University Of Washington

NIH Research Projects · FY 2025 · 2024-08

The discovery of BRCA1 and BRCA2 and other genes of homologous recombination repair, and the characterization of the roles of these genes in inherited predisposition to breast and ovarian cancer, have had a tremendous impact on cancer prevention and treatment. Yet family history remains one of the most important risk factors for these cancers, and a great deal of that risk remains unexplained. We propose two hypotheses.: (1) In some families, breast cancer is due to previously cryptic non-coding variation altering transcription and/or expression of known breast cancer genes (BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51C, RAD51D, and TP53); and (2) In some families, breast cancer is due to rare or private non-coding variants regulating expression of critical genes with no cancer-predisposing alleles in coding sequence. To test these hypotheses, we will deploy new technologies: long-read sequencing of genomic and cDNA; Fiber- seq identification of regions of open chromatin in cell types of choice; massively parallel reporter assays (MPRA) to compare effects of variant versus reference alleles for many sites simultaneously; and CRISPR inhibition (CRISPRi) of candidate regulatory regions. We propose to integrate these approaches to discover and characterize classes of non-coding pathogenic variation in 1253 extended families, severely affected with breast and/or ovarian cancer, but for whom no causal variants have been found by any current genomic technology. In Aim 1, we will use adaptive-sampling long-read genomic sequencing to determine the spectrum of all classes of rare non-coding variants in the extended TADs of the known breast cancer genes. In Aim 2. we will use long- read cDNA sequencing to characterize transcriptional consequences of the complex SVs and rare deep intronic SNVs co-segregating with breast cancer (from Aim 1). In Aim 3, we will use Fiber-seq analyses (already in hand) of fallopian tube epithelial cells and MCF10A mammary cells to identify candidate regulatory regions of breast cancer genes. Then, for rare variants in these peaks that are co-inherited with breast cancer in unsolved families, we will use MPRA to test allele-specific differences in reporter activity; and in parallel silence the regions with CRISPRi to evaluate their effects on gene expression. In Aim 4, we will extend Aim 3 genome-wide, to identify variants near other breast cancer-relevant genes that lie in open chromatin regions, that co-segregate with breast cancer in unsolved families, and that reveal allele-specific differences in regulatory activity. We expect to discover clinically meaningful genetic variation in non-coding regulatory regions, including in repetitive genomic neighborhoods particularly subject to complex rearrangement. We expect that this approach will be directly applicable to any complex diseases with an inherited genetic influence.

NSF Awards · FY 2024 · 2024-08

This project investigates how to more effectively enable the success and advancement of neurodivergent employees in the technology workplace. We do so by investigating specific success enablers grounded in an asset model of disability and considerate of the unique needs, interests, and strengths of neurodivergent individuals in the context of their work. Success enablers are a broader set of practices and work designs that include accommodations but are not limited to them. Success enablers include both support and, more importantly, changes to the workplace environment. The goal of SEEN Tech Professionals is to 1) empower neurodivergent professionals in the technology sector to determine, identify, and use what they deem as appropriate success enablers based on their intersecting identities and 2) build the capacity of organizations to systemize and normalize the use of success enablers more readily. The SEEN Tech Professionals project will improve technology workplace environments to advance equity for neurodivergent professionals by creating The Neuroinclusive Success Enablers Toolkit. The Toolkit compiles interventions designed to empower neurodivergent employees and build the capacity of their managers, HR professionals, and providers to leverage and normalize success enablers. The Toolkit will be freely available in accessible formats on the project website and distributed widely through the University of Washington and the project Advisory Group. The SEEN Tech Professionals research project investigates three central questions: 1) What are the most common success enablers neurodivergent individuals identify as critical in the workplace? 2) What role do managers play in identifying, normalizing, and systemizing success enablers to support their ND employees? 3) What are the organizational characteristics that support or limit the identification, implementation, and systemization of success enablers? Our work is based on Annabi and Locke’s Organizational Interventions Mitigating Individual Barriers (OIMIB) framework, grounded in the neurodiversity paradigm and critical disability studies and emphasizing intersectionality. Findings from this research will advance our theoretical and practical understanding of how neurodivergent technology professionals leverage various success enablers to address barriers they face and the role organizational interventions (e.g., Neurodiversity Hiring Programs) and managers play in facilitating the use of success enablers. Our project will be carried out in three phases over three years. The primary outcome of this study is to create high-impact interventions organized in a comprehensive Neuroinclusive Success Enablers Toolkit designed for technology employers and neurodivergent professionals. This award has been made in response to the NSF solicitation “Workplace Equity for Persons with Disabilities in STEM and STEM Education” (NSF 23-593). The project is funded by the Division of Equity for Excellence in STEM’s EDU Core Research program (ECR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT Zika virus (ZIKV) re-emerged over the last several decades to cause large outbreaks in the Asian Pacific and Americas. ZIKV disease, though usually mild, can occasionally cause severe neurological complications, including developmental defects in babies born to a ZIKV-infected person. Much remains to be learned about ZIKV host-pathogen interactions, including how the innate immune system fights the early stages of infection in human cells. Given their role in inhibiting viral replication, innate immune factors could help explain the heterogeneity in ZIKV clinical outcome and could lead to the development of treatments for ZIKV. The type I interferon (IFN) response plays a particularly critical role in the innate immune response to viruses, including ZIKV. IFN is secreted from virus-infected cells, binds its receptor on nearby cells, and sets off a signaling pathway that culminates in a transcriptional program turning on hundreds of interferon-stimulated genes (ISGs), some of which encode antiviral proteins against a given virus. While the IFN response is clearly important in restricting ZIKV, systematic studies to define which host genes are responsible for this potent effect are lacking. While several specific antiviral ISGs have been identified, there have not been broader attempts to define all host genes that contribute to IFN restriction of ZIKV, including non-ISGs that may play a regulatory role in the pathway. Our lab performed a CRISPR knockout screen to identify genes that contribute to IFN restriction of ZIKV. The screen approach successfully identified genes involved in IFN signaling (positive controls). IFI6, a previously described ISG and flavivirus restriction factor, was also identified, as was AMOTL2, a non-ISG in our cell type and a gene with no described role in innate immunity. Despite the fact that AMOTL2 is not itself IFN-induced, AMOTL2 knockout increased ZIKV replication in the presence, but not absence, of IFN. These findings confirmed the IFN-specific antiviral phenotype of AMOTL2 and led to the hypothesis that AMOTL2 acts upstream of ISG transcription in the IFN response. My preliminary results support this model and are the basis for the proposed studies. In Aim 1, the mechanism of AMOTL2’s viral restriction will be better elucidated. This will be achieved by first validating the specificity of the observed phenotype through rescue experiments, then assessing the involvement of AMOTL2’s binding partners YAP and TAZ in IFN restriction of ZIKV through double knockouts and co-IP assays. In Aim 2, I will test the breadth of AMOTL2’s antiviral phenotype across diverse viruses (Aim 2a) and with ZIKV infections in a biologically-relevant primary cell type (Aim 2b). The complementary results of my two aims will enhance our understanding of the IFN response against ZIKV and possibly other viruses.

