Oregon Health & Science University

NIH Research Projects · FY 2025 · 2025-09

Project Summary 78.2 million lower extremity fractures occurred globally in 2019, of which 5-10% resulted in delayed or failed healing. Further, fractures with vascular injuries have poor healing at higher rates (up to 46%), emphasizing the importance of oxygen delivery to the injured region during healing. Recent clinical studies found injury-associated anemia—hemoglobin (Hb) < 12 g/dL—is present in ~95% of lower extremity orthopaedic trauma patients. Due to decreased Hb, anemia is characterized by lower tissue oxygenation levels and increased systemic inflammation, both of which are linked to impaired bone healing. While blood transfusions remain the current standard of care for injury-associated anemia, they demonstrate less than 1% efficacy, whereas intravenous iron therapy (IVIT) achieves ~80% success in chronic anemia cases but remains unexplored in orthopaedic trauma management. To determine the impact of injury-associated anemia on bone healing, I developed a novel murine model of femur fracture with injury-associated anemia. Using this model, I demonstrated that moderate anemia (7 < Hb < 12 g/dL) reduces regenerating bone volume after injury, suggesting that anemia contributes to impaired bone healing. However, the underlying cause of impaired bone healing in the presence of injury-associated anemia and how to manage injury-associated anemia after injury have not been studied. Thus, the overarching objective of this study is to identify how injury-associated anemia affects bone healing processes and determine if IVIT treatment can facilitate successful bone healing. First, I will determine the impact of injury- associated anemia on temporal inflammation, hypoxia, and bone regeneration following fracture. In the second aim, I will establish the efficacy of IVIT in facilitating bone regeneration via resolution of orthopaedic injury- induced anemia. The research outcomes of this work will 1) provide insight into the biological effects by which injury-associated anemia impedes bone regeneration and 2) inform the translation of IVIT into the orthopaedic clinical setting to support bone healing. This work will inform clinicians on how management of injury-associated anemia can improve patient care after orthopaedic trauma. The proposed studies will be performed at Oregon Health and Science University, a world-class training environment with strong clinical collaborations. Drs. Karina Nakayama (sponsor) and Nick Willett (co-sponsor) have developed a comprehensive training plan to support my professional and technical development including skills in mentorship and scientific communication in addition to technical skills including multiparameter flow cytometry and dimensionality reduction techniques for multivariate analysis. Drs. Nakayama and Willett are engaged mentors with expertise in the research of musculoskeletal injuries and treatments. Dr. Willett offers access to cutting-edge biomedical innovation and collaborations at the University of Oregon. Dr. Nakayama, Dr. Willett, and my established history of productive collaboration through routine in-person and virtual meetings will ensure this proposal’s success. This research setting and their expert guidance will enable collaborative, impactful, & innovative research.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Proper nervous system function depends not only on functional interactions between neurons and glia, but also between different types of glia. This proposal is focused on defining fundamental properties of interactions between two major classes of glia in the vertebrate nervous system: astrocytes and oligodendrocytes. Astrocytes are the most abundant glial cell type in the human brain and are important for central nervous system (CNS) development and function. Astrocytes are elaborate cells that extend processes that contact synapses, neurons, blood vessels, and other glial cells in the CNS. Oligodendrocytes are the myelinating glia of the CNS; they wrap axons with myelin to facilitate rapid action potential propagation and provide vital trophic support to neurons. Our long-term goals are to understand how astrocytes and oligodendrocytes functionally interact. We know that astrocytes and oligodendrocytes are connected by gap junctions and that astrocyte factors can promote myelination and remyelination. Yet the cellular and molecular bases of these interactions are incompletely understood. Our preliminary data using in vivo imaging in zebrafish demonstrate a wealth of different forms of physical interactions between astrocytes and oligodendrocytes in the developing spinal cord. This proposal will use a combination of molecular, genetic, cellular, and proteomic tools to: fully define astrocyte-oligodendrocyte myelin interactions at the cellular and molecular levels; to define novel proteins at the astrocyte-myelin interface; to understand how oligodendrocyte development and myelination are impacted when astrocyte development is altered. Our work will provide exciting new insights into how astrocyte and oligodendrocyte interactions contribute to proper nervous system function in vivo and provide a foundation for understanding the properties of these cells in human disease states.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Sex differences in cancer have been clearly documented. In general, males have higher incidence rates and poorer outcomes compared to females. This difference is particularly pronounced in melanoma where females have a 38% survival advantage compared to males after adjusting for stage at diagnosis. Sex-based differences in immune function likely contribute to the sexual dimorphism observed in cancer. The female immune response has been characterized as more robust, mounting better responses to infection and vaccination than males. Further, androgens have been shown to be widely immunosuppressive. In multiple preclinical tumor models, including melanoma, androgen signaling through the androgen receptor (AR) has recently been shown to directly suppress anti-tumor T cell function. Notably, T cell function is only one aspect of the immune response. Conventional dendritic cells (cDCs) prime naïve T cells and serve to initiate and expand an antigen-specific, anti- tumor immune response. In melanoma, cDCs are required to generate a CD8+ T cell response against the tumor. However, how AR impacts anti-tumor function of cDCs is entirely unknown. Given the striking sexual dimorphism in immune system function and cancer, and the importance of cDCs in anti-tumor immune responses, there is a clear and urgent need to investigate the impact of AR signaling in cDCs. In preliminary studies in mice, we found that antigen-bearing cDCs from both male and female melanoma draining lymph nodes express AR. Importantly, we confirmed that human bone marrow derived cDCs (BMDCs) also express AR. To address the direct impact of AR-signaling on cDC function and anti-tumor immunity, we created a novel mouse model that specifically lacks AR in cDCs. Strikingly, these mice showed delayed melanoma tumor growth and significantly improved CD8+ T cell activation compared to AR-proficient control animals. Given our preliminary data, our central hypothesis is that cDC-intrinsic AR activity limits cDC function and impairs anti-tumor immunity. Here we propose to: i) identify the functional impact of cell-intrinsic AR signaling on cDC anti-tumor function using a novel mouse model where AR is deleted specifically in cDCs; ii) address the role AR in directly modulating expression of MHCI and use single-cell transcriptomics to elucidate the cDC-specific, AR-dependent, gene expression pathways driving immunosuppression; and iii) determine the efficacy of an AR-deficient, cDC-based, anti-cancer vaccine. We expect our work to contribute the mechanistic biology needed to understand how AR signaling in cDCs suppresses anti-tumor cDC function in males and females. This work will also provide key, preclinical, proof-of- concept data for targeting AR clinically in cDCs to improve cell-based vaccine efficacy. Broadly, these findings will improve our understanding of androgen-mediated immune modulation and support the integration of biologic sex as a variable in immunotherapeutic decisions.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY One of the challenges in treating post-traumatic stress disorder (PTSD) is its high comorbidity with other disorders, especially those involving reward and motivation. Treatment interventions for PTSD often consistent of cognitive-behavioral therapy, which involves learning coping strategies for the mental re-experiencing of traumatic memories. Preclinical work has attempted to model aspects of this treatment by focusing on extinction – a process that involves retrieving a traumatic memory and learning that the cues that evoke that memory are now safe. The challenge with extinction is that the learning that occurs during extinction is fragile; extinguished behavior returns with time, changes in context, or reminders of the original event. Pairing this extinction experience with pharmacological compounds that enhance memory may create a form of extinction that is persistent and resists relapse. We have found that drugs (histone deacetylase (HDAC) inhibitors) that promote histone acetylation may be especially powerful in creating a persistent memory for extinction. Although prior research on HDACs and extinction has been rigorous, the field has become stagnant at the behavioral level, with a major gap being that all of the focus has been on changes within an aversive or an appetitive system. That is, there is little to no understanding of how enhanced extinction effects operate in a model of PTSD comorbidities, where a traumatic experience results in persistent changes in appetitive behaviors. This is a major gap in the literature that our work attempts to fill by asking how potentiating extinction in a model of stress results in long-term changes in expression of appetitive behaviors that are persistently altered by that stressor. The two aims of our approach take the field of HDAC inhibition and extinction in novel and innovative directions. In Aim 1, we evaluate the persistent effects of acute systemic HDAC3 inhibition during extinction of a traumatic memory and in Aim 2, we evaluate the persistent effects of chronic site-specific HDAC3 inhibition within the infralimbic cortex during extinction of a traumatic memory. We evaluate these effects behaviorally, focusing on the reversibility of persistent changes that we have found to be induced by trauma – hyperresponsivity to mild stressors and alterations in appetitive motivation. We also evaluate the extinction effects at a molecular level, evaluating how HDAC3 inhibition alters specific loci within an extinction circuit. This work will have broad implications for pharmacological treatment strategies in preclinical models of PTSD.

