University Of Washington

NIH Research Projects · FY 2025 · 2021-07

Project Summary: Patients with bone dominant (BD) and bone only (BO) metastatic breast cancer (MBC) represent a large patient population1,2 who are often excluded from clinical trials using RECIST 1.1 as the primary response assessment because bone lesions are classified as non-measurable, non-target lesions3. Current blood-based biomarkers such as tumor markers (CA15.3, CA27.29 and CEA) have similarly shown limited utility in assessing response to therapy in patients with MBC. There is therefore an important need for better measures of therapeutic response for patients with BD MBC. EA1183 FEATURE is a prospective, multicenter clinical trial approved by the NCI and sponsored by ECOG-ACRIN designed to evaluate the value of serial FDG-PET/CT to assess response in BD MBC. The trial will test the ability of tumor metabolic changes to predict the clinically meaningful outcomes of progression free survival (PFS) and time to skeletal-related event (tSRE). Measurement and characterization of ctDNA provides an option of non-invasively evaluating both disease burden and emergence of genomic changes in tumor biology. We propose to integrate fluid-based tumor monitoring (by serial collection of circulating tumor DNA, ctDNA) and FDG-PET/CT imaging to determine if these biomarkers, separately or combined, can predict a response to therapy for in patients with BO or BD MBC participating in the EA1183 FEATURE trial. We will also assess the extent to which FDG-PET/CT, ctDNA, or both can predict PFS as early as 4 weeks into therapy. We hypothesize that integration of imaging (FDG-PET/CT) and fluid-based, liquid biopsy (ctDNA) assays may permit characterization of therapy response for patients with BO and BD MBC in advance of currently used methods, possibly as early as 4 weeks. This R01 proposal will provide support for additional objectives in EA1183, which are the aims of our proposal: 1.) to assess ability of qualitative and quantitative changes in serial ctDNA measures to predict PFS and time to SRE in patients with BO or BD MBC beginning new systemic therapy in EA1183; 2) to determine if early metabolic changes in bone metastases assessed by FDG-PET/CT at 4 weeks after start of systemic therapy predict PFS and tSRE in patients with BO or BD MBC; 3) to evaluate the relationship between changes in ctDNA and metabolic response as assessed by FDG-PET/CT and to test the combined ability of FDG-PET/CT and ctDNA at 4 and 12 weeks after the start of new systemic therapy to predict PFS and SRE. The outcome of this study will be the generation of robust response endpoints for BD MBC to provide access to clinical trials and guide clinic al practice for the large group of patients with this type of MBC.

NIH Research Projects · FY 2025 · 2021-07

Global health is a multi-disciplinary field that aims to improve health equity for individuals, families and populations worldwide. Nurse scientists are uniquely positioned to develop research to understand public health problems, inform health interventions, optimize health systems, and promote health within communities and across populations. Importantly, global health is concerned with problems that transcend national borders, both abroad in other countries and domestically in the United States. This global to local approach prioritizes international cooperation and collaboration from multiple disciplines to achieve health equity through evidence- based prevention and clinical care for those disproportionately burdened with illness and most in need of health services. The National Institute of Nursing Research supports global health research to achieve its mission of advancing nursing science to improve the health and well-being of all the world’s citizens. Accordingly, there is a need to train a cadre of nurse scientists who pursue scholarly careers to lead and contribute to research on global health issues. The Research in Nursing and Global Health (RiNGH) training program, coordinated by the University of Washington Center for Global Health Nursing and the University of Washington School of Nursing’s Office of Diversity, Equity and Inclusion will prepare pre-doctoral and post-doctoral trainees with the knowledge and skills to advance global health equity through team-based, interdisciplinary research. Trainees will benefit from a variety of learning experiences spanning coursework, research seminars, research residencies, independent research projects, professional development and career building workshops, and participation in scientific meetings and conferences. Trainees will have access to a group of established, committed investigators in Nursing and other health sciences disciplines with sustained programs of research related to global health and health equity to serve as mentors and guide their overall training. The specific aims of the RiNGH training program are to: (1) recruit and retain a diverse group of qualified, promising pre-doctoral and post-doctoral trainees to conduct research aiming to improve population health and healthcare globally and locally, using a health equity lens; (2) provide interdisciplinary, didactic research training in current and emerging theories, methodologies, and skills essential for conducting global health and health equity research; including the appraisal, design, and implementation of research approaches; (3) mentor trainees in principles and values of respectful community engagement and ethical conduct of research, especially in contexts involving marginalized, underserved populations abroad and domestically; (4) prepare future scholars for competitive research careers in academia, research centers, healthcare systems, government agencies, non-government organizations, foundations, or industry, through learning experiences and professional development as well as exposure to active research projects with foci in global health and health equity; and, (5) evaluate pre-doctoral and post-doctoral training program structures, processes, and outcomes on an ongoing and annual basis.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Big potassium (BK) channels, named after their "Big K+" conductance, are atypical potassium channels activated synergistically by voltage and calcium. Expressed in nearly all organs, BK channels have important roles in the function of the nervous, gastrointestinal, reproductive, visual, endocrine, urinary, lymphatic, skeletal muscle, and cardiovascular systems. BK channel activation requires the allosteric binding of calcium to the cytoplasmic C- terminal of the channel. Therefore, spatial localization at the plasma membrane and proximity to calcium sources are two critical factors regulating BK channel function. My previous work using super-resolution microscopy has demonstrated that rather to be homogeneously distributed at the plasma membrane, BK channels organize into large clusters. In addition, I demonstrated that these BK channel clusters organize into a higher hierarchical arrangement with voltage-gated calcium channels (CaV), which are their primary calcium source for activation. Combining electrophysiology and pharmacology, I established that this BK-CaV co-clustering is key for the activation of BK channels at physiological hyperpolarized voltages. Yet, the molecular mechanisms that mediate BK channel cluster formation and the co-clustering with specific CaV channels remain mysterious. Research in my lab now builds on my discoveries and focus on identifying the molecular mechanisms behind BK-BK and BK- CaV channel clustering and the role of clustering in the fine-tuning of BK function. Leveraging our expertise in super-resolution microscopy, quantitative fluorometry, ion channel biophysics, and lipid metabolism, we have identified three research areas that will advance our understanding of the mechanisms behind BK channel clustering. Research area 1 will focus on the study of the mechanisms mediating the formation and maintenance of BK-BK clusters – for this, we will systematically evaluate the role of the BK channel C-terminal and the role of BK β and γ auxiliary subunits. Research Area 2 will focus on the mechanisms mediating BK-CaV channel co- clustering – for this, we have identified AKAP150 and RIB2 as two strong candidates that interact with both channels and can underlie the co-clustering. Research Area 3 will focus in a poorly explored topic, the role of membrane lipid composition in the clustering of BK-CaV channels – I will leverage my expertise in the study of ion-channel modulation by phosphoinositides and cholesterol to lead the research on how these two signaling lipids are involved in ion channel clustering. Overall, this research program is designed to provide new insights into how BK channel clusters are formed and maintained near calcium sources. In the long-term, this proposal will open new avenues for the study of mechanisms modulating BK channel function in health and disease.