NIH Research Projects · FY 2025 · 2024-08

American Indians and Alaska Natives (AIAN) face some of the highest rates of substance use disorders and related mortality in the nation. AIAN may also face a host of mental health and substance use issues related to multigenerational exposure to historical trauma, personal trauma, poverty, and unemployment. Concomitantly, despite the effects of AIAN health disparities and social inequities, tribal communities have retained their language, culture, and community connectedness, which offer unique and important strengths that have yet to be fully integrated in tribal research efforts. The University of Washington Seven Directions (UW-7D) team proposes to partner with Drs. Melissa Walls (Professor, Johns Hopkins University Center of Indigenous Health (JHU-CIH) to provide a four-pronged approach to provide GATHER: Growing a Tribal Healing Effort through Research, designed to support N CREW funded research teams for the purpose of ensuring health equity within AIAN communities using a strengths-based, culturally grounded approach to grow tribal research in the areas of overdose, substance use, mental health, and pain research. The University of Washington Seven Directions (UW-7D) team will serve as the core support (Prong 1), supporting the documentation and monitoring of tribal research grants, along with building the initial relationships with tribal and other Native American Serving Organizations (NASO) research teams to develop trust, identify research capacity needs, readiness to engage in research and enhance research capacity of T/NSAO. In our technical assistance work, we recognize the importance of authentic relationship building, and this will inform our approach to working closely with T/NSAO research teams (Prong 2 – Technical Assistance Approach). JHU-CIH will provide the research support core (Prong 3), drawing from a wealth of experience and expertise to support the technical research capacity development and/or data enhancement capacity of T/NSAO grantees. They will provide training in grant writing, specific aims development, identification of study designs and measures, and data access and quality improvement. UW-7D and JHU-CIH will both serve as the translation and communication core (Prong 4), supporting the dissemination of opportunities, trainings on translating research findings to program content and other implementation materials, and supporting the annual convenings of T/NSAO research teams. GATHER’s purpose is to substantively contribute towards building research capacity among and provide instrumental research support to tribally led overdose, substance use, mental health, and pain research projects towards the improvement of the health and wellbeing of AIAN populations through fostering and growing Indigenized approaches to substance use research. The GATHER initiative aims to: 1) Conduct, stimulate, coordinate, and support collaborative research on the etiology and prevention of harmful alcohol use and related risks in the transition to adulthood, within an overarching developmental framework emphasizing social, cognitive, contextual, personality/temperament, and affective influences. 2) Provide the necessary administrative support and shared resources to facilitate the successful completion of component research projects contributing to the Central Theme. 3) Provide an administrative infrastructure, intellectual environment, and access to resources and initial support for young investigators and those new to the field of alcohol etiology and prevention. 4) Provide clinical and research training for students, fellows, staff, and faculty in the areas of cognitive, motivational, and behavior therapies targeting prevention and treatment of alcohol use and related risks. 5) Serve as a local, national, and international resource for dissemination of information and training to the public, trainees, and academic audiences, to foster the application of new knowledge to the reduction of harmful alcohol use and related risks in diverse EA populations. Benefits to AIAN Communities: This innovative multimethod approach will have important methodological, prevention, treatment, and environmental impact among AIAN communities, because it will a) offer key research supports for tribally-grounded substance use research; b) address research capacity needs at the individual, team, and community levels; c) provide groundwork to for a holistic, culturally informed research training and TA approach, and d) refine the methods of effective CBPR in tribal and NASO settings. Given the disproportionate effect of substance use, mental health issues, and lack of clinical capacity to effectively support pain management, and the far-reaching dissemination plan developed by the GATHER partnership, this innovative initiative has the potential for wide range impact among the funded and yet-to-be-funded N CREW research projects and across Indian Country in general. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions for the opioid crisis and overdose epidemic, including opioid use disorder and stimulant use disorder. The NIH HEAL Initiative bolsters research across NIH to address the national opioid public health crisis and improve treatment for opioid misuse and addiction.

NIH Research Projects · FY 2025 · 2024-08

The goals of the Northwest Genomics Center (NWGC) for All of Us (AoU) are to provide high-quality, cost-effective genotyping and whole genome sequence (WGS) data; to return genomic results for medically actionable and pharmacogenomic genetic variants; to enhance the scientific value of AoU by adding innovative -omic technologies including long-read sequence data. The NWGC brings together three internationally recognized Principal Investigators–Gail Jarvik, Chia-Lin Wei, and Evan Eichler–with decades of expertise in high-throughput clinical genomics. Together with their Co-Investigators—Christina Lockwood, Colin Pritchard, and Phil Empey—they have returned tens of thousands of variant interpretations to patients including AoU participants. Since August 2020, the NWGC has delivered genotype array data for 169,414 samples and genome data for 61,606 samples to the AoU Data Research Center (DRC). We have completed variant review and classification resulting in genomic return of results reports for more than 15,000 AoU participants including 411 medically actionable reports (i.e., American College of Medical Genetics pathogenic or likely pathogenic classifications). To advance the core business goals and objectives of AoU, the NWGC will generate high-quality genotyping array and whole genome sequence data for up to 50,000 AoU participants per year. In addition, we will interpret and classify variants for individuals who request genomic return of results (gRoR). We will continue to lead and support expanding gRoR content to participants (i.e., additional genes and medications) and leverage long-read -omics projects for investigators in the Researcher Work Bench (RWB). We will contribute our in-depth experience in sequencing technologies to support the FDA IDE supplemental validation plans for the NovaSeq X Plus platform and construct long-read resources in the discovery of pathogenic variants in medically- relevant genes and PGx targets. We will participate in analysis working groups and work with the DRC to improve genotype imputation and phasing tools, when funding is available, we will deploy innovative multiomics technologies to expand variant interpretation and functional annotation. The NWGC is ideally positioned to support and maximize the potential of AoU and its central mission to understand human genetic variation that impacts disease prevalence in the United States population.

NIH Research Projects · FY 2025 · 2024-08

Vestibular deficits are highly prevalent and cause debilitating symptoms. Degeneration and ototoxin- induced injury of vestibular hair cells (HCs) are underlying causes of some vestibular deficits. Remarkably, adult mammals, including humans, can replace some vestibular HCs once they are lost. However, in rodents and likely in humans, only one type of vestibular HC – type II (HCII) – is naturally replaced, and this degree of regeneration does not restore vestibulo-motor functions. Functional testing by our group and others’ provides strong evidence that the other type of vestibular HC – type I (HCI) – must be replaced to reverse vestibular deficits. HCI are very different from HCII; for instance, they have longer and thicker stereocilia (the mechanosensitive organelle of HCs), and they synapse on a single large calyx-shaped afferent terminal of a vestibular ganglion neuron (VGN) rather than on the VGN’s bouton terminals, as HCII do. These features are thought to endow HCI with functional specializations such as the ability to detect fast-onset, high-frequency stimuli. However, we understand very little about how these specializations develop or which molecular mechanisms control their genesis, differentiation, and maintenance in adult mammals. This lack of knowledge hampers scientists’ efforts to determine how to drive functional regeneration in mammals after HC loss. Recently, we found that conditional knockout (cKO) of the transcription factor Sox2 from normal HCII and from naturally regenerated HCII in adult mice drives many of them to partially convert toward the HCI fate, including establishing the long, thick stereocilia unique to HCI. Sox2 cKO from adult HCII also triggers VGNs to remove bouton terminals and extend a full or partial HCI-specific calyx terminal on the converting HCII. However, reprogramming and synaptic remodeling of regenerated HCII after Sox2 deletion are insufficient to recover vestibulo-motor functions. The objective of the proposed studies is to exploit these new findings to investigate molecular mechanisms by which HCIs establish and maintain their type-specific hair bundle and innervation phenotypes, which are critical for function. Aim 1 will use mouse multiomics and CUT&RUN to define gene regulatory networks by which SOX2 in HCII blocks acquisition of HCI features, and it will begin to define SOX2-independent mechanisms that promote the HCI fate. Aim 2 will use targeted gene deletion in mice to identify novel SOX2-regulated genes that are required to develop and maintain the stereocilia morphology of HCI. Aim 3 will use targeted loss and gain of gene function in mice to define novel SOX2- regulated genes that are necessary in HCI to establish and maintain the calyx-type terminal and will employ snRNAseq to identify new ligand-receptor pairs that may regulate VGN innervation of HCI and HCII. This project will engage experts from four laboratories to generate new fundamental knowledge about how vestibular HCs develop and maintain structures that are critical for sensory transduction and synaptic transmission, providing new insights for strategies to regenerate vestibular HCs in adult mammals.