NIH Research Projects · FY 2025 · 2025-09

The overall objective of this project is to conduct an in-depth analysis of pediatric prehospital emergency ventilation and identify a ventilation strategy associated with the best patient outcomes. Control of the airway and breathing is vitally important for children with a critical illness or injury treated in the Emergency Medical Services (EMS) system, though the best techniques are unknown. Ventilation delivery is a crucial component of prehospital resuscitation, ensuring adequate oxygen delivery to the brain and vital organs and appropriate clearance of C02 while minimizing the potential risks of overventilation, such as regurgitation and barotrauma. In EMS, clinicians typically use a self-inflating bag to give positive pressure ventilations, with manual control of the breath delivery. The existing data regarding the optimal ventilation rate for critically ill children treated in the EMS system is extremely limited, and current guidelines are based on expert consensus informed by small studies performed in hospitals. In this ancillary study, we will leverage the infrastructure of the Pedi-PART trial, an NHLBI-funded clinical trial carried out in the Pediatric Emergency Care Applied Research Network (PECARN). Pedi-PART compares pediatric airway management devices in over 60 EMS agencies. In the parent study, we will collect and archive physiologic data from the patient monitors used by these EMS agencies. In the proposed Pedi-VENT study, our team will apply sophisticated signal processing algorithms to the monitor data to determine the ventilation rate delivered by the EMS clinicians in the field. We will then apply target trial emulation methods to conduct analyses comparing pre-defined ventilation patterns based on the natural variability in current practice, similar to potential arms in a clinical trial. Though observational, our study will have the advantage of prospective data collection and standard definitions of exposure and outcomes with the parent trial infrastructure. The study aims are to 1) ETI) compare the effects of the airway strategy (BVM, SGA, on ventilation performance among critically ill children undergoing prehospital airway management, 2) examine the comparative effectiveness of different ventilation strategies on 30-day ICU-free survival, and 3) evaluate previous efficiently impact children how airway management strategies and ventilation trategies j ointly impact patient outcomes. No study has examined pediatric ventilation i n the EMS setting on this scale. We can conduct the study by leveraging an existing clinical rial led by members of our team. The results of this study will resuscitation guidelines for pediatric emergencies worldwide and help improve outcomes for critically ill treated in emergency care systems. s t

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Tuberculosis (TB) is caused by the pathogen Mycobacterium tuberculosis (Mtb) and its arsenal of secreted virulence factors, exhibiting one of the largest global disease burdens caused by a single infectious agent. TB is primarily a pulmonary disease with the latent form that affects 90% of infected individuals being asymptomatic, while the active form is characterized by a spectrum of clinical findings ranging from mild to severe, including cachexia, malaise, fever, and sputum production. A classic finding in the lungs of TB patients is the granuloma, a microenvironment of host immune cells recruited to infection foci. While the granuloma is believed to be host- protective by containing bacteria, concentrating immune responses, and preventing dissemination, granulomas may be detrimental as a fibrotic niche that is impermeable to T cells, favorable for Mtb to lie dormant, and impenetrable to antibiotics. These ineffective granulomas may contribute to latent TB in which true bacterial sterilization is indiscernible, leaving the disease incurable and patients at risk for activation. The host-pathogen interactions occurring within granulomas are thus important mechanistic targets for therapies but remain elusive. While previous granuloma histopathology has resulted in extensive descriptions of host microenvironment, the role of individual Mtb-expressed virulence factors in shaping these phenotypes has been underappreciated. This proposal aims to stain granulomas from non-human primate (NHP) lungs and human lymph nodes with an extensive panel of Mtb antigen-specific probes (i.e. commercial antibodies, novel in-house nanobody tetramers, acid-fast staining, and RNAScope) and advanced host multiplexing techniques (i.e. Phenocycler) to elucidate the localization, abundance, and molecular influence of Mtb factors on the granulomatous immune response and its success. Preliminary data show that Mtb bacilli are phenotypically heterogenous in terms of virulence factor expression, may localize far beyond the myeloid core where most bacilli reside, and secrete factors into host cells across the infected lung. The central hypothesis is that Mtb differentially regulates its virulence factor repertoire to manipulate immune dynamics, facilitating favorable granulomas that promote bacterial survival. This fellowship proposal also outlines a training plan customized for refining a rigorous physician-scientist in lung pathology and emerging/neglected infectious diseases. This includes an exceptional mentorship team with expertise across histopathology and TB, experimental NHP cohorts, nanobodies, and other reagents/resources unique to Oregon Health and Science University, and a training experience at the SAMRC Centre for TB Research in endemic South Africa. Completion of this proposal will result in 1) understanding Mtb heterogeneity in the infected lung, which may contribute to ineffective therapies and latent TB, 2) elucidating the role of virulence factors in granuloma progression, and 3) identifying novel Mtb-host interactions as therapeutic targets.