NIH Research Projects · FY 2024 · 2021-07

Summary The clinical gold-standard method for interrogating tissue specimens, slide-based (2D) histopathology, is based on centuries-old technologies with many inherent limitations. Recent technological advances have demonstrated the feasibility of achieving high-throughput slide-free 3D histology of biopsy and surgical specimens. In comparison to conventional slide-based histology, nondestructive 3D histology has the potential to provide a transformative improvement in diagnostic pathology performance for a number of reasons: (1) vastly greater (>100X) sampling of tissue specimens, (2) volumetric imaging of 3D cell distributions and tissue structures that are prognostic and predictive, (3) nondestructive imaging, which allows valuable biopsy specimens to be used for downstream biomarker assessment, and (4) a simplified process with cost benefits for healthcare institutions and payers. In recent years, we have developed a technology, open-top light-sheet (OTLS) microscopy, to enable high-throughput nondestructive 3D histology of ex vivo specimens. Our first generations of OTLS microscopes and imaging protocols demonstrated the ability to reliably image a variety of optically cleared clinical tissue specimens (surgical excisions and biopsies) in a nondestructive manner that does not interfere with conventional pathology methods. Here, we propose to develop a multi-resolution hybrid OTLS microscope (Aim 1), based on a novel non-orthogonal dual-objective (NODO) architecture, which will be superior in every regard to our previous systems, including resolution (and range of resolutions), imaging depth, and compatibility with nearly all clearing/labeling protocols and sample-holder materials (insensitivity to refractive-index mismatch). Furthermore, we will develop innovative pre-imaging methods to automate and standardize the tissue-labeling and clearing process for a robust fluorescent analog of H&E staining (Aim 2). Finally, we will develop post- imaging technologies for image-guided macro-dissection of thick tissues, which we will show has the ability to significantly improve the sensitivity of genomic assays (Aim 3). Collectively, our project aims are designed to extend current 2D pathology workflows into 3D to minimize clinical-adoption barriers. A rapid translational pathway exists through a 3D-pathology-services company (Lightspeed Microscopy Inc.) that has licensed our entire 3D pathology IP portfolio. As part of this larger translational effort, clinical studies are ongoing in our labs, along with development of AI-analysis methods for clinical decision-support (i.e. prognostication and prediction of treatment response). The instrumentation platform developed in this project will directly support a number of future disease-focused clinical studies to demonstrate the value of 3D pathology for the precision treatment of diverse conditions such as kidney disease, neurodegenerative diseases, and various forms of cancer.

NIH Research Projects · FY 2025 · 2021-07

Abstract The Cellular and Molecular Biology (CMB) training program offers doctoral candidates a multidisciplinary education in molecular and cellular biology at institutions located within the Seattle biomedical corridor. Administered out of the University of Washington (UW), this interdisciplinary program also supports predoctoral trainees at the Fred Hutchinson Cancer Research Center (Fred Hutch) and partner institutions. The primary objectives of the CMB program are to recruit a diverse group of enthusiastic and motivated students who are passionate about the biomedical sciences and to provide personalized training across a range of disciplines pertaining to basic and translational aspects of molecular and cellular biology. These talented individuals have the opportunity to be mentored by 63 faculty members who are experts in a range of disciplines. Students are drawn from seven participating graduate programs that include Biochemistry, Genome Sciences, Immunology, Microbiology, Molecular and Cellular Biology, Neuroscience, and Pharmacology. CMB trainees enter the program in Year 2 of graduate school after holistic selection through written and oral components of the annual application competition. Trainees complete mandatory coursework in biostatistics and fundamentals of molecular biology; participate in the Biomedical Research Integrity Lecture series; attend a monthly student organized research conference with speaking and networking opportunities; receive training in scientific rigor and reproducibility, gain scientific writing skills, participate in a peer mentoring program and take part in the annual CMB Training Grant retreat. Traditionally, the CMB program has successfully partnered with several minority advocacy groups to promote diversity on all campuses. We continue to expand the under-represented minority (URM) footprint and are now additionally emphasizing the recruitment and retention of students who are the first in their families to attend college (first generation) and students with disabilities. This innovative graduate training environment encourages trainees to pursue scientific excellence and endorses peer mentorship and the exploration of alternative career paths. The intended outcome is to nurture a diverse close-knit group of students who are equipped to become the next generation of scientific leaders.