NIH Research Projects · FY 2024 · 2024-08

Project Summary/Abstract Aging is associated with decline in skeletal muscle function even in healthy individuals, and the molecular mechanisms underlying this aging process are incompletely understood. The overall goal of the proposed project is to discover genomic alterations in skeletal muscle that are associated with aging, with the long term goal of creating a better understanding of mechanisms of aging in order to help prevent or slow down the aging related decline in muscle function. It is known that expression of hundreds of genes is altered in healthy muscle aging, we will dissect the aging muscle transcriptome in two ways beyond gene expression. Alternative splicing of messenger RNA creates diversity in the proteome and has been implicated in causing disease. Additionally, alternative splicing can cause exonization of previously intronic sequences, potentially leading to an amino acid sequence not previously seen by the immune system. Based on our preliminary data, Aim 1 will explore if alternative splicing of mRNA results in quantitative differential isoform changes associated with aging, resulting in increased expression of non-functional proteins that are relevant for healthy muscle function. Moreover, we will predict protein changes from alternative splicing and the potential for generation of novel peptide sequences. On the other hand, aging is associated with inflammatory changes (referred to as “inflammaging”). In Aim 2, we will investigate a potential source of inflammaging by quantifying expression of transposable elements in the aging muscle including LINE-1, HERV-K, and SINEs. All proposed analyses will be conducted on two existing datasets, including RNA-sequencing data from muscle biopsies of healthy individuals, which is ideal to isolate the effect of aging on the muscle transcriptome without influence from diseases more common in older individuals. Additionally, we will validate our results in a much larger and less selected RNA-sequencing data of hundreds of muscle biopsies, which will add confidence in the external validity of our results.

NSF Awards · FY 2024 · 2024-08

This award supports a CAREER project that engages in research and teaching activities related to the implications of neuroscience research.The project provides guidelines to stimulate greater collaboration between the neurosciences, academic communities, and the public. Ongoing results from this project will be made available to the public through open forum engagements and to university and high school students through classroom-based lectures to educate a wide audience about neuroscience’s benefits and challenges. This project uses a qualitative approach to map the relationships between overlapping neuroscientific epistemic communities—funding bodies, publication mechanisms, professional organizations, agencies, research labs, and bioethical domains. The project provides recommendations and educational resources for neuroscience educators and researchers. The project contributes to existing literature in Bioethics, Sociology, Science of Science, and Science and Technology Studies (STS) on the impacts of neuroscience research. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Mitochondria have retained a small circular genome (mtDNA) that encodes essential components of the elec- tron transport chain. Mutations in the mtDNA can cause devastating maternally inherited diseases, while the accumulation of somatic mtDNA mutations is linked to common pathologies. Although mtDNA mutations impact human health, the process(es) that influence their occurrence and level with the cell (i.e., heteroplasmy) remain under considerable debate, despite nearly 30 years of study. Using cutting edge sequencing methods, we have made key contributions to understanding the drivers of mtDNA mutagenesis in a variety of diseases and animal models. This includes the discovery that the types and frequencies of mutations varies considerably between organisms and tissues, evidence that deaminations arising from a single-stranded replication interemediate is the driver of mutations in most vertebrates, and strong evidence that selection of mtDNA is occurring in somatic tissues. Here, we aim to understand the cellular mechanisms that regulate mtDNA heteroplasmy and its intersection with mitochondrial quality control pathways. Testing hypotheses related to heteroplasmy and mtDNA selection has been difficult due to the reliance on bulk sequencing approaches. However, the advent of new methods, such as single-cell and ultra-high accuracy sequencing, has opened up the possibility of answering these questions. We will use a combination of these two technologies to directly assess how mitochondrial quality control mecha- nistically influences the occurrence and accumulation of mtDNA mutations. Specifically, we will pursue three main focus areas: 1) Using a modified form of scATAC-Seq adapted to quantify mtDNA mutations/heteroplasmy, we will perform experiments that will test hypotheses related to if/how mitochondrial quality control (mQC) mechanisms regulate mtDNA heteroplasmy in cells and thereby keeping deleterious mutations from exceeding a phenotypic threshold by small molecule interventions and manipulations genes known to be involved in quality control; 2) We have identified mutationally intolerant sites in mouse mtDNA. We will determine the specific molecular cause behind the presence of these apparent “immutable” sites by using cutting-edge base editing methods and then performing biochemical and in vitro assays to determine the impact of the induced mutation on mitophagy, mtDNA replication, oxidative phosphorylation, and other mitochondrial functions; 3) Develop single-cell ultra-high accu- racy long read sequencing that will allow for the simultaneous accounting of single-nucleotide variants, structural variants, and heteroplasmy. Such technology is needed in order to fully understand the biology of mtDNA mu- tations in disease. The long-term objective of our work is to define the cellular mechanisms that influence the occurrence of mtDNA mutations in the germline and somatic tissues. This work will contribute to an understanding of the molecular mechanisms that influence heteroplasmy which, in turn, could ultimately lead to the development of much needed treatments for diseases caused by mtDNA mutations.

NIH Research Projects · FY 2025 · 2024-08

Abstract/Project Summary Opioid use disorder is defined as chronic, maladaptive opioid use despite negative consequences. Prolonged opioid exposure can induce long-lasting changes to neural circuitry involved with reward and motivation, leading to drug cravings and tolerance to the drug’s rewarding effects over time. One brain region known to play a key role in opioid reward processing is the nucleus accumbens, which contains two subpopulations of GABAergic medium spiny neurons that express either the D1 or D2 dopamine receptor, as well as a variety of interneuron cell types and glia. Medium spiny neurons project to multiple brain regions, and µ-opioid receptors are also expressed on many neurons throughout the brain. Thus, it is reasonable to predict that chronic opioid exposure will be associated with global alterations in neuronal activity patterns. Improving our understanding of both the molecular and circuit-level mechanisms underlying opioid use disorder is crucial to facilitating the development of more effective treatments. To address this gap in knowledge, I plan to leverage a rodent model of chronic, voluntary oral fentanyl intake that I have developed and implemented. My goal for this training proposal is to use this paradigm to specifically assess what changes occur to gene expression and neural circuitry over periods of sustained opioid self-administration. In Aim 1, we plan to conduct single nuclei RNAseq in the nucleus accumbens of mice after chronic fentanyl intake and withdrawal. Aim 2 will utilize transgenic mice to visualize expression of the early immediate gene cFOS, a marker of neuron activity. Whole-brain clearing and light-sheet microscopy will be used to quantify the neural activation patterns associated with long-term fentanyl escalation and withdrawal. Together, these aims will lead to a more complete understanding of the impact of chronic opioid exposure, which may inform the development of novel therapeutic interventions for opioid use disorder.