NSF Awards · FY 2025 · 2025-09

Metabolic diseases, such as diabetes, affect millions of people worldwide, profoundly impacting health, quality of life, and healthcare costs. However, traditional approaches to dietary management rely on generalized guidelines and intermittent blood tests, which limit their effectiveness in capturing the real-time metabolic changes necessary for personalized care. To address this, researchers at Purdue University and Oregon Health & Science University (OHSU) are developing NOURISH, an innovative digital twin technology that continuously tracks multiple metabolic indicators using wearable biosensors. By pairing these real-time measurements with advanced computational models, NOURISH simulates whole-body metabolism in individual patients and provides tailored dietary recommendations. The initial validation of these sensors will focus on healthy volunteers with controlled metabolic challenges, providing foundational data necessary for future clinical applications. NOURISH aims to significantly improve personalized dietary interventions and metabolic health outcomes. The project will also provide interdisciplinary training opportunities in advanced technologies and prepare a diverse and skilled workforce to meet critical national needs for healthcare innovation. The NOURISH project directly aligns with the NSF’s Foundations for Digital Twins (FDT-BioTech) program by developing advanced biosensors and integrating them into a physics-informed whole-body metabolism digital twin (WBM-DT). The team will retrofit FDA-approved continuous glucose monitors (CGMs) with tellurene-based nanosensors, enabling real-time measurement of multiple clinically relevant biomarkers, including glucose, lactate, β-hydroxybutyrate, branched-chain amino acids, glutamate, glutamine, acetoacetate, and glycerol. Sensor validation will be conducted initially in healthy adult volunteers using mixed-meal tests and standardized metabolic challenges. Data will be assimilated using an ensemble Kalman filter and Bayesian uncertainty quantification (UQ) to calibrate mechanistic metabolic models that accurately simulate systemic body-level metabolic fluxes. This framework will be combined with probabilistic AI-based control algorithms to deliver precise nutritional guidance tailored to individual physiological states. Additionally, large-scale, bias-audited synthetic cohorts will validate the model's accuracy, reliability, and fairness across diverse populations. These foundational methodological advancements will provide essential regulatory science toolkits for precision nutrition, facilitating broader biomedical applications for managing metabolic and chronic diseases. All training and outreach activities are designed to be open and accessible to participants, regardless of their protected characteristics, thereby aligning with the NSF’s commitment to inclusive participation in STEM. 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 · 2025-09

PROJECT SUMMARY/ABSTRACT Up to 30% of menstruating individuals experience heavy menstrual bleeding (HMB) at some point. HMB is associated with complications such as iron deficiency anemia and increased requirements for medical and surgical interventions, as well as decreased quality of life and missed work or school. While HMB is associated with structural anomalies such as fibroids, bleeding disorders and anticoagulation, in many cases there is no known cause. Prior studies have demonstrated a positive association between increased tissue plasminogen activator (tPA), an anticoagulant factor, and plasminogen activator inhibitor-1 (PAI-1), a procoagulant factor, and HMB. Such factors, along with other important pro- and anticoagulant factors, such as von Willebrand factor (VWF) and tissue factor pathway inhibitor (TFPI) are expressed by endothelial cells. We hypothesize that endometrial endothelial cells (EECs) regulate uterine hemostasis through the up and down regulation of coagulation factors and that differences in such factors contribute to HMB. We propose to investigate the role of tPA, PAI-1, TFPI, VWF multimers and EECs in uterine hemostasis through (1) quantification of tPA, PAI-1 and TFPI in the menstrual blood of women with and without HMB on each day of menstrual bleeding, (2) comparative analysis of VW multimer distribution in the same samples and (3) single cell RNA sequencing of endometrial biopsy samples, including EECs, with attention to coagulation factors. The results of this study will be used to generate testable hypotheses regarding management options for HMB. Dr. Samuelson Bannow has generated data supporting the important contribution of coagulation factors to differences in menstrual bleeding by demonstrating increased risk of HMB in women who use oral anticoagulants associated with higher peaks of activated anti-factor X effect compared to those with lower peak effect. Her primary mentor, Dr. Alison Edelman, has extensive experience with clinical and translational studies of menstrual bleeding and her co-mentor, Dr. Les Myatt, has expertise in women's health and basic science techniques such as cell culture and RNA sequencing. Her advisors will provide additional expertise and guidance in interpretation of RNAseq data and biostatistics. The proposed training will enable Dr. Samuelson Bannow to acquire clinical and research expertise in women's health, gain experience with translational and basic scientific research methods, and develop skills in granstmanship and scientific communication. Importantly it will also allow her to develop the necessary research infrastructure and program for future, independently funded studies in the pathophysiology and management of excessive uterine bleeding.