NIH Research Projects · FY 2025 · 2021-07

Abstract: Opiate addiction extorts a tremendous toll on society, but a mechanistic understanding of how repeated exposure to opioids such as heroin ultimately results in compulsive drug-taking and -seeking behavior in some individuals, but not others, is still not known. A longstanding idea is that enduring changes in neural circuit function occur because of drug-induced gene expression changes in certain brain cells. This facilitates subsequent drug-taking and -seeking behaviors in vulnerable individuals. Unfortunately, identifying cell-type specific alterations following drug use (typically performed in established animal models of addiction), is generally a slow and tedious process as changes in gene expression following in vivo drug exposure are typically assayed in series, within heterogeneous brain regions, in an a-priori hypothesis driven fashion (i.e. previous knowledge predicting a specific gene may be involved). This dramatically limits the throughput of data collection and likely complicates the subsequent interpretation as gene expression patterns data are typically captured from thousands to millions of homogenized cells. Given that the nervous system is composed of highly heterogeneous tissue, re-assessing cell type specific gene expression changes in an unbiased manner from 1000's of individual cells is desperately needed. Here, we propose to combine our expertise in order to generate comprehensive datasets aimed at understanding how single-cell gene expression, circuit connectivity, and neural activity patterns are impacted by previous drug-taking behavior. These data will provide a much-needed cellular atlas and resource for the addiction neuroscience community and will likely lead to the identification of many novel cell type, gene expression changes, and ensembles that can be leveraged for future study.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Over one million new cases of tuberculosis (TB) and 239,000 TB-related deaths occur in children each year. Young children, especially those with HIV, are more likely to present with disseminated or extrapulmonary TB and paucibacillary disease, often missed by respiratory sampling. Non-sputum, biomarker-based diagnostic tools for rapid TB detection and treatment response in children, using easily obtained specimens are urgently needed. Exosomes are small (30-100 nm) membranous extracellular vesicles (EVs) originating from endosomal cell compartments; those secreted by M. tuberculosis (Mtb) or Mtb-infected macrophages appear to play a significant role in Mtb pathogenesis. Our collaborators have developed a rapid and sensitive nanoplasmon-enhanced scattering (nPES) assay which directly detects Mtb-exosomes (Mtb-EVs) from as little as 1 L of serum. Proof- of-concept nPES assays performed with Mtb markers LpqH (19-kDa Mtb lipoprotein) and LAM distinguished adult TB from at-risk patients and normal controls, and among pediatric TB cases (including HIV+) and controls with high sensitivity and specificity. We propose using archived specimens and clinical data from the Pediatric Urgent Start of HAART (PUSH) Study (NCT02063880) and a new proposed prospective cohort of children suspected of TB with high HIV prevalence to evaluate performance of nPES detected Mtb-EVs for pediatric TB diagnosis (Aim 1), treatment response (Aim 2), and evaluation of a point-of-care platform (Aim 3). In addition to assessing conventional diagnostic performance measures, we propose to use advanced epidemiologic methods (Bayesian latent class analysis) given the context of an imperfect reference. Additional evaluation in adult TB patients and healthy controls including household contacts (adults and children) and recently BCG-vaccinated infants without TB is proposed. We hypothesize nPES detected Mtb-EVs will 1) have similar diagnostic performance to the reference of Xpert/culture among children with confirmed TB without the need for sputum, and identify additional children missed by respiratory sample, 2) will provide a useful surrogate marker of treatment response with decline in quantitative levels during successful treatment, and 3) will maintain performance with a point-of-care platform. Using cryopreserved samples from a well-characterized cohort of children with HIV who underwent intensive TB evaluation and a prospective cohort of children suspected of TB with high HIV prevalence provides opportunity for efficient evaluation of a novel diagnostic with potential for clinical impact to improve pediatric TB diagnosis.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Heart disease is the leading cause of death in the U.S., often driven by irreversible cardiac tissue damage leading to heart failure. Cardiac tissue does not naturally regenerate, and thus is an important area of focus for tissue engineering. Previous development into engineered heart tissue (EHT) "patches" with some functional architecture such as pre-patterned vasculature and alignment has shown promising results when implanted into small animal models. Moreover, injected stem cells and implanted cardiac "sheets" have shown some success in restoring some cardiac function when implanted into infarcted hearts of large animals or humans. Despite these successes, scaling EHT that incorporates important functional features such as vascularization and alignment to a physiological thickness (cm-scale) remains a major engineering challenge. The overarching goal of this proposal is to generate in vitro physiologically thick heart tissue patches that can ultimately be implanted into patients to replace damaged tissue. Two key considerations for recapitulating native tissue are (1) cardiac tissue is highly vascularized, and (2) alignment of cells and extracellular matrix within each physiological layer is critical to function. We address these challenges utilizing novel open microfluidic patterning approaches. Our open microfluidic technological advancement offers unique benefits to EHT; for example, it is compatible with virtually any hydrogel, including standard extracellular matrix material such as collagen and fibrin, used extensively for EHT. It is also compatible with specialized stimuli-responsive engineered hydrogels, opening up possibilities for complex engineered tissues with spatial and temporal control. Further, the flow of precursor fluid is driven by passive surface tension forces; thus, sensitive stem-cell- derived cells are not exposed to shear stress from extrusion through a needle or photochemical crosslinking, which are requirements for other tissue fabrication techniques such as 3D bioprinting. Finally, a large area (cm- scale) can be patterned with a single pipetting step, making this fabrication approach ideal for generating large (cm-scale) tissues. In this proposal, I apply these unique attributes of open microfluidic pattering to EHT. Specifically, the ability to pattern enzymatically degradable gels through a background of standard cell culture ECM materials such as collagen or fibrin enables the patterning of complex vasculature in three dimensions. I will also take advantage of previously demonstrated modular stacking of open microfluidic devices and suspended microfluidics to generate aligned EHT patches. In these patches, the tissue is anchored on either end, inducing ECM remodeling and alignment. Each layer is generated and aligned independently. Then, they are stacked together at an angle from the previous layer, creating a multilayered tissue mimicking the heart's helical tissue fiber alignment. As such, I will address in two independent aims, vascularization and tissue alignment of physiologically thick EHT using open microfluidics.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Lower respiratory tract infections, including pneumonia, are the most common cause of infection-related death worldwide. γδ T cells are highly conserved leukocytes possessing innate-like immune response characteristics. Enriched in the mucosa, γδ T cells may play an outsized role in regulating pulmonary inflammatory responses and have been proposed as a basis for novel immunotherapies. However, data concerning the inflammatory role of pulmonary γδ T cells during pneumonia in humans are limited. Dr. Shelton Wright is a pediatric intensive care physician-scientist whose goal is to obtain the necessary training to become an independent translational investigator of pulmonary host defense. He has established a translational research program that has identified a critical role of γδ T cells in pulmonary melioidosis, a highly lethal cause of pneumonia. Based on these findings, he has developed a central hypothesis that host resistance to pulmonary melioidosis is dependent on an IL-17+ γδ T cell inflammatory response. Dr. Wright's research goal is to determine the mechanisms underlying the host γδ T cell inflammatory response during severe lung infection. Using pulmonary melioidosis as a model for severe lung infection, Dr. Wright will test his hypothesis through the following 3 Aims: 1) Determine the role of γδ T cells in lung inflammation during pulmonary melioidosis in vivo; 2) Identify differential human γδ T cell inflammatory responses to B. pseudomallei infection in the ex-vivo lung compared to peripheral blood; and 3) Characterize γδ T cell activation in patients with early pulmonary melioidosis. The Career Development Plan for this grant proposal will provide Dr. Wright with critical training in pulmonary immunology, transcriptomics and high containment pathogen research. Dr. Wright has leveraged the outstanding training infrastructure at the University of Washington and its affiliates to bring together a wide array of experts in these fields to provide targeted mentorship for this proposal. Dr. Wright's multidisciplinary team of mentors will greatly complement his proposed research project along with a carefully selected didactic program. The data generated by his proposal and the skillsets developed from his training program will significantly facilitate Dr. Wright's goal of developing into an independent translational physician-scientist investigating the mechanisms of pulmonary host defense during severe lung infection.

