SCI, yet current treatments produce limited improvements in movement abilities. Recent clinical and experimental evidence suggests that electrical stimulation of the nervous system can be an effective therapy for a variety of neurological disorders. Here we will extend study of a novel application of electrical stimulation for rehabilitation of forelimb motor deficits in a rat model of cervical SCI. The strategy is designed to enhance the function of spared neural pathways by directing Hebbian plasticity through targeted stimulation of volitionally activated neural circuits. Results will lay the foundation for a future clinical trial using spinal stimulation in human subjects. Previous data demonstrated the effectiveness of multi-channel, intraspinal stimulation triggered by activity of forelimb muscles to improve motor performance in rats with chronic SCI. Specific Aim 1 seeks to complete the characterization of the interplay between stimulation and physical rehabilitation in order to establish principles for combining the therapies. Specific Aim 2 will examine possible causes underlying dramatic differences in recovery in female and male rats treated with the closed-loop stimulation. Specific Aim 3 will determine if activity-dependent epidural stimulation, a much less invasive and easier to translate to clinical practice approach than intraspinal stimulation, is as effective in facilitating recovery.

NIH Research Projects · FY 2024 · 2021-08

ABSTRACT The rapid spread of multi-drug resistance has created a great need for new combination therapies to treat a variety of conditions, including infectious diseases and cancer. In one pressing example, multidrug resistant tuberculosis (TB) affects about 500,000 people each year and novel drug regimens are sorely needed. However, identifying new regimens has been daunting in part due to the inability to prioritize among a very large number of possible drug combinations. To address this need, we have generated an experimentally grounded, machine learning algorithm, INDIGO-MTB, which predicts the synergy or antagonism of TB drug combinations with high accuracy. Here we propose to adapt INDIGO-MTB into a multifactorial pipeline to dissect combinatorial drug efficacy and drive preclinical regimen development for TB. We will build in and validate the ability to predict drug interactions under stressful environmental conditions that mimic TB infection, and extract molecular mechanisms of drug interactions. We will then combine synergy and efficacy measurements to create new regimen rankings, which we will validate both in vitro and in a mouse model of TB infection. Altogether, our work will establish a tool for rapid assessment of TB drug combinations and a framework for applying this approach to other conditions where new multidrug therapies are needed.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY One of the most pressing public health priorities for the COVID-19 pandemic is the development of an effective and inexpensive therapeutic. The long-term goal of this proposal is to develop such COVID-19 treatments, as well as the methods needed to rapidly create such molecules as soon as any new pathogen is identified. The central hypothesis is that computational design can be used to quickly create proteins with potent antiviral activity and others that suppress “cytokine storms” associated with advanced infection. Such countermeasures, if rapidly developed and deployed, could save millions of lives during an outbreak until vaccines are developed. The specific aims are to: 1) overcome current limitations in the discovery and development of protein therapeutics by creating methods for the de novo design of hyper-stable miniproteins that bind tightly to vulnerable binding sites on the SARS-CoV-2 Spike glycoprotein, including the receptor binding domain (RBD) of the ACE-2 cellular receptor and the fusion peptide region; 2) Enhance the avidity of such anti-Spike minibinders through genetic fusion of multiple copies, or through rational design of higher-order oligomers to create drug compounds that are less prone to viral mutagenic escape; 3) Apply the same minibinder design pipeline to create cytokine receptor antagonists of key cytokines IL-6 and IL-1β likely involved in acute respiratory distress syndrome (ADRS) associated with COVID-19 mortality; 4) Assess the efficacy of antiviral and anti-interleukin minibinders by several routes of delivery (intravenous, intranasal and subcutaneous) in rodent models of COVID-19 and assess immunogenicity in order to identify those designs best suited for further preclinical development. As proof of principle, the first anti-Spike minibinders have already been designed, were found to bind to SARS-CoV-2 Spike RBD, and were found to neutralize live virus with activities rivaling the most potent known antibodies. This proposal is innovative because it seeks to apply powerful emerging methods in the computational design of new protein therapeutics to the COVID-19 pandemic. The proposal is significant because it would be the first example of computational protein design yielding potent and entirely de novo antiviral and anti-inflammatory therapeutics for an active pandemic. Ultimately, rapid minibinder design methods have the potential to generate treatments for future pandemics, as well as for many other common and neglected diseases and conditions.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy bacteriophage nucleic acids containing sequence complementarity to the guide RNA. CRISPR systems harbored by different bacteria can be extremely diverse and use different strategies to neutralize infecting phages. CRISPR systems are not well-represented in traditional model bacteria. As such, their function has typically been studied by heterologous overexpression in E. coli. Accordingly, we have limited knowledge of the complex interactions that arose from the co-evolution of CRISPR-Cas systems with their natural hosts and the phages that infect them. Research in my laboratory focuses on establishing natural models to investigate the interfaces between CRISPR-Cas immunity, bacterial host physiology, and phage infection. While most of the six CRISPR types use Cas DNases to recognize and cleave phage DNA, the type VI CRISPR system uses the nuclease Cas13 to cut RNA instead. I have developed a natural bacterial host of the type VI CRISPR system, Listeria seeligeri, and a collection of its phages as a tractable model for studying how this system protects against infection. Once Cas13 engages target viral RNA, it becomes activated as a non-specific RNase, resulting in widespread cleavage of both phage and bacterial RNA and the abortion of the phage lifecycle. Thus, infected cells with type VI immunity do not lyse, and fail to produce viral progeny, but stop growing and become dormant. The goals of this proposal are to (i) determine which transcripts cleaved by Cas13 trigger entry into dormancy; (ii) understand how L. seeligeri cells survive the dormant state, and resuscitate themselves once the phage has been eliminated, and (iii) discover and characterize endogenous regulatory mechanisms controlling Cas13 activity in L. seeligeri and its phages. The results generated by this research will provide fundamental insights into the molecular biology of RNA-targeting CRISPR systems and aid in their development as biotechnology tools. Finally, Cas13-induced cellular dormancy bears similarity to a phenomenon termed persistence, in which subpopulations of pathogenic bacteria stop growing and become transiently tolerant of bactericidal antibiotics during human infection. Therefore, the studies proposed here could reveal general mechanisms by which persistent bacteria survive antibiotic exposure and re-enter the growth cycle, which would represent attractive targets for therapeutic intervention.