NIH Research Projects · FY 2026 · 2025-09

PROJECT SUMMARY Ophthalmology heavily relies on multimodal electronic health record (EHR) data, such as clinical notes, structured data, and imaging, to diagnose, treat, and monitor patients. However, inconsistencies in documentation and the lack of standardized datasets pose significant challenges to leveraging this data for research and clinical applications. Developing robust AI-driven tools that integrate and standardize multimodal EHR data can transform ophthalmic research and improve patient outcomes. This project focuses on advancing the development of AI models, including natural language processing (NLP) and large language models (LLMs) to address these challenges. Specifically, the project will develop tools to enhance data quality, extract key clinical concepts, and standardize multimodal datasets for ophthalmology research. These tools will support tasks such as predicting disease progression, improving clinical workflow efficiency, and enabling data harmonization for multicenter research.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY Necrotizing enterocolitis (NEC) is a devastating inflammatory disease that affects the intestines of premature infants. There is a major gap in our understanding of the factors that contribute to the pathophysiology of NEC, including no cure for this often deadly disease. This proposal aims to help fill the knowledge gap by defining a role of the developmentally expressed mRNA-binding protein IMP1 in intestinal mucus composition and survival during NEC. A healthy, mature mucus barrier is essential to protect the intestinal epithelium from inflammation. Our preliminary work shows that Imp1 expression promotes survival during NEC with concomitant upregulation of Qsox1, an enzyme involved in the final glycosylation step (sialyation) of mucus, which translates into the detection of less immature mucus in mice expressing Imp1 during NEC pathogenesis. Roles for IMP1 in NEC and intestinal mucus composition are not known. Understanding how Imp1 regulates Qsox1 and modulates mucus maturation could open new pathways for therapeutic development in NEC. Our long-term goal is to leverage post-transcriptional regulation of intestinal epithelial damage response to improve outcomes for infants at risk for or with NEC. The overall objective of this proposal is to delineate mechanisms of Imp1-mediated survival and intestinal mucus glycosylation. Our central hypothesis is that Imp1 promotes Qsox1 expression to enhance the mucus barrier and promote survival during NEC. Our specific aims are to 1) determine the impact of Imp1 expression on intestinal mucus composition throughout NEC pathogenesis and 2) define the requirement for Qsox1 in Imp1-mediated survival in NEC. Our approach will use Imp1 genetic mouse models combined with an experimental NEC-like intestinal injury model to define the impact of Imp1 on intestinal mucus, including glycosylation, permeability, ultrastructure, and intestinal proteomics during NEC. Key findings will be confirmed in human NEC tissue. We will define Imp1 target mRNAs in the NEC intestine and the Imp1 binding site within Qsox1 via crosslinking RNA immunoprecipitation. We will crossbreed Imp1 overexpressing and Qsox1 knockout mice to determine if Qsox1 is required for Imp1-mediated NEC survival. This research is innovative because it 1) links Imp1 to survival in NEC, 2) connects Imp1 to mucus regulation via Qsox1; 3) will define new molecular mediators of mucus composition in NEC. At the end of the project, we expect to 1) establish Imp1 as a regulator of mucus in NEC; 2) generate novel data connecting Imp1 to Qsox1 regulation and intestinal mucus; 3) use proteomics to uncover mechanisms by which Imp1 promotes survival in NEC; 4) define new pathways that could be leveraged for NEC prevention or treatment. Positive impact: This research will define a role for intestinal mucus in NEC and Imp1 as a new regulator of mucus maturation during inflammation, opening new lines of therapeutic investigation.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY In 2019, Escherichia coli (E. Coli) alone was responsible for nearly 1 million deaths worldwide. Difficulties in vaccination and therapeutic development have exposed the need for research on innate immune mechanisms in the intestinal epithelium considering E. coli is usually contracted via an oral route. Among other important defenses, enteropathogenic bacteria like E. coli activate an innate immune sensor termed the NAIP-NLRC4 inflammasome. This leads to inflammatory cell death termed pyroptosis and extrusion of the infected epithelial cell into the lumen of the intestines, which prevents further dissemination of bacteria and maintains the integrity of the epithelium. Several other inflammasomes have been implicated in enteropathogenic infection, however functional redundancies and steady state roles have not been clarified. I propose to study which inflammasomes from this family of sensors are activated and protective during E. coli infection beyond NAIP-NLRC4. Our lab has found that infection with the E. coli murine model, Citrobacter rodentium (C. rodentium) in the absence of the NAIP-NLRC4 inflammasome and the non-canonical inflammasome Caspase 11 still induces pyroptosis. This pyroptosis is accompanied by “specks” of the inflammasome adaptor ASC, indicating that another inflammasome may be active in intestinal epithelial cells (IECs). This project will assess the contribution of another inflammasome, NLRP1, to the host response. NLRP1 is a prime candidate as it is expressed and protective at other barrier tissues, can be activated by bacterial effectors, and has been shown to form ASC specks. This also implies strong evolutionary pressure on the pathogen to circumvent these redundant sensing pathways. I will inquire if E. coli can inhibit multi-inflammasome activation by specifically targeting downstream outcomes of inflammasome activation, such as extrusion. I found that the C. rodentium actin nucleating effector, Tir, reduces the host cell's ability to extrude, a highly actin dependent process. I hypothesize that the NLRP1 inflammasome activation in the intestinal epithelium is protective during C. rodentium infection, however C. rodentium effector Tir is able to circumvent the inflammasome by inhibiting IEC extrusion. To investigate this, my first aim will evaluate the role for NLRP1 in inducing pyroptosis during live imaging of primary colonic monolayer infection by C. rodentium, and the temporal characteristics of the extrusion. Furthermore, using NLRP1 knockout mice I will investigate the contribution of NLRP1 to maintaining protection from C. rodentium in vivo. In my second aim, I plan to evaluate the characteristics of extrusion including timing and actin structure, following infection with a C. rodentium mutant Tir strain. This work will use cutting edge stem cell derived organoid and imaging tools to reveal inflammasome pathway redundancy during enteropathogenic infection and the countermeasures C. rodentium takes to quell this host function. With guidance from my sponsor and cosponsor Dr. Rauch and Dr. Merritt, and the resources at Oregon Health & Science University, I will be able to accomplish these aims and work toward my career goals.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY SARS-CoV2 infection has impacted millions of people worldwide. An estimated 5-10% of individuals continue to experience complex, long-term symptoms even after recovering from acute SARS-CoV2 infection. Long COVID has been linked to various risk factors, including the reactivation of persistent viral infections like Epstein-Barr virus (EBV) and other human herpesviruses. Understanding the timing of persistent virus reactivation is an important step in devising effective mitigation strategies. Moreover, causal relationships between herpesvirus reactivation and long COVID remain unclear. Current diagnostic tools are insufficient and there remains an unmet need for early predictive and prognostic biomarkers. Notably, human herpesviruses such as EBV express numerous viral microRNAs throughout infection and SARS-CoV2 itself encodes a miRNA-like small noncoding RNA. Additionally, SARS-CoV2 infection dramatically disrupts the host miRNA environment. Given that miRNAs are highly stable in circulation and have demonstrated utility as biomarkers for various clinical conditions, we hypothesize that herpesvirus-encoded miRNAs could serve as diagnostic and/or prognostic indicators of SARS- CoV2-related illnesses. Our preliminary studies on SARS-CoV2 cases uncovered an increased prevalence of circulating EBV miRNAs in symptomatic individuals. In this project, we will evaluate circulating viral and host miRNA signatures in SARS-CoV2 infections. Outcomes of these analyses will shed light on miRNAs that can be used as systemic biomarkers to aid in stratifying patients and identifying those with active herpesvirus infections.