NIH Research Projects · FY 2025 · 2024-08

The emergence of antibiotic resistance poses a global medical threat. The discovery of new antibiotics from naturally-occurring secondary metabolites or from high-throughput screening of chemical libraries has failed to match the pace of resistance mechanisms. Therefore, new approaches for rapidly developing antibiotics are required to address this global medical need. We propose to integrate computational peptide design, deep learning, and high-throughput chemical synthesis to create a general framework for the structure-guided design of new antibiotics. Our recent advances in physics-based and deep-learning-based peptide design algorithms enable the design of structured peptide macrocycles with atomic-level accuracy. We will extend and implement these computational methods to design inhibitors of a validated target for antibiotics — the bacterial type I signal peptidase (SPase I). The secretion and correct folding of critical extracellular and periplasmic proteins in gram-positive and gram-negative bacterial requires the cleavage of their preproteins by SPase I. Naturally-occurring inhibitors of SPase I, arylomycins, show a limited spectrum of activity due to sequence variations in SPase I from different pathogenic bacteria. We propose to implement parallel strategies to design broad-spectrum inhibitors of SPase I. In one strategy, we will leverage the promising interactions between arylomycin and conserved regions of the SPase I active site as starting points and extend these interactions into macrocyclic inhibitors by sampling different backbone conformations and amino acid sequences inside the SPase active site. In a parallel de novo design approach, we will first identify promising docked conformations of individual amino acids to the SPase I pocket and then graft those interactions on pre-enumerated and predesigned sets of millions of structured cyclic peptides. The de novo design approach will be more widely applicable and enable targeting proteins that do not yet have a natural inhibitor identified. We will filter the tens of millions of computational models in silico to find the best design models for experimental validation. The promising design models will be chemically synthesized and screened for binding to SPase I as a massively parallel macrocycle library with tens of thousands of members. We will further test the computationally-designed macrocycles for SPase I binding, inhibition of the proteolytic activity, inhibition of bacterial growth across multiple species, toxicity, and the accuracy of the designed binding mode. The overall workflow will be implemented as iterative cycles of design-synthesis-test-learn to enable simultaneous optimization of potency, bacterial growth inhibition, stability, and other drug-like properties. Overall, SPase I inhibitors developed in this project will be promising candidates for further development as antibiotics. The computational and experimental methods developed during this project will be broadly applicable to targeting other bacterial proteins for designing new antibiotic candidates or inhibitors that can address antibiotic resistance mechanisms.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Chronic pain affects between 40 to 100 million people in the US and managing this pain with opioids has increased the incidence of opioid substance abuse and death from overdose. There is an urgent and unmet need for effective chronic pain medications with lower misuse potential. Δ9-tetrahydrocannabinol (THC) is the most abundant pharmacological agent found within Cannabis and acts via the endocannabinoid (eCB) signaling system to produce analgesia. Thirty-eight US states have approved legal medical Cannabis for the pharmacological treatment of pain and anxiety, alongside recreational use in many cases. However, the precise neuropharmacological and physiological mechanisms of THC use for pain treatment, and the circuit mechanisms that underlie the pain-relieving properties of THC are unknown. The basolateral amygdala (BLA) is a critical brain region important for encoding affective information from incoming stimuli and is a key component in pain processing. The BLA has increased neuronal activity during chronic pain, and glutamatergic neuronal ensembles detect and report noxious stimuli to cortical structures that impact pain perception. The BLA has enriched expression of the receptor sensitive to eCBs and THC, cannabinoid 1 receptor (CB1R). The central hypothesis of this proposal is that THC acts as a partial agonist at CB1R to decrease excitatory BLA neuronal activity and pain behaviors during chronic pain. This proposal directly addresses the 2022-2026 NIDA Strategic Plan Goal 1.1 to, “Expand our understanding of the biological mechanisms underlying drug use, addiction, diverse treatment responses…” and the NINDS 2021-2026 Strategic Plan to“… extend progress in prevention for neurological diseases beyond stroke, building on basic research advances in epilepsy, TBI, neurodegenerative diseases, chronic pain...” Aim 1 will determine whether THC acts within the BLA at CB1Rs to alter glutamatergic neuronal activity to promote analgesia during chronic pain. In this aim, we will multiplex classic neuropharmacology and dual-color fiber photometry with a machine learning-based behavioral tracking platform to directly correlate local THC-action and neural activity with naturalistic chronic pain behaviors. We then propose in Aim 2 to decode how and where THC alters specific BLAglu neuronal ensemble activity during chronic pain. We will use in vivo 2-photon calcium imaging, a CRISPR site-specific CB1R deletion strategy, and a head-fixed behavioral paradigm to observe glutamatergic BLA neuronal ensembles during pain. These findings will provide foundational knowledge informing the cannabinoid, substance abuse, neuropharmacology, and pain fields. For this proposal, I will gain training and expertise in the use of pharmacological, novel biological and high resolution computational techniques and learn valuable career development skills through a wide variety of scientific, intellectual, and mentored opportunities. This F31 proposal is specifically tailored to my personal training needs and career aspirations that will prepare me for a career as an independent academic neuropharmacologist.