NIH Research Projects · FY 2025 · 2021-08

Abstract Children who survive severe traumatic brain injury (sTBI) live with profound impairments that alter their development and future possibilities. Worldwide, TBI is the leading cause of death and disability among children and adolescents. In the US, the annual incidence of pediatric TBI is greater than MS, HIV/AIDS, spinal cord injury, and breast cancer combined1. Although not strictly pediatric diseases, this comparison illustrates the magnitude and importance of the pediatric global health epidemic we are addressing. Our primary focus for scientific investigation is to conduct a high quality randomized controlled trial addressing a critical TBI management question: Does using a protocol with information from intracranial pressure (ICP) monitoring to direct treatment of children with sTBI improve outcomes vs an aggressive management protocol based on imaging and clinical examination alone? This follows on our adult ICP study2 which found no outcome differences and has occasioned re-thinking of treatment guidelines for sTBI patients >13. A separate study is essential because children are not simply small adults and some treatment approaches carry age-related additional risks. Thus, study findings will inform US and global clinical practice. This trial will be conducted in 7 Latin American pediatric ICUs where infrastructures and practice patterns are optimal for strong internal validity and resources represent trauma care in the developing world. The successful adolescent/adult BEST TRIP trial, which collected high-quality data in similar environments (cited > 900 times) underscores the feasibility of this approach. Specific Aim: In a Phase III randomized superiority trial in 428 children with sTBI from 7 Latin American pediatric trauma centers, test the effect on outcomes of management of sTBI guided by a protocol using information from ICP monitors vs. management using a protocol that uses imaging and clinical exams to guide treatment. Hypothesis #1: Children with severe TBI whose acute care treatment is managed using a protocol based on data from ICP monitoring will have significantly lower mortality and better quality of life and global outcome at 6 months post-trauma than those whose treatment is managed with a protocol based on imaging and clinical exam. The primary measure of functional recovery is the PedsQL at 6 months. A secondary measure is GOSE-Peds. Hypothesis #2: Incorporating ICP monitoring into sTBI patient care of will minimize secondary complications, decrease length of stay in ICU and decrease brain-specific treatments. Specific Aim: We will train personnel in centers new to research in how to conduct high-quality scientific studies, and will extend the training for the personnel with whom we have been working, solidifying previous capacity- building efforts, and initiating new efforts.