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Placental dysfunction causes more than half of preventable stillbirths in the United States. Placental dysfunction also causes preeclampsia, fetal growth restriction, and gestational diabetes, which can represent near misses for stillbirth. Stillbirths can be prevented by identifying pregnancies at increased risk and using increased fetal monitoring and sometimes early delivery before stillbirth occurs. Current ways of assessing pregnancy risk misses the majority of people who go on to have a stillbirth. Existing biomarkers of placental health fail to detect most placental dysfunction occurring later in pregnancy. Focus on nutritional health and allostatic load could improve pregnancy risk assessment, as they are both associated with cardiometabolic risk factors for placental dysfunction. Allostatic load is the inflammatory state caused by chronic exposures to stress, such as racism. Assessing allostatic load and maternal nutrition helps address inequities in stillbirth. Dietary intake represents a potentially modifiable risk factor and thus a target for intervention to improve pregnancy health. To reduce the incidence of stillbirth, we must improve 1) our understanding of how allostatic load contributes to placental dysfunction, and 2) the role of nutrition in placental dysfunction. Without this knowledge, we are limited in assessing pregnancy risk and identifying impactful interventions to prevent stillbirth. Our long-term goal is to improve prediction and prevention of stillbirth. We propose rigorous evaluation of the impact of allostatic load and perinatal nutrition on placental dysfunction. The main objective of this project is to identify the effect of allostatic load and dietary patterns on pregnancy outcomes and placental health. The secondary objective is to advance the field of placental biology using novel assessments of placental function in order to improve detection of placental dysfunction during pregnancy. Aim 1: Measure incremental benefit of adding allostatic load biomarkers to classic placental biomarkers in predicting risk of clinical placental dysfunction. Aim 2: Determine the association between perinatal nutrition (ultra-processed food consumption and Healthy Eating Index) and clinical placental dysfunction. Aim 3: Determine association between key biomarkers of placental health and allostatic load and novel measures of placental tissue function and stress. To test these aims, we will enroll 495 pregnant participants prior to 14 weeks' gestation and following them prospectively through pregnancy. We will conduct dietary assessments and serial blood draws to assess allostatic load, cardiometabolic, and placental biomarkers at three time points; two prenatal ultrasound assessments of fetal and placental development; and placental histology, exosome, and trophoblastic functional evaluations to determine the role of allostatic load and nutritional contributors in placental dysfunction. This work has the potential to reduce preventable stillbirth through better risk assessment and improved placental health.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY With more than 500 million people affected worldwide and the number of cases steadily rising, diabetes presents a significant public health challenge. Thus, there is an urgent need for developing effective strategies for diabetes management to mitigate the burden of the disease on individuals and healthcare systems. Because most of the day-to-day care in diabetes is handled by patients, self-management is critical to maintain adequate glycemic control and prevent diabetes-related complications. However, self-management is a complex and involves continuously monitoring glucose levels, adhering to recommended treatment and medications, and adjusting diet and physical activity. The increasing availability of digital technologies and advancements in artificial intelligence (AI) offer new opportunities to improve decision support systems by enabling the monitoring and analysis of multi-modal health to identify digital biomarkers that provide insights into the various factors influencing diabetes, leading to a more comprehensive understanding of the disease and its management. Previous research works have focused on identifying digital biomarkers of glycemic control from glucose management data (i.e., glucose, insulin, nutritional, and physical activity data). The goal of this project is to expand the scope of past studies by exploring digital biomarkers that can capture clinically relevant glucose metrics, physiological states, and individuals’ behaviors to predict glucose and patient-reported outcomes. By leveraging physiological signals such as heart rate, step count, sleep data, glucose management data, and patient-reported outcomes from individuals with type 1 and type 2 diabetes, we aim to develop an AI-based framework to automatically identify biomarkers that can be used to better understand diabetes and its management and to inform the development of patient-centered decision support tools to both simplify diabetes management and improve glycemic control. This 2-year project involves collaboration between data scientists and clinical investigators to complete 2 proposed aims. Aim 1 involves (1.1) engineering biomarkers from multi-modal health data that relate to glycemic control, individuals’ behaviors, and patient-reported outcomes; (1.2) developing neural network-based data fusion models to predict glucose outcomes and patient-reported outcomes (e.g., sleep quality, fear of hypoglycemia, and diabetes distress surveys scores) using multi-modal digital biomarkers; and (1.3) using explainable AI and domain knowledge to identify the most relevant biomarkers. In Aim 2, (2.1) we will develop a random forest-based algorithm for building patient-centered decision support tools to provide actionable recommendations on what a person with diabetes can do to improve glycemic control considering the user’s specific goals and preferences. (2.2) We will demonstrate our approach in type 1 and type 2 diabetes in silico.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Tuberculosis (TB) caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb) is the leading cause of infectious disease morbidity and mortality worldwide. Immune system sampling of the intracellular environment for Mtb antigens is crucial to control infection and prevent active tuberculosis. Major Histocompatibility Complex, Class I-Related molecule (MR1) is a conserved nonclassical antigen presenting molecule expressed in several cell types that presents Mtb derived riboflavin metabolites to MR1-restricted T cells (MR1Ts). MR1 antigen presentation to this subset of CD8+ T cells is poised to play a crucial role in controlling Mtb infection; MR1Ts are enriched in the lung and rapidly release pro-inflammatory cytokines and apoptotic factors to kill infected cells. Our laboratory has found that endosomal trafficking facilitates MR1 antigen presentation, yet the exact mechanisms by which MR1 loading and trafficking to the cell surface occurs are not known. The specific purpose of this project is to decipher molecular mechanisms of endosomal trafficking to ultimately inform our understanding of MR1 antigen presentation. We have found that endosomal calcium signaling mediates MR1 antigen presentation during Mtb infection. Interference of two-pore calcium channels (TPCs) via siRNA-mediated knockdown specifically decreases MR1 presentation of Mtb antigens. Nicotinic acid adenine dinucleotide phosphate (NAADP) facilitates endosomal calcium release by activating TPCs; treatment with a selective, membrane-permeant NAADP antagonist or knockdown of an NAADP binding protein important for TPC activation also decrease MR1 presentation of Mtb antigens. NAADP is synthesized from NADP, and this reaction requires catalysis by ADP-ribosyl cyclase. Cluster of Differentiation 157 (CD157) is one of few proteins that possesses this enzymatic activity. The central hypothesis of this proposal is that Mtb infection triggers CD157-mediated NAADP generation to facilitate MR1 antigen presentation of the Mtb-infected cell via the TPC. To test this hypothesis, I will infect antigen presenting cells with Mtb and assess MR1 antigen presentation using a human MR1T cell clone. Experiments in Aim 1 will further elucidate the role of TPC calcium release in MR1 antigen presentation via T cell IFN-γ release ELISpot assays, and characterize interactions of TPCs with MR1 and Mtb via live-cell imaging. Aim 2 will identify the source of NAADP and establish where NAADP generation occurs in relation to MR1 antigen presentation of Mtb-infected cells. This proposed work will have broad implications for how endosomal calcium sensing facilitates recognition of Mtb-infected cells. The results from this research will elucidate key, novel pathways that underpin the immune response to Mtb, which have potential to inform tuberculosis vaccine development.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Cell turnover and active secretion release extracellular (also so-called cell-free) RNA, DNA, and vesicles into circulation. The presence of these extracellular analytes in bodily fluids has enabled broad applications in oncology, transplantation, obstetrics, and metabolic and neurological degenerative diseases. Analyzing cell-free DNA (cfDNA), cell-free RNA (cfRNA), and extracellular vesicles (EVs) provides clarity in understanding disease states for clinical management. Major technical challenges in cfDNA, cfRNA, and EV analyses are associated with their complexity, low abundance, and preanalytical variation. My lab develops new tools to address these challenges in high-throughput deep characterization of cfRNA, cfDNA, and EVs in bodily fluids. First, we will develop tools to reliably decompose and control for technical variability, RNA degradation, and complexity of cfRNA. To address technical variability, we will identify an intrinsic cfRNA reference gene set using non-negative matrix factorization. To reliably determine the cell-type-specific footprint of cfRNA, we will develop a k-mer-based classification at the individual sequencing read level, leveraging highly abundant and commonly expressed genes. To overcome the fluctuation and a high dropout rate caused by cfRNA low abundance and degradation, we will build a computational framework for anomaly detection and biomarker combination using topic modeling. Second, we will introduce a new method to induce the epigenetic contrast of cell-free nucleosomes (cf- nucleosomes) directly in body fluids through polyamine-mediated phase separation. We aim to measure and map cf-nucleosome pseudo-accessibility at sites that differ for cell states such as active and repressive chromatin marks, transcription factor binding sites (TFBSs), regulatory loci, gene bodies, and 3D genome structure. Third, we will develop a new technique to dissect the heterogeneity of EVs at the single vesicle level using proximal barcode transferring. To enhance barcode transferring efficiency, we propose using bridge amplification and DNA walking. To improve background correction and address the scarcity of proteins detected on individual EVs, we will develop a computational framework utilizing an innovative mixture model approach that accounts for noise and is robust against measurement dropout. In summary, we are developing methods to analyze low-abundance and complex circulating cfRNA, cf-nucleosomes and EVs in accessible bodily fluids. Our non-invasive approaches will enable the investigation of natural longitudinal disease progression and adaptive responses to interventions in human, non-human primates, and animal models.