NIH Research Projects · FY 2024 · 2024-07

The purpose of this Ruth L. Kirschstein National Research Service Award (NRSA) Individual Pre-Doctoral Fellowship in Nursing Research (F31) application is to provide research training for Ms. Ahrens, a third-year doctoral student at the University of Washington School of Nursing. The long-term goal of this training is for Ms. Ahrens to develop into an influential independent researcher in a research-intensive academic setting with a research focus on improving Intensive Care Unit (ICU) delirium prevention and management for patients who speak a language other than English (LOE) to improve health equity for LOE patients in critical care. ICU delirium is a consequential sequela of critical illness that affects up to 70% of ICU patients and is associated with increased length of stay, hospital costs, and risk of death. The primary preventative and mitigation strategy for ICU delirium is the ABCDEF bundle, which includes: Assess and treat pain; Both spontaneous awakening trial and breathing trial; Choice of sedation; Delirium assessment; Early mobility; and Family engagement. Due to a language barrier, LOE critically ill patients may be at increased risk of not receiving full ABCDEF bundle components, as four out of the six bundle components are reliant on effective communication for task completion and accurate assessments. Previous literature finds that professional medical interpreters are used less than 20% of the time when they are necessary in medical settings. Critically ill patients have additional barriers to professional medical interpreter use, and additional tools to facilitate cross-language communication are needed. The contributors to health disparities for LOE patients in ICU delirium care have not been previously studied. To address this gap, the proposed study will use qualitative and mixed-methods implementation science design to investigate determinants of fidelity to the ABCDEF bundle for LOE critically ill patients, guided by the health equity implementation framework and the implementation fidelity framework. Aim 1 of the study will qualitatively assess the facilitators and barriers to ABCDEF bundle fidelity for LOE patients from 32-48 key stakeholders in ICU delirium prevention, including healthcare workers who implement the ABCDEF bundle in the ICU at the University of Washington Medical Center and LOE ICU survivors who received ICU delirium prevention care. Aim 2 of the study will assess the feasibility, acceptability, and appropriateness of the VidaTalkTM application as a method to enhance communication between healthcare workers and linguistically diverse patients while performing ABCDEF bundle tasks. The findings from this study and associated training will provide foundational knowledge to generate a program of independent research for Ms. Ahrens with the goal of improving health equity for LOE patients in critical care.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT (DESCRIPTION) Brief Alcohol Screening and Intervention for College Students (BASICS) is an evidence-based harm-reduction approach developed in the early 1990’s to address problematic alcohol use among college students. Since then, there has been a proliferation of trials supporting its efficacy and effectiveness in diverse college populations and it has become the basis for the majority of current brief motivational interventions with individual-focused strategies shown to reduce high-risk drinking behavior and/or negative drinking-related consequences. Despite this, several challenges and research gaps remain in our understanding of potential barriers faced by college campuses that are currently implementing BASICS. Additionally, it is unclear whether most schools are delivering BASICS with adherence to its main components and principles or whether adaptations to the intervention are routinely applied due to feasibility considerations1. The proposed research aims to use a mixed methods approach to identify factors influencing implementation outlined by the Exploration, Preparation, Implementation, Sustainment (EPIS)2 framework and the implementation outcomes acceptability, appropriateness, and feasibility outlined by the Implementation Outcomes Framework3. Recently, the International Town and Gown Association (ITGA) financed the implementation of BASICS training with 52+ campuses across the United States, which consists of an intensive brief training model as well as providing technical assistance from BASICS experts based out of the University of Washington. Leveraging this large-scale implementation, the proposed F31 study will examine which factors influence successful integration of BASICS within diverse campus environments. This training grant will also allow the applicant to develop a depth and breadth of knowledge of BASICS and related evidence based AOD interventions as well as expand and deepen knowledge of implementation science theory, mixed-methods data collection, evaluation, and analysis methodology. Data collection through surveys and interviews will allow for a comprehensive understanding of contextual factors that impact implementation of BASICS. Additionally, the proposed research will identify descriptive characteristics of BASICS implementers and universities to better understand circumstances under which the intended mode of delivery of the intervention is likely to be adapted or adhered to. Findings will contribute to the existing literature on brief interventions for alcohol use on college campuses, offering insights into the complexities of large-scale implementation efforts. Knowledge generated by this research will inform future efforts to optimize integration of BASICS and similar programs, ultimately promoting better public health outcomes and improved student well- being at universities nationwide.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY The overall goal of this training proposal is to employ quantitative imaging to noninvasively characterize tumor microenvironmental heterogeneity in triple negative breast cancer (TNBC) for the prediction of treatment response and outcome. TNBC is an aggressive breast cancer subtype with notable diversity in disease biology and clinical presentation. Recently-approved immunotherapies introduce exciting new avenues for neoadjuvant treatment of TNBC; however, response to immunotherapy is variable and treatment can entail significant adverse effects and financial cost. Variable response to therapy can be attributed in-part to heterogeneity of the tumor microenvironment, affecting therapeutic delivery and efficacy. Through an innovative approach known as “habitat imaging”, multiparametric magnetic resonance imaging (mpMRI) can be used to spatially resolve local microenvironments within a lesion into distinct tumor subregions, or habitats. For the research component of this proposal, we propose to use quantitative breast imaging and informatics techniques to identify tumor habitats and whole-lesion habitat signatures for TNBC patient stratification. We hypothesize that imaging-derived habitat signatures identified prior to treatment can aid in stratifying TNBC patients with increased probability of achieving pCR and/or decreased risk of recurrence. We will test this hypothesis through the following specific aims: Aim 1 (K99) To retrospectively identify tumor habitat signatures from pretreatment mpMRI to stratify TNBC patients and predict treatment outcome; Aim 2 (R00) To prospectively employ habitat imaging using hybrid positron emission tomography (PET)/MRI for improved TNBC patient stratification. Successful completion of my research aims will provide a clinically-translatable methodology for improved understanding of an individual’s lesion physiology that could guide personalized treatment strategies for optimal patient outcome. To provide me with the necessary training to successfully carry out these research aims, this proposal outlines a mentored-training plan with three areas of focus: 1) strengthen my expertise in clinical breast cancer research, 2) receive additional training in computational pathology, and 3) obtain educational training and hands-on experience with PET/MRI to prepare for research in the independent R00 phase and beyond. This training program will be executed under the direct mentorship of NIH-funded researchers and clinicians specializing in medical oncology, nuclear medicine, pathology and radiology, and take place within the well-equipped and established cancer research environment at the University of Washington and Fred Hutch Cancer Center. As outlined in my career development plan, funding from this proposal will be used to dedicate time for educational workshops and training seminars, along with regular meetings with my mentorship team. Together, this training and research proposal will ensure that I am well-prepared to achieve my career goal to become an independent investigator focused on the translation of innovative imaging techniques to clinical practice to further personalize breast cancer care.

NSF Awards · FY 2024 · 2024-07

This project aims to serve the national interest by enhancing learning and promoting understanding of lab concepts through the innovative use of digital twin technology - the integration of traditional simulation-based engineering models with real-world remote 3D hardware models. Students spend a significant amount of time using programmable devices such as microcontrollers and microprocessors. A common challenge for them is the visualization of those devices as part of a real-world system rather than just circuit boards with switches and blinking LED lights (Light Emitting Diodes). This 36-month IUSE Level 2 Engaged Student Learning project, titled “Remote Experimentation and Digital Twinning for Accessible and Innovative Learning (REDTAIL)", promotes the democratization of immersive access to advanced STEM devices and equipment to students located in different cities, states and even countries. This is particularly significant for students from underrepresented and underserved communities, where the project is expected to have a substantial impact. REDTAIL, led by the Remote Hub Lab research group at the University of Washington, is to be leveraged to design, test and evaluate an innovative digital twin infrastructure for students of electrical engineering, computer engineering, and computer science. The project offers an ecosystem that is expected to overcome the limitations of traditional lab sessions by enabling students to interact with real devices in simulated real-world scenarios, thereby enhancing their grasp of fundamental engineering concepts. REDTAIL makes immersive learning globally accessible through an established network by LabsLand, a global network of remote laboratories that connects institutions to equipment located worldwide. The project's diverse team includes Navajo Technical University, LabsLand, and the University of Wuppertal and Hochschule Bonn-Rhein-Sieg University in Germany. Researchers will develop a REDTAIL Simulation Repository (RSR), which will host the simulations from within the Cloud. In order to ensure that communication between the simulation and the actual device has no latency, each simulation comprises two parts: one running on the web (visualization of simulation) and another in a controller physically connected to the target device. Additionally, researchers will develop an open source REDTAIL Development Kit (RDK) to be used by third party developers for creation, test and upload of simulations to the RSR repository. REDTAIL is centered around innovative pedagogy, blending remote experimentation with 3D simulations to enrich curricula, making them more engaging and closely aligned with real-world scenarios. Beyond its immediate scope, REDTAIL offers scalability and an open-source framework, making immersive learning globally accessible through the LabsLand network. The research planned will contribute to fundamental knowledge in three areas: 1) the integration of remote experimentation into engineering curricula; 2) the effective combination of remote experimentation with 3D simulation representation; and 3) the impact of these innovative technologies and pedagogies on faculty teaching and student learning. Project evaluation will involve collection and analysis of data at the participating institutions for measurement of student performance, engagement, satisfaction, and the overall educational impact of the REDTAIL infrastructure and teaching approach. Insights derived from evaluation will be used to further improve and enhance the ecosystem. The university's Office of Educational Assessment researchers will work with the principal investigator (PI) and project team to develop formative and summative evaluation instruments to monitor and assess the impact of the intervention. The NSF IUSE: EDU Program supports research and development projects to improve the effectiveness of STEM education for all students. Through the Engaged Student Learning track, the program supports the creation, exploration, and implementation of promising practices and tools. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-07