NIH Research Projects · FY 2026 · 2021-06

Project Summary Opioid abuse has reached epidemic proportions in the United States and is responsible for more than 40,000 overdose deaths each year 1. In particular, synthetic opioid addiction has proven to be extremely difficult to combat, in part because it generates powerful opponent processes in the user. Each dose of synthetic opioid produces rapid and potent euphoria that is strongly associated to drug cues, while withdrawal and abstinence from synthetic opioids induce severe distress. Avoidance of withdrawal and subsequent exposure to drug cues are key deterrents to long-term abstinence. The Lateral Habenula (LHb) is an exciting target for studying neuronal facilitation of relapse, as LHb activity is correlated with both stress evasion and the encoding of motivational value 6. LHb activity during acute withdrawal may motivate relapse via stress avoidance mechanisms, while during abstinence cues that are normally paired with drugs go unrewarded, inducing activity in the LHb. This activity may motivate drug seeking via a process akin to reward prediction error. Under the primary mentorship of Drs. John Neumaier, Michael Bruchas, Paul Phillips, and Charles Chavkin, this K99/R00 Pathway to Independence award will allow me to obtain training in cutting edge in-vivo calcium imaging and optogenetics, and oral self-administration model development. This training will allow me to elucidate the roles of the major LHb output pathways in motivating avoidance of fentanyl withdrawal and cued reinstatement. During the mentored phase of this grant I will be trained to use GCaMP6-based in-vivo calcium imaging to record LHb neurons that project specifically to the ventral tegmental area (VTA), the rostromedial tegmentum (RMTg), or the dorsal raphe nucleus (DRN) during naloxone-precipitated withdrawal, conditioned place aversion testing, and fentanyl reinstatement. I will also be trained to combine in-vivo calcium imaging with red-shifted optogenetic inhibition in order to leverage simultaneous circuit imaging and recording, and test causal relationships between neural pathway activity and behavior. Finally, I will be trained in novel oral fentanyl self-administration (SA) model development, which will be invaluable to my independent research career as a behavioral neuroscientist. During the independent phase of the award, I will combine this training with my prior expertise to determine if activity in each LHb pathway is necessary for expression of withdrawal related negative affect, withdrawal induced CPA, or cued reinstatement to fentanyl seeking. I will do so by simultaneously inhibiting LHb projections and recording from each LHb target region (VTA, RMTg, or DRN) to determine the effect of inhibition on behavior and monoaminergic nucleus activity. In summary, the research proposed in this Pathway to Independence Award will elucidate the role of individual LHb pathways in motivating avoidance of withdrawal and cued reinstatement, as well as their role in the expression of negative affect. This award will provide training in new technical tools, grant writing, lab management, mentorship, and other skills necessary to establish an independent research program capable of producing high impact studies and training the next generation of scientists.

NIH Research Projects · FY 2025 · 2021-06

Project summary/abstract NACC (as U01 AG016976, at University of Washington) has been active since 1999. The existing NACC infrastructure is described in the Facilities and Resources Section of this application. The broad goals of the past funding cycles are consistent with those of the current U24 RFA — that is, to serve as: a.) the central hub for organizing and enabling communication within and outside of the ADRC program, including annual meetings and steering committees; b.) a national data resource, collecting data from the Alzheimer's Disease Research Centers (ADRCs) as well as affiliated data and sample repositories; and c.) a facilitator of current and future AD/ADRD research. NACC has considerable experience and success in reaching these goals, and is positioned for continued success in a rapidly advancing field. New data and methods are appearing in areas such as biomarkers, neuropathology, and tests for early detection. Through the strategic adoption of technological advances, we will build on these accomplishments at an accelerated pace. Building on our already significant capabilities, we will: promote and broaden communication within and outside of the ADRC program; expand informatics capabilities of NACC consistent with FAIR principles; conduct and support methodologic and applied research, leveraging deep expertise in biostatistics and data science; and support early-career research scientists by providing peer-reviewed competitive funding for several “junior investigators” each year. We aim to continue to improve our approach in each arena by combining time-tested approaches with the new tools and innovations we are developing to meet the changing scientific, technological, and communication needs of the NIA ADRC program and the field.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Pregnant women are highly vulnerable to influenza A virus (IAV) and are at increased risk for maternal death, preterm birth and stillbirth. Universal influenza vaccines (UFV) are thought to be possible if conserved regions of influenza virus are targeted and appropriate immune responses generated. However, relevant animal models are lacking in which to test such a vaccine, particularly for pregnant women. This proposal is focused on investigating maternal and placental immune responses to IAV in a pregnant nonhuman primate (NHP) model to understand the viral-host factors driving enhanced maternal disease. Our central hypothesis is that an aberrant Th17 response during an acute IAV infection leads to a broadly dysfunctional innate and adaptive immune response that prevents viral clearance and enhances risk for maternal death and stillbirth. Th17 cells produce high levels of the inflammatory cytokines IL-17 and IL-2218,19 with early Th17 polarization considered to be critical for IAV resolution; aberrant and/or late activation of the Th17 pathway by IL-23 in murine models is thought to impair viral clearance and promote lung injury.20,21 Our preliminary data in a pregnant NHP model of an acute IAV H1N1 infection demonstrates pneumonia in all animals by Day 5 post-IAV inoculation. In pregnant NHP, influenza disease scores were higher than non-pregnant animals with notable extra-pulmonary organ injury (myocarditis, white matter injury). Pregnant NHP demonstrated a nearly absent early Th17 CD4+ T cell response in whole blood and PBMC coupled with a marked increase in Th17 cells in the lung at peak immunopathology compared to non-pregnant animals. Inflammatory cytokines and chemokines in the lungs and bronchoalveolar lavage fluid (BAL) were also greater in pregnant versus non-pregnant animals. In Aim 1, non-pregnant and pregnant pigtail macaques will be challenged with either IAV H1N1 A/CA/04/09 or H3N2 A/Texas/71/2017 (N=8, each group) and undergo blood and BAL sampling until necropsy at Day 5 (peak immunopathology). In Aims 1A and 1B, we will determine pregnancy- specific immune correlates of IAV disease by evaluating the frequency of Th17 CD4+ T cells and a broad spectrum of innate/adaptive immune responses (i.e. immune cell subsets, cytokines/chemokines, Type I/III interferons) in the blood, BAL and lung. In Aim 1C, we will evaluate antiviral responses in the placenta linked to adverse pregnancy outcomes (e.g. cytokines/chemokines, NLRP3 inflammasome activation, CD8+ T cells). In Aim 2, we will use bulk and single cell RNA-sequencing to define changes in the transcriptome within PBMC, BAL, lung and placenta with a focus on Th17 transcriptional networks and antiviral innate immune pathways. In summary, the preliminary data indicates an aberrant Th17 response in pregnant animals, which is critical to promoting viral clearance and preventing lung injury. These studies will be the first to comprehensively analyze innate/adaptive immune responses during an acute IAV infection to elucidate the pathogenesis of severe lung disease in pregnant women. Results from these studies are critical for IAV pandemic preparedness to enable testing of efficacy and safety of new UFV.