NIH Research Projects · FY 2026 · 2025-09

Project Summary Opioids play a frontline role in acute pain management in the clinical setting. Prolonged use of opioids causes multiple adaptations including tolerance—a phenomenon in which increasing opioid dosage is required to maintain drug efficacy. The pharmacological component of opioid tolerance is largely driven on the cellular level and is the result of cellular protein networks terminating the activity of the mu opioid receptor (µOR). A critical step in this regulatory process is membrane trafficking. High efficacy opioids cause the µOR to rapidly traffic from the cell surface to internal cellular structures called endosomes. At endosomes, the µOR is either sorted back to the cell surface for continued activity (resensitization) or sent to the lysosome for destruction (downregulation). A long-standing model is that downregulation can drive pharmacological opioid tolerance through proteolysis of opioid receptors outpacing new receptor synthesis. One challenge in testing this model is that we don’t know the identity of key cellular protein networks regulating mu opioid receptor trafficking at endosomes. Here we leverage our recently developed proteomic and genomic methods for identifying cellular proteins which regulate opioid receptors. In Aim 1 we focus on the trafficking of the µOR at endosomes and, using biochemical and cellular techniques, test the hypothesis that the endosomal Retromer complex rescues mu opioid receptor from lysosomal degradation in a process called recycling. We will use functional genomics to identify potential druggable targets—kinases and ubiquitin-ligases—that regulate µOR through endosomes, and test the related hypothesis that opioid-induced downregulation of the µOR can be blocked by engineered “super-recycling” variants of the receptor. In Aim 2 we test the hypothesis that endosomal recycling and the endosomal Retromer complex controls three different phases of µOR signaling: endosomal signaling, resensitization, and the development of pharmacological opioid tolerance at the cellular level. Together, these studies seek to define a critical regulatory process controlling µOR function and identify cellular proteins which could be targeted to control the development pharmacological opioid tolerance.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Approximately 120,000 young children are newly infected with HIV-1 each year. In the absence of antiretroviral therapy (ART), infants have a mortality rate of up to 50% within the first 2 years of life, but administration of ART reduces morbidity and mortality and is now implemented early in life for most children. Unfortunately, even with ART, children under 5 years old are at higher risk of mortality than older children or adults on ART. While a number of factors may contribute to this increased mortality, a hyporesponsive immune system likely plays an important role. Children living with HIV-1 have been shown to have diminished antibody responses, with a lack of HIV-1 seroconversion after early ART and poorer antibody responses than their uninfected peers to common childhood vaccinations. The overarching goal of our research is to improve HIV- and vaccine-specific antibody development in children living with HIV-1 by targeting the differentiation of T follicular helper (Tfh) cells, which provide critical survival and differentiation signals to B cells. Tfh differentiation is limited in infants compared to adults, and our preliminary analysis of pediatric clinical samples identified elevated Helios expression in infant CD4+ T cells as a potential regulator of Tfh cell development. CRISPR knock out of Helios in naïve CD4+ T cells from cord blood or healthy adult blood before in vitro Tfh differentiation revealed a negative correlation between the frequency of Helios+ cells remaining and subsequent Tfh cell differentiation. We hypothesize that administration of a Helios degrader, DKY709, to target Helios expression in CD4+ T cells will improve Tfh cell differentiation and subsequent antibody development after vaccination or infection. Due to the uniquely high expression of Helios in infant CD4+ T cells but not adults, we will use the infant rhesus macaques model of HIV to determine if Helios degradation by DKY709 will improve the magnitude and durability of antibody responses to 1) vaccines administered to SHIVSF162P3 infected or naïve animals and 2) SHIVSF162P3 after early ART administration. Together these studies will provide important preliminary data about the capacity of Helios degradation to improve Tfh differentiation and humoral immune responses in infants, particularly in the context of SHIV infection. Importantly, the results from either aim will provide critical information that can be used as the basis for future research proposals. The candidate is currently a Research Assistant Professor in the Vaccine and Gene Therapy Institute, which is located on the Oregon Health & Science University West Campus with the Oregon National Primate Research Center. This provides a highly collaborative environment in which to do HIV research with nonhuman primate models. Building on her previous research studying immune responses in clinical cohorts, this proposal will further her training in using the infant SHIV infection model to assess immune responses to therapeutic interventions and in developing clinical trials to translate research findings into infants. Completion of the career development plan and innovative research study will provide a strong foundation upon which to build a successful independent research career in pediatric HIV-1 immunology.

NIH Research Projects · FY 2026 · 2025-09

Photoreceptors are specialized sensory cells that convert light stimuli to neuronal signal. Diminished responses to light suggest cell damage and death, which lead to poor vision and blindness. Preserving and restoring photoreceptors are meaningful therapeutic targets for retinal degeneration. Diagnosing, managing, and drug development require accurate and reliable assessment of visual function. This multidisciplinary program will develop innovative ophthalmic imaging technologies and novel objective, quantifiable functional biomarkers for cone photoreceptors. The prospect of a sensitive tool for detecting early vision changes and over a short period of time represents a paradigm shift in ophthalmic tests and trial design. Aim 1. We will develop next generation optical coherence tomography (OCT) technologies to enable functional photoreceptor imaging over a wide retinal area and at high topographical resolution. OCT split-spectrum amplitude-decorrelation optoretinography (SSADOR) is an integrated hardware-software approach that can measure photoreceptor light response without the need for electrode contact, subjective feedback, or highly complex instruments. OCT SSADOR enables co- registered analysis of photoreceptor function and retina morphology, which can help understand disease mechanisms and to facilitate longitudinal observations. Innovations in ultrahigh speed OCT and SSADOR will lead to a clinically accessible tool for assessing photoreceptor function over the macula, which is responsible for fine central vision and associated with quality of life. Aim 2. We will develop novel objective, sensitive, and reliable biomarkers of cone mediated visual function. Advanced software algorithms can reduce SSADOR measurement variability. We will use parallel computing technique to reduce processing time and enable real time feedback to guide clinical imaging, further reduce variations and improving yield. Highly accurate and sensitive SSADOR cone functional measurements can identify subtler impairments and detect changes early in disease progression. This study will characterize the measurement repeatability of SSADOR biomarkers. Additionally, we will establish a normative reference database to allow for inter-subject comparison and facilitate clinical interpretation. Aim 3. We will investigate cone photoreceptor impairment and loss associated with inherited retinal disease (IRD), a heterogenous family of vision threatening conditions. This pilot clinical study will compare SSADOR functional biomarkers in two independent cohorts of IRD patients and age-matched normal controls in global, regional, and local scales. Measurements will be correlated with clinical diagnoses of disease severity and visual function (e.g., best corrected visual acuity, retinal sensitivity, and electroretinogram). We will follow IRD patients longitudinally to monitor disease progression and identify fast progressors. If successful, this program will develop imaging biomarkers of visual function that can be practically used in the clinics for a wide range of retinal diseases. This will save vision by guiding medical intervention through active progression monitoring, and by facilitating therapeutic trials with sensitive and reliable visual outcome endpoints.