NIH Research Projects · FY 2026 · 2021-08

PROJECT SUMMARY Heart failure (HF) is a costly and complex health condition affecting millions of Americans. HF care is often fragmented which negatively affects quality, safety, and patient-centered outcomes. Structured Interprofessional Bedside Rounds (SIBR) is a model of care developed to bring interprofessional team members together with patients and families using a structured format to collaboratively arrive at a daily care plan. The SIBR model is characterized by four core components: an interprofessional approach, utilization of a rounding structure, intentional patient and family engagement, and development of a shared daily care plan. A growing body of evidence associates SIBR implementation with improvements in team and patient outcomes. The hypothesized mechanism through which SIBR operates is that having a predictable structure leads to improvements in communication, fewer gaps in care, and more consistent utilization of evidence-based approaches. These changes are thought to lead to improvements in patient outcomes such as, length of stay, readmission rates, patient-centeredness of care, and safety/adverse events. Despite frequent improvements in outcomes following SIBR implementation, an evidence gap exists as to the role that fidelity (adherence) to the SIBR model plays in how and why this model works and the extent to which outcomes can be further improved if fidelity is higher. To address this knowledge gap, this proposal leverages a timely opportunity to study SIBR fidelity and its relationship to care and outcomes among patients with advanced HF at the University of Washington Medical Center, where a SIBR model has been the standard of care for 4+ years. The central hypothesis is that higher- fidelity SIBR will be associated with better outcomes. To test this hypothesis, I will carry out a prospective cohort study to achieve three specific aims: (1) identify associations between SIBR fidelity and patient outcomes, (2) determine the extent to which SIBR fidelity predicts time to initiation and completion of an evidence-based “Advanced HF Work-Up Pathway”, and (3) examine patient and family experiences of care quality and safety in the context of higher- and lower- fidelity SIBR. These aims will lay the groundwork for an initial multi-site R01 to study SIBR in practice and a future R-level grant to implement and evaluate an optimized SIBR model. Through this work, I will obtain formal training in HF outcomes research, advanced implementation science study designs, and patient-oriented clinical research methods. I will be mentored by an expert team of NHLBI-funded researchers, Bryan Weiner (primary mentor, implementation science), Randall Curtis (communication and palliative care), Brenda Zierler (interprofessional collaboration and clinical pathways), and Kevin O'Brien (advanced heart failure care). The combination of mentorship, coursework, and experiential learning will position me to become an independent investigator using scientifically rigorous approaches to evaluate the implementation and outcomes of evidence-informed patient- and family-centered models of care.

NIH Research Projects · FY 2025 · 2021-08

The US is currently experiencing an opioid epidemic with opioid overdose, opioid use disorder (OUD) and Emergency Department (ED) visits related to opioids on the rise. The ED is at the forefront of this public health emergency and often a place where patients with OUD come for treatment of medical problems, both related and unrelated to opioid use. Medications for OUD (MOUD) and linkage to care from the ED are a critical piece in addressing this crisis. Patients in the ED with OUD present with a complex constellation of substance use, psychiatric and medical comorbidity, and often experience fragmented healthcare delivery. Comprehensive disease management strategies, such as the Collaborative Care model, hold great promise for addressing the treatment needs of patients with OUD in the ED. The Emergency Department Longitudinal Integrated Care (ED-LINC) intervention derives from the Collaborative Care model and is a multi-component, longitudinal intervention for patients with OUD and related comorbidity that is initiated during an ED visit. The ED-LINC clinical trial will randomize 500 patients with OUD seeking care at two Washington State EDs to the ED-LINC intervention (n=250) or usual care control (n=250) conditions. Patients randomized to the ED-LINC intervention will receive; 1) overdose education; 2) brief bedside intervention targeting motivation to engage in outpatient care; 3) a patient-centered approach to MOUD using a treatment decision support tool; 4) longitudinal and proactive care management which will proceed for three months along with weekly caseload supervision of the interventionist by a mental health professional allowing for stepped-up care targeting opioid use and associated comorbidity. ED-LINC is supported by the Emergency Department Information Exchange (EDIE) platform, which is a novel care coordination tool that collects data from hospitals in over 30 states and packages this information for emergency providers in real-time alert notifications. Further features of EDIE include creation of tailored care plans. The trial incorporates pragmatic outcome assessment elements, including the use of EDIE and statewide administrative data to assess important outcomes on the intent-to- treat sample. The ED-LINC trial tests the hypotheses that over time, patients randomized to the ED-LINC intervention, when compared to patients randomized to usual care, will demonstrate: 1) significant reductions in self-report illicit opioid use; 2) significant increases in MOUD initiation and retention, and 3) significant reductions in ED utilization. Secondary analyses will explore the impact of methamphetamine use comorbidity and sex on intervention treatment effects. The study team also plans to conduct an implementation process assessment using novel mixed methods to understand the potential for sustainable implementation of intervention elements. Data derived from this formative evaluation will be used to prepare for the presentation of study results at a policy summit in the final year of the trial which will allow for rapid dissemination and potential policy changes related to care for patients with OUD from the ED.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Glucagon and insulin are pancreatic hormones that control the glycemic response to fasting and feeding. Canonically, glucagon is secreted in the fasted state where it stimulates hepatic glycogenolysis and gluconeogenesis in order to raise glycemia. Conversely, insulin is secreted in response to elevated blood glucose to stimulate glucose uptake in peripheral tissues. These canonical, opposing effects of glucagon and insulin on the liver overlook the long understanding that amino acids in mixed nutrient feeding stimulate the islet a-cell to secrete glucagon. Moreover, our recent work has shown the importance of glucagon-stimulated insulin secretion to maintaining glucose tolerance. Therefore, the expected response to a physiologic mixed nutrient meal is the co-secretion of glucagon and insulin into portal circulation. The preliminary data presented herein support the notion that glucagon and insulin work cooperatively, not antagonistically, to control prandial glucose metabolism. We hypothesize that glucagon and insulin co-secretion controls hepatic glucose metabolism to allow glucose to reach the periphery before being stored in the liver. Successful completion of this project will alter our fundamental understanding of hepatic glucose metabolism and my provide insight for novel therapeutic targets for the treatment of metabolic disease. Importantly, completion of the proposed aims will support several new technical and conceptual developments that will provide a foundation for a career as an independent investigator.