NIH Research Projects · FY 2025 · 2021-06

Project Summary: The malaria control program on Bioko Island, Equatorial Guinea was among the vanguard of highly intensive and highly successful malaria control programs in sub-Saharan Africa. Intensive malaria control began in 2004 under the Bioko Island Malaria Control Program (BIMEP) manages commodity distribution, surveillance, monitoring, and evaluation to eliminate malaria from Bioko Island. After initial success, the program has documented slower progress, and malaria persists through residual local transmission by vectors and frequent travel to mainland Equatorial Guinea resulting in malaria importation. There is a significant need to develop a methodology that would allow BIMEP to improve malaria control through spatial targeting and rapid development of an evidence base to reduce residual transmission and guide elimination efforts across transmission contexts. A practical solution, called adaptive vector control, that combines elements of integrated vector control and adaptive management. The overall goal of this proposal is to develop adaptive vector control as a rigorous and quantitative methodology to help programs understand residual transmission, build an evidence base, and identify strategies to suppress residual transmission and eliminate malaria. The specific goals of adaptive vector control are to quantify residual transmission in the urban setting of Malabo, Bioko Island the capital of Equatorial Guinea, where 90% of the residents of Bioko Island live, and use that evidence to guide vector control through an iterative, structured policy process. We will use existing evidence from surveillance, monitoring and evaluation to develop, validate, and analyze dynamic models of mosquito aquatic habitats, mosquito population dynamics, and malaria transmission in the city. We will use the models to design adaptive sampling and adaptive studies to reduce uncertainty about programmatic decisions, and through simulation-based analytics, we will help the program to improve spatial targeting of indoor residual spraying and larval source management. Finally, we will use the methods to build an evidence base to support enhanced vector control with novel vector-based interventions to help BIMEP eliminate malaria. The challenges of reducing malaria incidence in Malabo and on Bioko Island are similar to the challenges faced elsewhere in sub-Saharan Africa, and adaptive vector control is one way of addressing the problems of urban vector control in the African context.

NIH Research Projects · FY 2026 · 2021-06

Agonists of toll like receptors (TLRs) are promising anticancer vaccine adjuvants because of their ability to induce proinflammatory cytokines necessary to generate a robust immune response. However, currently available TLR agonists suffer from a number of limitations including self-regulatory immunosuppression and unfavorable local pharmacokinetics resulting in poor availability within dendritic cells. Further, current TLR agonist-based anticancer vaccines generate a robust cytotoxic CD8 T cell response but not CD4 Th 1 helper T cell response, which is critical for inducing effective, long-term antitumor immunity. We will address these important challenges through a synergistic combination of drug discovery and drug delivery efforts. Our team has developed a suite of highly substituted imidazoquinolines, which activate TLR7 and/or 8 and induce significantly higher levels of cytokines compared to imiquimod, an FDA approved TLR7 agonist. Our studies show the balance between proinflammatory and immunosuppressive cytokines can be tuned through structural modifications. Encapsulation of these novel agonists in acidic pH responsive nanoparticles (NPs) resulted in robust activation of CD4 and COB T cells as well as natural killer (NK) cells, leading to a stronger anticancer immune response than free agonist or that encapsulated in non-pH responsive NPs. Importantly, intradermal delivery of NP vaccine using a hollow microneedle platform led to an enhanced Th1 immune response, which is essential for effective induction of long-term antitumor immunity. We will build on these exciting findings and further optimize the new agonists for efficient encapsulation in pH responsive NPs, tune the NP properties for improved targeting of dendritic cells following delivery via hollow microneedles, and investigate potentiation of NK cell-mediated antibody-mediated cellular cytotoxicity. The Specific Aims of this revised R01 grant application include: Aim 1: Design and synthesize TLR7/8 agonists that are optimized for NP encapsulation Aim 2: Optimize pH responsive NP formulation for hollow microneedle-assisted ID delivery Aim 3: Determine anticancer efficacy of NP vaccine following hollow microneedle-assisted ID delivery We expect our studies will identify new design principles and delivery strategies for TLR agonists that overcome the limitations of current anticancer vaccines and further advance the field of cancer immunotherapy.

NIH Research Projects · FY 2025 · 2021-06

Abstract The U.S. faces a relentlessly growing syphilis epidemic that is concentrated among men who have sex with men (MSM) and transgender (TG) persons. We confront that epidemic with diagnostic tools that have barely changed in over half a century as we continue to rely on serological tests that have a window period from infection to test positivity of 3-5 weeks. This application proposes studies that will use a new, industry- developed experimental transcription mediated amplification (TMA) assay for Treponema pallidum (Tp) both to improve our understanding of the natural history of syphilis, and to assess whether adding TMA testing to serological screening can identify seronegative persons with syphilis in the earliest stage of infection, before serological tests become positive. Our preliminary data suggest that 10% of MSM with syphilis are seronegative, but TMA positive in pharyngeal or rectal specimens. This application builds on that finding. We will initially define the optimal specimen types for TMA testing among persons with active syphilis by comparing TMA positivity in pharyngeal vs. oropharyngeal specimens, and in whole blood vs. serum (Aim 1). Throughout the project, we will test remnant serum, rectal and pharyngeal specimens collected from STD clinic patients diagnosed with syphilis to define how often Tp RNA is present at different anatomic sites in persons with different stages of infection (Aim 2). Finally, we will enroll 3350 MSM and TG STD clinic patients in a prospective study to determine whether adding Tp TMA testing of blood, pharyngeal and rectal specimens leads to earlier identification of syphilis in persons with negative serological tests (Aim 3). For this aim, we will enroll seronegative patients without clinical evidence of syphilis. Participants will receive standard clinical evaluation and treatment at enrollment and will provide the study with serum, rectal and pharyngeal specimens, which the research team will freeze. Twelve weeks after enrollment, we will test these specimens using the TMA, and will contact persons with positive TMA results for interview and repeat serological and TMA testing in order to determine if study subjects developed syphilis or seroconverted since enrollment. Findings from these studies will define how often persons with different stages of syphilis are likely transmissible, and will determine whether screening that integrates serology with TMA testing is superior to standard serological testing alone.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Nonsteroidal anti-inflammatory drugs (NSAIDs) are indicated for first-line treatment of many conditions ranging from pain to heart disease, but therapeutic use of the entire medication class can be precluded by reported allergy. While NSAIDs represent the second most commonly reported class of drug allergies, it is estimated that <20% of reported NSAID-induced reactions are consistent with allergic hypersensitivity (eg, urticaria, angioedema, anaphylaxis), with the rest representing non-allergic side effects or intolerances (eg, gastrointestinal upset, bleeding, nephrotoxicity). Allergy over-reporting and lack of reaction clarification can lead to unnecessary drug avoidance, and increased use of second line, more costly, and less effective alternative medications, with resultant downstream adverse effects on patient outcomes. The primary objective of this proposal is to determine the impact of reported NSAID allergies on prescribing patterns and clinical outcomes in distinct populations and to define predictors for true NSAID-induced allergic hypersensitivity. The proposed study has three Aims: (1) to assess the impact of reported NSAID allergy on postoperative opioid prescribing and sustained opioid use in a large electronic health record-derived cohort of patients undergoing common surgical procedures; (2) to evaluate differences in aspirin administration and adverse cardiovascular outcomes in patients with acute coronary syndromes with and without reported NSAID allergy; (3) to use a well-defined cohort of subjects who have undergone allergist-observed NSAID drug challenge to determine patient- and drug-specific risk factors for NSAID-induced hypersensitivity. Dr. Li’s research plan will be supported by coursework and training in clinical and epidemiological study design, advanced biostatistical methods for research with large datasets, informatics, patient recruitment, and drug-challenge protocols, and the exceptional scientific environment at the University of Washington. This K23 proposal is well-aligned with NIH initiatives to reduce unnecessary opioid prescribing and NIAID support of drug allergy research, and will position the candidate to submit an R01 application during the award period and to establish herself as an independent patient-oriented investigator with a focus on improving the care and health of patients with drug allergies.