NIH Research Projects · FY 2025 · 2025-09

In this proposal, we will define cell states in the kidney, and specifically along the thick ascending limb, distal convoluted tubule, and connecting tubule in health, and during stress. In preliminary work, we have used an technique called INTACT to enrich specific cell types, allowing us to study their transcriptome in unparalleled detail. We have combined this approach with immunohistochemistry, allowing us to find new cell types in these tubule segments. We recently used this approach in a publication defining the distal convoluted tubule Here, we will use new mouse lines that allow us to enrich connecting tubules to test how potassium stress and diuretic drugs alter cell state, leading to both homeostatic adaptation and to dysfunction. We will then examine thick ascending limb cells, where our preliminary data suggest the presence of two previously unrecognized cell types. One of these generates a transepithelial voltage and mediates salt reabsorption. The other is electrically neutral, but mediates the reabsorption of calcium and magnesium. We will then test whether water stress and calcium stress activate these cell types selectively. To achieve this, we have planned the following Specific Aims: Aim 1: Define the role of cell types and states in DCT and CNT function Characterize cell types along DCT and CNT Determine how dietary K+ intake alters cell state to modulate Ca2+ excretion Determine how diuretic drugs alter cell state to modulate Mg2+ and Ca2+ excretion Aim 2: Define the role of cell types and states in TAL function Characterize TALa and TALb cells and develop a new physiological model Determine how water stress and arginine vasopressin affect TALa cell states and function Determine how calcium stress affects TALb cell states and function