NIH Research Projects · FY 2024 · 2021-08

Abscesses are infected walled-off fluid collections of pus and bacteria and represent a ubiquitous global healthcare problem. They are common sequelae of surgery, infections, or disease, and can affect any part of the body. Current standard of care includes hospitalization, antibiotics, and drainage of the abscess with a catheter. The bacteria in pus are susceptible mechanical damage with High Intensity Focused Ultrasound (HIFU), which generates localized cavitation and is a potential noninvasive means to treat abscesses. The significance of this proposal is that treatment of abscesses using ultrasound therapy under ultrasound guidance is better for the patient because it is minimally- or non-invasive, has less procedural pain, doesn’t require that the patient live for up to several weeks with a drain inserted, requires no catheter management, there is no potential tract for new infections, and no need for CT/fluoro imaging radiation. Furthermore, ultrasound treatment will reduce the need for antibiotics, and thus reduce the potential for drug resistant, life-threatening microbes.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Human herpes simplex virus 2 (HSV-2) is a sexually transmitted pathogen affecting 500 million people worldwide. Most of the global burden of HSV-2 disease results from the virus’s ability to establish chronic infections, characterized by recurrent anogenital ulcerative disease. The severity of symptoms experienced by those with chronic HSV-2 infection is highly variable, a feature of the disease which remains unexplained. While prior research has focused primarily on the role of the host in mediating the severity of chronic HSV-2 infection, evidence from studies both in animal models and humans suggests that the virus may also play an important role. The objective of this proposal is to improve our understanding of the pathogenesis of chronic HSV-2 infection by investigating the impact of viral factors on the severity of symptoms experienced by infected persons. The proposal consists of three aims. In Aim 1, Dr. Casto will perform a genotype-phenotype association study to scan the HSV-2 genome for genetic variants that are associated with disease severity. This aim will use genome sequence data for 100 HSV-2 samples from individuals with severe HSV-2 disease (cases) and for 100 samples from individuals with mild disease (controls). Some genetically-mediated traits are determined by many variants each with small effect. Such variants can be difficult to identify in genotype- phenotype association studies, so Dr. Casto will also assess for other intrinsic differences among HSV-2 samples that correspond to disease severity. In Aim 2, she will assess for differences in viral gene expression that are associated with disease severity by comparing the abundance of individual viral transcripts between viruses from cases and from controls grown in vitro. She will also compare viral transcript abundance in biopsies of HSV-2 lesions from cases and controls. In Aim 3, she will assess whether viruses from cases cause more severe disease in an animal model than viruses from controls. The results of this work will provide the first insights into the role of viral factors in determining the severity of chronic HSV-2 disease, insights that will help dictate the future direction of research in this field. Dr. Casto is an Acting Instructor/Senior Fellow Trainee in the Division of Allergy and Infectious Diseases at the University of Washington. She and her mentors have devised a comprehensive career development plan that includes: 1) Regular meetings with mentors to discuss research progress and career development; 2) Acquisition of the skills needed to perform in vitro and in vivo experiments with HSV; 3) Focused coursework that augments Dr. Casto’s skills in biostatistics, bioinformatics, and data analysis; 4) Attendance of seminars and workshops to develop Dr. Casto’s abilities in scientific communication and laboratory management; 5) Participation in local, national, and international conferences which will allow Dr. Casto to present her research. By the end of this award, Dr. Casto will transition to independence as an investigator, running her own lab dedicated to understanding genotype-phenotype connections for infections due to HSV and other viruses.