NIH Research Projects · FY 2025 · 2021-06

SUMMARY Immunglobulin µ (IgM) is an evolutionary old class of antibody that is the first antibody produced in response to an infection. Several IgMs are present as natural antibodies and are vital for immunity during the early stages of development. Unlike any other class of antibody, a single copy is sufficient for activating the classical complement system, and several monoclonal IgMs have shown massive potential for the treatment of cancers. The large size, high degree of glycosylation, and structural heterogeneity of IgM has rendered them refractory to structural studies and to date the molecular mechanisms of how antigen binding activates IgM to initiate the complement cascade remain murky. In contrast, a detailed understanding of the structure and various interactions of immunoglobulin g (IgG) have been fundamental to their advancement as the premier molecular platform for biotherapeutics. Accordingly, a similar level of understanding of IgM will be critical for their development as a new class of biotherapeutics. This proposal aims to apply structural mass spectrometry techniques, electron microscopy with complementary structural approaches, and biophysical tools to study the structural changes within IgM that govern activation of the complement cascade. Various types of IgM-antigen complexes will be prepared and analyzed using hydrogen/deuterium exchange and X-ray foot printing with mass spectrometry to track the local structural changes within IgM upon binding different presentations of antigen (Aim 1). Electron microscopy and small angle X-ray scattering will be used to visualize and track large-scale structural transitions in the antigen-IgM complexes (Aim 2). The full combination of techniques will be used to study how IgM recruits and activates the initial component, C1, of the complement cascade (Aim 3). The molecular mechanisms of how IgM recognizes antigen and recruits complement will provide a foundation for modulating the immune system for a new paradigm in biotherapeutics.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY / ABSTRACT Acute respiratory infections are the leading cause of mortality among neonates globally, particularly among low birthweight infants from pregnancies complicated by prematurity or placental insufficiency. Maternal immuniza- tion, the vaccination of pregnant women to enhance antibody transported across the placenta to the fetus, is utilized increasingly as an infection prevention strategy to protect neonates from pathogens such as influenza, and may be critical as new vaccines against SARS-CoV-2 are developed. This proposal aims to better eluci- date transplacental antibody transfer in low- and high-risk pregnancies, specifically those with prematurity or placental insufficiency, which is not known. It also describes a research training program that will allow me to become an independent physician-scientist with a translational research program focusing on infectious diseases and vaccines in pregnancy. This proposal builds upon my training in high-risk obstetrics and global health, and provides a detailed plan to improve my knowledge of virology, immunology, vaccinology, cohort studies and statistical analysis. It incorporates the expertise of an outstanding mentorship team, including experts in infectious diseases, immunology, pathology, statistics, and computational biology who are dedicated to the success of this project and the development of my career as an independent clinical researcher. The first aim of this proposal involves establishing normal and high-risk pregnancy cohorts and quantifying transplacental antibody transfer in the context of pregnancies resulting in both normal and low birth weight infants, including those complicated by placental insufficiency leading to small for gestational age infants or prematurity. I will compare quantity of transplacental antibody transfer of total IgG, influenza- and SARS-CoV- 2- specific antibodies, and expression of neonatal Fc receptor in placentas. I will also compare neonatal immunity against influenza among women who did and did not receive influenza vaccine during pregnancy, allowing the opportunity to develop multivariable models and adjust for multiple clinical covariates. For my second aim, I will characterize antibody profiles and transplacental transfer in the same cohorts using a systems serology approach to determine biophysical and effector functions of maternal and fetal immunology. Through accomplishing the aims in this proposal, I will contribute significantly to our knowledge of maternal immunizations, maternal and neonatal immune interactions and neonatal immunity against influenza and SARS-CoV-2. Improved understanding and characterization of transplacental antibody transfer in the context of maternal immunizations may provide insight in the optimization and individualization of vaccine delivery and timing in high-risk pregnancies and, ultimately, has the potential to improve neonatal survival globally. This proposal will allow me to develop a unique set of cross-disciplinary skills and expertise in order to transition to independence as a physician scientist with a larger research program specializing in respiratory infectious diseases and vaccines within the context of low- and high-risk pregnancies.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Despite significant advances made in understanding the genetic basis of the more than 10,000 estimated Mendelian conditions in recent decades, further scientific collaboration and discovery is required to identify the full set of Mendelian diseases and their underlying genetic causes. To build on the success of the Centers for Mendelian Genomics by using further technological, methodological, and data-sharing innovations, NHGRI is establishing a new Mendelian Genomics Research Consortium. As Data Coordinating Center for this Consortium, we propose to further the discovery and characterization of Mendelian conditions by achieving four main aims. (1) Managing the release of molecular and phenotypic data generated or collected by the Consortium's Research Centers, featuring semi-automated and scalable quality control procedures that will validate incoming data against collaboratively-established standards and requirements. We will facilitate data sharing within the Consortium and the broader scientific community by ensuring that curated data are submitted to appropriate repositories including AnVIL, ClinVar, and Matchmaker Exchange. (2) Supporting program outreach through a Consortium website that highlights program progress and discoveries and enabling connections among researchers, clinicians, and families. We will also oversee education, training, and mentorship opportunities. (3) Coordinating logistics for the Consortium, including organization of virtual and in-person meetings and administrative support for all Consortium-wide activities. (4) Overseeing an Opportunity Fund, administering up to 5 awards in each of years 2-5 of the Consortium, to support novel and scalable approaches to functional follow-up on candidate variants. To achieve these four main aims, we propose innovative approaches, including leveraging emerging cloud-based systems for the storage and analysis of human genetic data and integrating international data sharing standards to ensure that data is findable, accessible, interoperable, and reusable (FAIR). Furthermore, we bring a unique blend of expertise across the fields of data science, cloud systems, biostatistics, bioethics, clinical genetics, and Mendelian discovery to the Consortium, and our application rests on 13 successful years of experience and expertise serving as Coordinating or Analysis Centers for 7 large-scale genetic and biomedical projects. Through support and coordination of this Consortium, we will help yield the benefits of identifying the genetic basis of Mendelian conditions to support diagnosis, therapy, and further delineation of syndromes. Further public health benefit will accrue via biological insights into Mendelian diseases that also advance our understanding of related common, complex diseases.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT The genetic basis of >2,920 Mendelian conditions (MCs) remains unknown, and hundreds of novel MCs are described each year. Our group has, in partnership with 2,379 investigators from 656 institutions in 55 countries, assessed 15,387 samples from 5,675 families and has, over the past decade, identified genes for 1379 MCs, including 915 novel discoveries. The translation and impact of these discoveries on diagnostics and clinical care has been immediate and substantial. Additionally, we have developed multiple new analytical tools including CADD, PRIMUS, CoNIFER, SMRT-SV, RV-TDT, as well as methodological innovations including MIPs, smMIPs, and approaches for low input exome and genome sequencing (ES/WGS). We are also deeply committed to open data sharing with rolling submission of exome and genome data to the AnVIL (1,439 deposited); development of a MatchMaker Exchange node (http://MyGene2.org) that enables public sharing of genotype and phenotypic data among families, researchers, and clinicians; and creation of a public data browser (http://geno2mp.gs.washington.edu) that links de-identified, individual-level genotypes from over 18,000 exomes/genomes to individual phenotypes. In this application, we build upon these successes to establish the University of Washington Mendelian Genomics Research Center (UW-MGRC) with the overarching goal to maximize novel gene discovery for MCs, with an emphasis on canonical MCs that have gone unsolved using ES/WGS, and noncoding variants underlying MCs. To this end, we will develop novel approaches to inform variant interpretation and functional validation for the human genetics community at-large and disseminate results, data, and tools openly. We will capitalize on immediate access to sequence-ready samples from ~300 MCs (>26,000 samples), 1,500 samples suspected of harboring a causal noncoding variant for a MC, and an aggressive sample solicitation plan in partnership with industry, academic centers, and other NIH programs. We propose three specific aims: (1) maximize novel gene discovery for MCs by solicitation, sequencing, and analysis of families with unexplained (i.e., no known underlying gene) MCs; classic MCs considered high priority by the clinical genetics community and that have been recalcitrant to gene discovery efforts; and cases that remain unsolved after prior exome or genome sequencing. (2) Develop new strategies for gene discovery for unsolved MCs caused by variants that are difficult to detect or of unknown functional effects (e.g., structural variants, repeat expansions, cryptic splice, regulatory, etc.), and/or unusual modes of inheritance, and, in doing so, characterize the genetic architecture of pathogenic noncoding variants underlying MCs. Implement high- throughput screening and targeted follow-up functional studies to prioritize and validate assertions of pathogenicity of candidate noncoding variants. (3) Take a leadership role to openly and publicly, when feasible, share sequencing and rich phenotypic metadata, methods, and knowledge, to empower investigators worldwide and accelerate the pace of gene discovery.