NIH Research Projects · FY 2025 · 2025-08

Cardiovascular diseases (CVD) are the number one cause of premature death in American women, but are highly preventable chronic illnesses. Compared with men, women are disproportionally at risk for developing CVD, which presents and progresses differently in women. However, female-specific risk factors (such as endometriosis and hypertensive disorders of pregnancy) for CVD are not included in clinical tools used to guide prevention and treatment of CVD. The overarching goal of this career development award proposal is to support the applicant in developing the necessary skills and training in informatics, epidemiology, and preventive cardiology to lead the advancement of risk assessment for CVD in women. Commonly used CVD risk-prediction tools are biased towards undertreatment for women. This may include the newly released CVD risk-prediction tool from American Heart Association called the PREVENT Equations, which do not include female-specific CVD risk factors. The overall research objectives of this application are to 1) test sex-differences in the predictive performance of the PREVENT Equations, 2) determine if female-specific risk factors are available in electronic health record data and if they accurately reflect participant-reported history, and 3) use best-subset statistical modeling to examine if adding female-specific CVD risks to traditional CVD risk factors improves our ability to predict CVD in women. The central hypotheses are that the PREVENT Equations are valid measures of CVD risk in men and women, female-specific data in the electronic health record is discordant from self-reported data, and that using robust statistical modeling will identify one or more female-specific CVD risk factors that improves model performance when added to traditional CVD risk factors. The first aim of the proposed research is to test the predictive performance of the PREVENT Equations using the NIH-supported All of Us Research Program data. Using sex-stratified data, discriminative statistics are expected to validate the PREVENT Equations predictive ability for time-to-first cardiovascular event among both women and men. The second aim is to assess the availability and quality of female-specific CVD risk factors in the All of Us electronic health record data using comparative statistics. The third aim is to measure the incremental value of female-specific CVD risk factors on the traditional CVD risk factors using the All of Us Research Program data. We will use best-subset logistic regression modeling to find the best fitting model for female-specific CVD risk prediction. The proposed research is significant in addressing gaps in clinical assessment of CVD in women, and innovative by engaging the relatively new NIH All of Us Research Program and leveraging informatics for accurate identification of CVD risks in the electronic medical record to improve women's CVD health. The proposed research and mentored training plan included in this award will support the applicant in their long-term career goal to become an independent investigator focused on reducing the burden of cardiovascular disease among women.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Post-translational ubiquitin signaling is emerging as a key component of both immune defense as well as bacterial virulence. Though they lack a canonical ubiquitin system of their own, pathogenic bacteria have evolved secreted `effector' proteins to manipulate ubiquitin signals, thereby gaining access to host cell processes including targeted protein degradation, trafficking, and inflammation. The far-reaching importance of ubiquitin signaling across many cellular processes stems from its ability to form a diverse set of polymeric chains that signal for distinct outcomes. The complexity of ubiquitin signaling vastly outweighs our understanding of its regulation and cellular outcomes. While the signaling roles for some types of polyubiquitin are known (e.g., protein degradation directed by Lys48-linked polyubiquitin), the functions of many so-called `atypical' subtypes have remained a mystery despite decades of research. Remarkably, we and others have recently identified a series of bacterial effector proteins that have evolutionarily converged upon the specific regulation of atypical polyubiquitin signals linked through Lys6, suggesting this post-translational signal must play a central role within the host-pathogen interface. In a fascinating paradox, we find that bacterial pathogens like enterohemorrhagic Escherichia coli (EHEC) have evolved to upregulate Lys6 polyubiquitin, while others such as Legionella pneumophila have evolved to downregulate it. Based on our preliminary data and clues from outside the context of infection, we propose that bacteria hijack Lys6 polyubiquitin as an accelerated proteasomal degradation signal. We propose to test this hypothesis from three perspectives: 1) understanding the molecular basis of Lys6 polyubiquitin specificity, 2) defining the roles of bacterial E3 ligases that form Lys6 polyubiquitin signals during infection, and 3) identifying the molecular pathways restricted by bacterial deubiquitinases that remove Lys6 signals. Our study benefits from multiple conceptual and technical innovations, including the ability to rewire bacterial E3 ligases between Lys6 and Lys48 specificity, allowing detailed studies of their effects on protein degradation and bacterial virulence. In addition, by removing the ability of L. pneumophila to restrict Lys6 polyubiquitination, for the first time we can identify upstream and downstream regulators of Lys6 signals, as part of what could be a new arm of cell-autonomous immunity. The insights this work will generate for the fields of bacterial pathogenesis and post-translational signaling are highly significant, and could lead to new opportunities for anti-virulence therapeutics. Over the next five years we will open a window into ubiquitin signaling at the host- pathogen interface in order to understand the evolutionary benefits of regulating this poorly studied, atypical Lys6 polyubiquitin signal.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Bacteria contain a vast diversity of metabolites due to rich accessory genomes 100 times greater than that found in humans. Most of these metabolites are unknown metabolites and are often excluded from current metabolite studies that use standard metabolomics references limited to common human, drug or industrial chemicals, providing an incomplete perspective in these investigations. There is currently no oral bacterial metabolite reference library available for the study of dental disease by metabolomics. The overarching goal of this research plan is to establish a novel 13C-labeled oral bacterial metabolite reference library with known oral bacterial strains and to propose an unprecedented integrated workflow approach combining the 13C-labeled oral bacterial metabolite library with clinical samples to select targeted unknown metabolites to be analyzed for differential LC-MS, transcriptomics and genetic modification leading to full characterization of unknown oral metabolites related to dental caries or health. In Aim 1, a 13C-labeled oral bacterial metabolite reference library for Streptococcus mutans and Streptococuss gordonii will be created using SWATH LC-MS, with dual peaks identified and curated to improve rigor and reproducibility in oral metabolomics studies. Under Aim 2, the newly generated oral bacterial metabolite reference library will be evaluated for feasibility and optimized for its application in the study of clinical saliva and plaque samples for the study of caries versus health. The goal of Aim 3 will be to establish the unprecedented workflow using the 13C-labeled oral bacterial metabolite library combined differential metabolomics, transcriptomics analysis, and genetic modification leading to isolation, purification and full characterization of previously unknown bacterial metabolites relevant to caries versus health. Creation of a 13C-labeled oral metabolite reference library is crucial for the generation of quality oral metabolomics analysis by allowing for differentiation and removal of noise or artifacts, quantification of labeled metabolites, preliminary identification of unknown compounds, normalization across multiple laboratories/equipment, corrections for ion suppression and, by our novel approach, will allow unknown metabolites to be linked back to specific oral bacteria for full structural elucidation. Most importantly, this approach will allow the inclusion of oral bacterial metabolites previously missed, overlooked, or excluded in other studies. This research will resolve many of the issues of quality metabolomics in oral studies, while limiting the unknowns to target for isolation, purification and characterization to manageable numbers. Solving these challenges in oral bacterial metabolomics will greatly advance the field of oral metabolomics investigations, leading to novel natural products discovery that can be used as treatment or prevention of oral (e.g., childhood dental caries) or other human disease.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Congenital hyperinsulinism (CHI) is an infantile/childhood disorder causing life-threatening hypoglycemia, brain damage, and developmental defects due to persistent over-secretion of insulin by pancreatic β-cells. CHI most commonly results from loss-of-function mutations in the pancreatic KATP potassium channel subunits, Kir6.2 and SUR1, encoded by KCNJ11 and ABCC8, respectively. KATP channels homeostatically couple insulin secretion and plasma blood glucose. Loss of KATP activity can result from loss of expression and/or loss of channel gating function. A subset of CHI patients retains sufficient residual KATP function to be treated successfully with a KATP channel opener, diazoxide. However, diazoxide is ineffective in numerous CHI patients who harbor mutations that impair KATP expression at the β-cell surface. These include missense mutations that disrupt folding, assembly, and/or trafficking of the channel to the plasma membrane, and nonsense mutations that introduce premature termination codons (PTCs) and prevent translation of full-length functional proteins. To avoid brain damage from severe hypoglycemia, patients carrying such mutations may require total pancreatectomy, which causes life-long insulin-dependent diabetes and digestive complications. Current gene replacement therapy methods using adeno-associated virus vectors are not suitable for the large SUR1 subunit, which harbors the great majority of KATP trafficking/nonsense mutations. Thus, there is a critical need for novel therapeutic strategies for diazoxide-unresponsive KATP-CHI. In this R21 application, we address this unmet challenge by exploring novel therapeutic concepts. Specifically, we hypothesize that (1) pharmacological correctors can rescue trafficking-impaired KATP channel mutants to restore insulin secretion regulation, and (2) Anti-Codon Engineered (ACE)-tRNAs can suppress PTCs to restore expression and function of KATP channels bearing nonsense mutations. We propose to establish cell and animal models of CHI-KATP mutations and conduct pilot studies needed to advance these new therapeutic concepts in two independent Aims. In Aim 1, we will generate and characterize human β-cell line and mouse models of CHI-KATP trafficking mutations and test the ability of pharmacological correctors to restore channel expression and function as well as normalize insulin secretion. In Aim 2, we will conduct a high-throughput screening to identify SUR1-PTCs amenable to ACE-tRNA rescue in a heterologous expression system, followed by generation of human β-cell lines harboring such mutations to evaluate insulin secretion correction by ACE-tRNAs. The proposal’s premise is built on a rigorous review of the literature and strong preliminary and published data from the investigative team members who have complementary expertise. The proposal is innovative as it will generate new tools and knowledge to advance new therapeutic concepts for CHI. Successful outcomes may transform the landscape of CHI clinical therapy and potentially impact discovery of therapeutics for other congenital diseases caused by similar molecular def ects.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY/ABSTRACT The Assisted Reproductive Technologies (ART) Core at the Oregon National Primate Research Center (ONPRC) supports a diverse research portfolio of studies conducted by internal investigators, foundation missions, and academic and industry partners. The ART Core provides advanced technology and exceptional expertise on nonhuman primate (NHP) gamete and ovarian follicle function, contraception, fertilization, early embryogenesis, implantation, fetal development, reproductive toxicology, and customization of NHP genomes for biomedical model development. NHP models of human disease are in high demand as the translational data obtained from studies can potentially revolutionize drug discovery and disease therapy development, supporting the many disciplines of the overall research community. Of particular relevance to this funding opportunity, the ART Core is home to a unique and nationally recognized repository, funded by an R24 mechanism, that contains cryopreserved biospecimens from multiple NHP species across multiple sources, which are accessible on demand to investigators. Currently, these biospecimens are stored in liquid nitrogen (LN2) across multiple free- standing open access containers that must be manually and diligently monitored by ART Core staff with LN2 refilled daily. Sample tracking is maintained through manual cataloguing and data entry. This type of manual storage system makes the samples susceptible to variations in temperature, spontaneous release of the vacuum, and samples being misidentified or lost. We therefore seek funding for a self-contained, automated cryogenic storage system that has multiple superior features including automatic and redundant LN2 monitoring and fill, temperature monitoring with alerts, automated sample placement and retrieval, inventory management and documentation, barcoding of samples, and partitioning of inventory. Such a system would allow the ART Core to ensure sample security and long-term preservation for the ONPRC/OHSU community and the larger biomedical research community, particularly in our mission to safeguard rare and valuable biospecimens to continue support for emerging, existing and future research programs.