This project seeks to address a key challenge which involves extracting information from paleoclimate proxies with resolution as fine as one month (coral records) to several decades (sediment cores). Accordingly, most efforts to date have focused on annually resolved proxies, or separate ocean and atmospheric Paleoclimate data assimilation (DA), to mitigate this technical difficulty. This potentially limits understanding the continuum of climate variability and has important implications for near-term climate forecasts, adaptation, and planning. This project uses a different approach to connecting seasonal to millennial scales, and ocean to atmosphere. Specifically, the researchers aim to develop and implement a new 4D variational framework for multiscale proxy assimilation. The project leverages recent data syntheses of proxy observations to inform the reanalysis and use independent, borehole-based thermometry, as well as a recent synthesis of documentary data for validation. The new monthly-resolved dataset will be applied to modeling and understanding tropical cyclone statistics and their relationship to large-scale dynamics using new deep-learning weather models, and also characterize the sources of climate variability on seasonal to centennial scales. The potential Broader Impacts includes support for graduate students, development of visualizations tools for K-12 education and citizen science, and publicly available data and useful for the paleoclimate and climate dynamics communities. The project will produce a new set of global climate fields bridging seasonal to centennial timescales. A major benefit to the climate community could be a unified testing ground for interannual to decadal climate forecasts, which are presently limited by the shortness of the instrumental record. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Opioid use has reached epidemic proportions and is now the leading cause of preventable death in the United States. Beyond the risk of death, opioid use disorder causes an incalculable amount of suffering as it devastates the lives of individuals and their communities. The heightened motivation for drug rewards compared to non- drug rewards stems in part from changes in the brain circuity associated with motivation. A greater understanding of this circuitry may lead to targeted interventions that can attenuate drug motivation. Decades of research demonstrate that the lateral hypothalamus (LHA), and its modulation of the dopamine system, plays a crucial role motivation. Multiple species of animals will work tirelessly to obtain electrical stimulation of the LHA, and lesions of the LHA reduce motivation across a broad spectrum of behaviors. Recent research has revealed complexity in the function of the LHA, as subpopulations of neurons can drive discrete or even opposing behavioral phenotypes. Despite the established role in motivation broadly, the role of the LHA subpopulations in motivation for opioids remains largely unknown. This proposal seeks to investigate the behavioral function and spatiotemporal signaling dynamics of multiple LHA subpopulations and downstream dopaminergic signaling during the development and expression of motivation for opioids. During the K99 period, I will receive world-class training in the theory of opioid pharmacology and self-administration, techniques for longitudinal recording using 2-photon imaging, and advanced analysis of the relationships between neuronal signaling dynamics and behavior. In Aim 1 (K99), I will determine the causal behavioral function of multiple LHA subpopulations during motivation for opioids using transient inhibition or excitation of neurotransmitter defined neuron populations. In Aim 2 (K99), I will determine the relation between bulk signaling dynamics in the LHA subpopulations and downstream dopamine signaling using fiber-photometry and the single cell dynamics of LHA subpopulations using 2-photon imaging throughout the development and expression motivation for opioids. By recording throughout the experiment, I will have the power to characterize bulk and single-cell signals based on activity across each stage of opioid self-administration. In Aim 3 (R00), as I transition to developing my own research lab, I will investigate the caudal circuit effects of transient stimulation of LHA subpopulations during motivation for opioids. Altogether, the results of the proposed aims will deepen our understanding of the role of the LHA in mediating motivation for drugs of abuse and related neuronal circuitry. The training I will receive throughout the K99/R00 period will facilitate my development into an independent researcher investigating the behavioral function and spatiotemporal signaling dynamics related to substance use disorder.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Pregnancy-capable Veterans experience an elevated risk of adverse reproductive health outcomes relative to the general population due to a high prevalence of comorbid mental, physical, and psychosocial conditions influenced by trauma, combat exposure, and other experiences. Veterans' unique trauma and stress experiences may influence the development or severity of other health conditions that pose issues for reproductive health outcomes and care access, such as autoimmune conditions. Pregnancy, in particular, can introduce elevated risks for people with autoimmune conditions because many are managed with teratogenic medications, and pregnancy may exacerbate disease activity. Access to ongoing contraceptive care is especially vital for people with autoimmune conditions because it allows them to mitigate the potential health risks of pregnancy and control when they want to achieve pregnancy. These conditions often require regular, ongoing care from multiple providers, which may lead to gaps in access and quality of wanted contraceptive care. Subspecialists, who often provide most care for this population, may have limited expertise in contraception and fear the possible implications of pregnancy for their patients. The underlying elevated risk of adverse reproductive health outcomes coupled with the unique challenges of autoimmune conditions may be further compounded by provider and facility-level characteristics for Veterans who use Veterans Health Administration (VA), where pregnancy-capable Veterans remain a minority and availability and quality of contraceptive care are variable. The VA is an ideal setting for studying contraceptive care delivery for this population due to the system’s size, comprehensive electronic health record (EHR), and diverse patient population. The proposed mixed methods study aims to examine the potential multi-level quality and access barriers to contraceptive care delivery to Veterans with autoimmune conditions using three different data sources. First, differences in documented procedural or prescription contraception will be investigated for Veterans with the most prevalent autoimmune conditions compared to those without these conditions using structured EHR. Hierarchical modeling will identify facility, provider, and patient-level factors associated with the documentation of a method. Second, novel natural language processing software and qualitative analytical methods will be applied to unstructured EHR clinical notes to characterize contraceptive care for Veterans without a documented method to explore contributors to the lack of documentation, such as poor care quality or lack of need. Third, the findings from the first aims of the project will be further contextualized by conducting qualitative interviews with Veterans with autoimmune conditions with and without documentation of contraception to characterize access and quality barriers to receiving desired contraceptive care. The results from these aims can provide evidence of potential issues with contraceptive care delivery for Veterans with autoimmune conditions and offer actionable steps for improving VA policy and practice related to this care.