NIH Research Projects · FY 2024 · 2021-06

PROJECT SUMMARY Coenzymes and antioxidants mediate hundreds of biochemical reactions and are fundamental to cellular and mitochondrial function. The redox coenzymes undergo reversible oxidation and reduction reactions, while the balance between oxidized and reduced forms drive important cellular functions including regulation of ion channels, cell signaling, cell survival and death. Cellular dysfunction associated with these coenzymes has been implicated in many diseases including cancer, heart failure, diabetes, obesity and aging. Given the importance of NAD metabolites and their declining levels in aging, numerous clinical trials of nutritional supplementation of the coenzymes' precursors are currently underway. Despite the immense interest and the need to determine cellular and subcellular levels of these metabolites, no reliable method exists currently for their simultaneous and comprehensive measurement. The major challenge is that these molecules are unstable and their levels are sensitive to sample harvesting, extraction and measurement conditions. As a result, errors involved with their measurement using conventional methods often outweigh biological variations and potentially lead to incorrect inferences. To overcome these challenges, and building on our preliminary studies of methodological developments using nuclear magnetic resonance (NMR) spectroscopy, in this proposal, we seek to develop methods to reliably measure the coenzymes and antioxidants in blood, cells, tissue as well as two subcellular components: mitochondria and cytoplasm. Further, we seek to develop methods to measure these coenzymes in live cells and mitochondria in real time. We will also translate the protocols and measurements to the widely used mass spectrometry platform for broader dissemination. The proposed project has three main aims: (1) Develop methods to reliably measure the coenzymes and antioxidants in blood, tissue, cells and two subcellular components: mitochondria and cytoplasm using NMR spectroscopy; (2) Develop methods to measure the coenzymes and antioxidants in live cells and mitochondria in real time using NMR spectroscopy; and (3) use NMR spectroscopy to guide the translation of the methods for analysis of the unstable (coenzymes and antioxidants) as well stable metabolites to mass spectrometry for wider applications in the metabolomics field. The outcome will provide robust methods to analyze important coenzymes and antioxidants in a broad range of biological sample types that can be used by many researchers. The new methods will also provide novel avenues for investigation of live cells and mitochondrial metabolism in real time. These developments will impact numerous areas of biomedical science.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ ABSTRACT Meeting reproductive health needs of women living with HIV (WLWH) is essential to help women prevent unintended pregnancies, safely conceive, and eliminate mother-to-child HIV transmission (MTCT). Reproductive life planning is complex for WLWH, who are faced with making decisions about antiretroviral treatment selections and potential for drug interactions with contraceptive methods, planning for safe conception, and STI prevention. While family planning (FP) use is high among WLWH, discontinuation of FP is common among women who desire pregnancy prevention, and is an important driver of unmet need for FP and subsequent risk of unintended pregnancy and adverse maternal and child health outcomes. Many programs in sub-Saharan Africa integrate FP service delivery into routine HIV care, but HIV care providers face challenges with implementing these models of care. HIV care providers may be ill equipped to ensure WLWH receive high-quality, rights’ based reproductive health counseling and services due to lack time, training, resources, and skills. Prior studies on integrating FP services into HIV care consistently cite implementation challenges. Mobile health technology may provide a low cost solution to augment counseling services, strengthen health care systems, and alleviate demands on HIV providers. We hypothesize that providing comprehensive counseling and two-way SMS communication, will 1) improve delivery of integrated HIV and reproductive health care services, 2) reduce contraceptive discontinuation rates, 3) be acceptable and feasible to implement, and 4) be cost-effective and contribute to prevention MTCT efforts. We will adapt a unique two-way SMS platform (Mobile WACh) that combines automated bulk SMS messaging and dialogue with a health care provider for a new population, new environment, and new outcomes for long-term impact. The Mobile WACh platform will be customized to provide continuous reproductive life planning counseling for WLWH. We will test the combined intervention in a cluster randomized controlled trial among women receiving HIV care at 10 facilities in Kenya (330 per facility). We propose to evaluate the effect of the counseling and SMS communication intervention, Mobile WACh Empower, on reproductive health outcomes. In Aim 1, we will determine the effect of the Mobile WACh Empower intervention on FP discontinuation, dual method use, and unmet need for FP over 2 years. In Aim 2, we will evaluate acceptability, feasibility, and scalability of implementing Mobile WACh Empower under real- world settings, from both WLWH and provider perspectives in focus group discussions. In Aim 3, we will construct a mathematical model to measure health and economic impacts of Mobile WACh Empower, including cost- effectiveness of the intervention per pregnancy and MTCT averted. This trial will evaluate a novel intervention to address a crucial gap in provision of integrated reproductive health and HIV care, and has the potential to make a significant contribution to global goals of universal access to FP and elimination of MTCT.

NIH Research Projects · FY 2026 · 2021-06

There are key gaps in understanding gene regulation in all organisms, including humans, which impede the interpretation of the many disease- and trait-associated genetic variants that reside in regulatory elements. I will address three of these gaps: (i) the context-dependent function of regulatory elements that can repress or enhance gene expression; (ii) the contribution of active regulatory elements in transposons to host genome expression in development and in response to environmental change; and (iii) the consequences of variable ploidy for gene regulation and genome architecture in cell types relevant to human disease. To add sequence and epigenome context to regulatory landscapes, I will leverage newly published long-read, single-molecule methods that can resolve regulatory activity along chromatin fibers, even in single cells. These methods can detect active regulatory elements in individual transposons and in individual polyploid cells resulting from endoreduplication. To enable whole-organism and environmental context, I will explore and contrast these gaps in human cells and in maize plants. The human and maize genomes are similar in size, with similar numbers of genes and regulatory regions, yet maize plants can be assessed as whole organisms across generations and exposed to conditions that alter the activity of regulatory elements and transposons. In the first project, I will build on results from our massively parallel reporter assays (MPRAs) testing regulatory elements from four plant genomes, including maize, for repressive and enhancing activity. I will combine these and publicly available human MPRA data with the rich and contextual information from maize and human long-read single-molecule data to identify features – including sequence characteristics, protein occupancy and native epigenome neighborhood – to model regulatory element function in human cells and maize tissues. Because there are many more active retrotransposons in maize than in human, in the second project, I will examine the effects of retrotransposons on host genome regulation across maize development, in different tissues, and in response to stress. Once I have identified mechanisms of transposon-driven host genome expression in maize, I will take a targeted approach to identify their conserved counterparts in human cells. Plants show distinct and homogeneous endoreduplication states whereas human cell populations show variable and heterogeneous endoreduplication. Thus, in the third project, I will adapt the long-read, single-molecule methods to assess regulatory elements and genome organization in endoreduplicated cells, first in plants and then in human liver cells and tissue. My approach of contrasting these knowledge gaps in both systems should yield novel and unexpected insights.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY: Worsening population-level trends in cardiometabolic health highlight the profound need to move away from traditional disease models focused on the remediation of downstream cardiometabolic risk factors to instead focus on relevant upstream exposures. Relevant upstream exposures include early life adversities (ELA), a unique subset of social determinants of health that occur early in life and are hypothesized to become biologically embedded, thereby shaping life course trajectories of health and disease risk over time. A robust literature shows ELA exposures confer prospective risk for cardiometabolic disease, yet few—albeit promising—studies have examined whether early intervention in ELA-exposed children may lessen this risk. Review of relevant literatures suggests generally that 1) earlier intervention is more effective; 2) intervention benefits are greatest in families most in need; 3) parenting is a mechanism through which early intervention benefits are transmitted; and 4) existing parenting interventions, deemed successful with respect to parent-child behavioral and relationship outcomes, are candidates for testing in relation to child physical health outcomes. Building on this foundation, the proposed study represents a unique and time-sensitive opportunity to extend the aims of an existing RCT in which a parenting intervention—Promoting First Relationships® (PFR) versus no intervention—was implemented as an adjuvant to depression treatment in a sample of low income, postnatal women. The purpose of the proposed study is to determine whether benefits of the PFR intervention, originally designed to impact parent- child behavioral and relationship outcomes in infancy may extend to the child’s cardiometabolic health in early childhood. It is hypothesized that the PFR intervention will augment an upstream resiliency factor—parenting quality—at an early period of vulnerability, potentially benefiting the child’s cardiometabolic health. Expected intervention effects on the more distal child cardiometabolic health outcomes are hypothesized to be partially attributable to changes in the more proximal intervention-related targets, including maternal sensitivity, parent understanding, mother-child relationship quality, and child self-regulation. The proposed study seeks NIH funding to support the return of 214 mother-child dyads (85% of 252 total families) who participated in the original RCT. The majority of families (80%) belong to a minority race/ethnic group, 32% are Spanish-speaking, and all are considered low income. Families will complete two home-based visits between child’s age 5-6 and 7-8 years. These visits will entail assessments that parallel measures in the original study regarding parent-child behavioral and relationship outcomes but will also include health-focused assessments in domains known to predict long- term risk for disease, including cardiovascular health, metabolic health, and inflammation. Health indicators will be derived from data sources including anthropometric and blood pressure assessments, a dried blood spot collection, activity and sleep monitoring, and interviewer-administered questionnaires. Results will extend broadly to vulnerable families at disproportionate risk for poor cardiometabolic health.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT The hippocampus is critical for capturing rich, multimodal representations of experience and facilitating the long-term storage and later recall of these experiences. During sleep and pauses in behavior, the hippocampus can “replay” prior experience – reactivating the neural ensemble corresponding to the original experience in a time-compressed manner. During sleep, such replay is thought to underlie memory consolidation, while during behavior, replay is thought to additionally serve a more prospective role: contributing to planning or deliberation by retrieving stored memories in order to inform upcoming decisions. However, the content of replay neither solely reflects recent experience nor reliably predicts future behavior, leaving it unclear how exactly the representations of experience that are replayed relate to upcoming choices. Understanding the relationship between replay and behavior is particularly critical because abnormalities in replay and sharp wave ripples (SWRs; the network activity signature of replay) have been observed concurrent with impaired memory-dependent behavior in aging and diseases of aging. Establishing how replay content changes with aging, and whether these changes cause deficits in memory-guided behavior, has the potential to generate new therapeutic strategies to prevent or reverse memory impairment. In order to define how replay contributes to memory-guided decision-making in normal cognition and in the context of age-related memory impairment, we have developed a neurofeedback-based operant conditioning paradigm that targets SWRs. This paradigm provides rapid feedback contingent upon real-time detection of SWRs at a specific point during each trial of a spatial memory task, and results in substantially increased occurrence of SWRs in a trial phase- specific manner. Consequently, subjects experience more replay at the required trial phase, which occurs immediately prior to the choice point of a memory-dependent task. In addition to demonstrating that replay can be enhanced by neurofeedback, this behavioral paradigm provides an increased opportunity to link the content of replay with subsequent behavior. This paradigm lays the foundation for the three aims of this proposal: to define the relationship between replay and memory-guided behavior, to assess how this relationship changes with age, and to adapt the operant conditioning strategy to directly counter age- related replay dysfunction. I will complete these aims with the guidance of an exceptional mentoring team led by Loren Frank and including Carol Barnes, Uri Eden, and Karunesh Ganguly. During the mentored phase of the award at UCSF, I will conduct the proposed real-time feedback studies, gain expertise in using state-space models to capture and quantify replay content, scale experiments to efficiently examine larger cohorts of young and aged animals, and focus on professional development in order to facilitate a successful transition into an independent faculty position at an academic institution.