NSF Awards · FY 2024 · 2024-07

Metabolism is the set of essential chemical reactions in all organisms, including humans, which sustain life. The reactants are called metabolites and represent thousands of distinct compounds, many of which are essential to biological function. Metabolic homeostasis describes the processes that maintain the concentrations and control the rates of the constitutive metabolic reactions. Although much is already known about specific reactions in this process, recent experiments demonstrate an unexpected behavior: the periodic oscillations of central metabolites, including ATP, the molecular unit of cellular energy, as well as other nucleotides (NTPs and dNTPs). The focus of this proposal is understanding both how (the mechanism by which these oscillations are generated) and well as why (the biological rationale) these oscillations occur. Learning what attributes of regulatory control are optimized by the cell is a challenge of central importance to understanding the emergent principles that describe the function of living systems. The widespread detection of regulatory oscillations would change our fundamental mechanistic understanding of cellular regulation, and characterizing their dynamics could become a novel tool for the discovery of regulatory interactions. The project will pursue four aims that will help understand the mechanism by which these oscillations are generated, as well as the biological rationale for these oscillations to occur. Aim 1: Model regulatory oscillations. The team of investigators will model nucleotide homeostasis to explore the phenomenology of these regulatory networks and determine under what conditions oscillatory behavior is expected. We will analyze the tradeoffs between regulatory precision, rapidity, and overshoot. Aim 2: Characterize fork-velocity oscillations. Key evidence for metabolic oscillations comes from the PI’s recent observation of replication-fork-velocity oscillations. We will test the proposed regulatory mechanism for these oscillations by measuring their response to regulatory perturbations. Aim 3: Measure cell-cycle-dependent transcription of metabolic enzymes. The PI predicts that metabolic oscillations should result in the cell-cycle-dependent expression of many metabolic enzymes in nearly all bacterial systems. The team of investigators will test this hypothesis using RT-qPCR, RNA-Seq, and fluorescence microscopy. Aim 4: Measure cell-cycle dependent metabolite levels. The team of investigators will directly test for the existence of metabolic oscillations using two independent approaches: NTP and dNTP levels will be measured in synchronized cells using Liquid Chromatography Mass Spectroscopy (LC-MS) and ATP levels will be measured using a biosensor in single cells using fluorescence microscopy. In addition to these scientific aims, this award will support a number of educational activities. It will support the training of graduate and undergraduate students working in an interdisciplinary environment that combines physics, chemistry, engineering, and biology. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY The objective of this proposal is to define the impact of maternal viral infections and inflammation on immune programming within the fetal brain and lymphoid organs in a well-defined non-human primate (NHP) model. Discoveries in fetal human immunology have primarily come from human cord blood with few studies of fetal blood or the innate immune capacity of major organs. The link between innate immune activation and fetal brain injury is unknown but is of major translational significance to guide novel therapies for fetal protection. Our central hypothesis is that the fetal innate immune programming induced by a maternal ZIKV infection or other infectious triggers initiates a program of cellular stress-response in the fetal brain. This connection between the fetal immune programming and cellular stress-response pathways in the fetal brain has never been studied across gestation or with a suite of diverse and highly sophisticated immunologic tools and platforms. Our preliminary studies in a microculture ex vivo model using human fetal tissues demonstrate active regulation of the innate immune response by Sendai virus (SeV; a model virus inducing innate immune activation) and Zika virus (ZIKV) by 24 hours post-infection. Our preliminary data reveals that 3 days after a maternal ZIKV infection in our NHP model, there is a strong correlation in the fetal brain linking the innate immune response with induction of cellular stress and autophagy. In this proposal, we will use the NHP model to obtain a complete collection of fetal blood and major tissues from the first and third trimesters to interrogate maturation of fetal innate and adaptive immune programming in the fetal brain and major lymphoid organs, which we will link to cellular stress in the fetal brain. In Aim 1, we will use an ex vivo microculture model to determine innate immune pathways activated by model viruses (ZIKV, SeV), Type I IFN (IFN-β), IL-6 and TNF-α in the first and third trimesters within NHP fetal brain and lymphoid organs (fetal brain, placenta, spleen, thymus, blood). In Aim 2, we will use a pregnant NHP model of an acute ZIKV infection to link the profile of fetal innate and adaptive immune activation with production of alarmins and induction of the autophagic response in the fetal brain. In both Aims, we will employ diverse immunologic tools (CyTOF, Nanostring nCounter, Luminex, ELISA, immunohistochemistry, bulk and single cell RNA-Seq) producing high-dimensional data that can be leveraged using bioinformatics to reveal gene networks of innate immune programming that direct viral and inflammatory injury of major fetal organs in early and late gestation, which can inform therapeutic approaches for fetal neuroprotection in the setting of inflammatory preterm birth or congenital viral infection.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY/ABSTRACT Children under 5 years of age account for ~50% of the 1.2 million new cases of pediatric TB each year, but are least likely to be diagnosed, and are at highest risk of death without prompt treatment. Young children are more likely to present with disseminated or extrapulmonary TB and paucibacillary disease, often missed by respiratory sampling and our currently available diagnostics. Non-sputum diagnostic tools for TB detection and treatment response in young children, using easily obtained specimens are urgently needed. Our team has successfully developed an ultra-sensitive CRISPR-based approach (CRISPR-TB) that detects M. tuberculosis (Mtb) cell free DNA (cfDNA). In pilot evaluation with repository blood samples, CRISPR-TB demonstrated high sensitivity (94%) and specificity (95%) among adults and children with TB and their asymptomatic household contacts in Eswatini. Among hospitalized Kenyan children with HIV (median age 2 years), CRISPR-TB detected 100% with microbiologically confirmed TB, and an additional 85% with clinically diagnosed TB (i.e. missed by respiratory-based diagnostics). We now propose to expand CRISPR-TB evaluation in a large prospective cohort of 400 children with suspected TB (majority <5 years) to evaluate CRISPR-TB diagnostic performance (Aim 1) including longitudinal evaluation for treatment response (Aim 2), and extend evaluation to urine samples (Aim 3). Additional cohorts of adults with confirmed TB and their asymptomatic household controls, and recently BCG-immunized asymptomatic infants will be evaluated. Exploratory aims will pilot a POC platform, assess utility of CRISPR-TB to identify early incipient or subclinical disease in participants with missed diagnosis at baseline who develop incident TB, and estimate “true” prevalence of pediatric TB using Bayesian latent class analysis given imperfect reference tests. We hypothesize CRISPR-TB will have similar/improved diagnostic performance to Xpert on respiratory samples among children with confirmed TB without the need for sputum, and identify additional children missed by respiratory samples, will provide a useful surrogate marker of treatment response with decline in quantitative levels during successful treatment, and be adaptable to urine samples.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT The overarching goal of this collaborative and translational research on hepatitis B (HBV) is to discover a safe and effective long-acting antiviral (ARV) therapy that can provide sustained viral suppression. The current daily and chronic oral antiviral dosing for HBV patients causes pill fatigue and leads to fluctuating drug levels. Both aspects lead to viral rebound and increases the risk of liver cancer. The introduction of Cabenuva, a long-acting HIV drug product containing two injectables (cabotegravir and rilpivirine two different products used in combination), has been a game-changer for the AIDS community, addressing pill fatigue and other barriers. However, patients co-infected with HBV and HIV are not benefiting fully from this long-acting HIV therapy. To mitigate the risk of HBV rebound and progression to cirrhosis and/or liver cancer, there is an urgent need for a long-acting HBV product that can provide sustained viral suppression, alleviate pill fatigue, and ensure consistent antiviral coverage for both people with HBV and HBV-HIV. The proposed translational research aims to leverage the expertise and collaborative capacity of the investigative team to transform current best, oral antiviral drugs that is short acting into a long-acting HBV therapy. With extensive resources and a proven track record in preclinical and clinical antiviral research and product translation, our goal is to identify a lead and backup product candidate suitable as monthly self-injectable long-acting HBV therapy. The intent of the HBV therapeutic candidate it is for use alone or in combination with existing long-acting HIV therapies like Cabenuva. The proposed approach utilizes a well-established platform technology to develop innovative and new long-acting anti-HBV products, with the potential to achieve longer-lasting viral suppression and improve HBV therapeutic outcomes. Our research plan incorporates a set of success matrices and aims based on a defined target product profile (TPP), with guidance from an external scientific advisory board (SAB). The research aims are: (1) identifying lead and backup antiviral compositions and processes through molecular-level drug-lipid (excipient) interaction studies to define the characteristics of long-acting products, (2) evaluating and defining pharmacokinetic profiles of long- acting dosage forms in vivo to identify lead and backup candidates, (3) identifying preferred user characteristics for long-acting HBV therapy among people living with HBV, and (4) verifying antiviral activity through dose-response pharmacokinetic evaluation in a primate model and a woodchuck hepatitis model. This five-year translational research progress from in vitro drug-lipid interaction studies to the evaluation and selection of a lead and backup long-acting anti-HBV therapy for preclinical and clinical development. The study results will inform early discussions with the FDA and the FDA guidance received will serve as the foundation for seeking NIH to support the manufacturing of GMP materials and GLP toxicokinetic studies, thereby advancing our overarching goal of developing a novel long-acting HBV therapy for individuals living with HBV.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Introduction: Jason Coult, PhD, is a scientist whose career goal is to become an independent computational investigator improving survival from out-of-hospital sudden cardiac arrest (OHCA) through research of novel OHCA resuscitation technologies that incorporate clinical understanding. His K01 proposal, “Artificial Intelligence to Improve Resuscitation following Out-of-Hospital Cardiac Arrest”, seeks to develop smarter, deep learning-based defibrillator algorithms that can improve OHCA survival by guiding personalized resuscitation treatment. Candidate: Dr. Coult is a recently- appointed Research Assistant Professor at the University of Washington Department of Medicine. He completed a PhD in bioengineering in 2019, and has expertise in signal processing, artificial intelligence (AI), and OHCA research. Career development and mentorship: Dr. Coult has convened an interdisciplinary team of mentors and advisors with expertise in animal and human clinical resuscitation, cardiac electrophysiology, prehospital emergency medical services (EMS), AI and deep learning, computational arrhythmia models, biostatistics, and defibrillators. The training plan cultivates necessary skills in deep learning and biostatistics, advances understanding of clinical resuscitation, provides career guidance, and facilitates Dr. Coult’s progression to become a successful independent investigator. The proposed work will take place under Thomas Rea MD (primary mentor) at the University of Washington. Proposed research: OHCA is a leading cause of mortality. Survival is possible though generally poor. Resuscitation currently follows a fixed, one-size-fits-all protocol that requires CPR interruption at regular intervals to determine the patient’s cardiac rhythm, assess vital status, and apply treatment. This research will use large retrospective datasets of human OHCA defibrillator recordings, Dr. Coult’s expertise and mentorship/advisory group, and emerging deep learning methods to achieve the following aims: 1) to design deep learning-based AI algorithms that can identify the specific OHCA rhythm (asystole, ventricular fibrillation (VF), organized rhythm) and estimate the “vitality” (morphologic phenotype associated with clinical outcome) of these rhythms during ongoing CPR, 2) to characterize the effect of drug interventions on measures of vitality, and 3) to apply rhythm and vitality features to predict shock-refractory VF patients (requiring ≥ 3 shocks) during CPR, enabling preemptive antiarrhythmics or other strategies to mitigate subsequent shock failure. Summary: The proposed research will advance resuscitation towards a precision strategy that aligns treatment with an individual patient’s real-time physiology, providing the potential to improve OHCA survival. The training and mentorship will foster the development of necessary AI-related technical skills, deepen clinical understanding of OHCA and resuscitation, and help inform next- step, R01-funded research, collectively supporting Dr. Coult’s transition to an independent investigator.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT [30 lines] The continued growth of syphilis cases in the United States is associated with significant maternal-fetal morbidity and mortality, health disparities for racial/ethnic minorities, and men who have sex with men (MSM). Rising case counts have led to a shortage of penicillin, the treatment of choice for the etiologic agent of syphilis, Treponema pallidum subsp. pallidum, and the only approved therapy for pregnant patients and neonates. Penicillin shortages have led to increased reliance on doxycycline for treatment and prophylaxis, but patient compliance is suboptimal, particularly for doxycycline treatment which requires twice daily dosing for > 14 days. These conditions - high case counts, increased use of doxycycline, and potential incomplete treatment – are ripe for selection of doxycycline resistant T. pallidum strains. A previous second line treatment, single dose azithromycin, is no longer recommended due to high rates of resistance (> 79%) observed among MSM. However, susceptible strains may circulate among non-MSM populations where resistance rates have historically been much lower (11%) suggesting there are still patients who could benefit from azithromycin therapy. Unfortunately, there are no clinical assays that detect T. pallidum resistance to either drug. Such assays would serve the dual purposes of public health surveillance and informing therapeutic antibiotic selection, particularly for patients unlikely/unable to comply with multiday doxycycline treatment. Resistance to azithromycin is mediated by single nucleotide polymorphisms (SNPs) in the 23S rRNA gene and doxycycline resistance is predicted to emerge as SNPs in the 16S rRNA gene. Resistance-conferring SNPs are amenable to molecular detection by allele-specific nucleic acid amplification assays. We have previously developed and validated in our clinical laboratory a sensitive and specific Reverse-Transcription Loop-mediated isothermal Amplification (RT-LAMP) molecular test for T. pallidum and are currently validating the assay in a multiplexed, point-of-care (POC) format. In this project, we propose to build upon our existing assays by developing complementary approaches to detect antibiotic resistance in T. pallidum. The deliverables will be a POC allele-specific LAMP for drug resistance (AS- LAMP) multiplexed with organism detection and an internal amplification control, and a parallel next- generation sequencing (NGS) assay for high-complexity clinical laboratories. In Aim 1, we propose to develop and validate both a POC AS-LAMP and an amplicon-NGS assay for detection of SNPs mediating azithromycin resistance. In Aim 2, we propose to develop and validate an amplicon-NGS assay for detection of doxycycline resistance and to develop a panel of AS-LAMP primers that can be deployed following detection of emerging doxycycline resistance genotypes. After primer and protocol optimization, we will validate the assays following established procedures for deploying clinical assays in CLIA- and CAP-certified laboratories. Our goal is to generate inexpensive, rapid POC testing for T. pallidum drug resistance to guide optimal therapy during a single patient encounter and to improve public health surveillance for drug resistant strains.