Duke University

NIH Research Projects · FY 2024 · 2023-09

A cancer diagnosis requires patients and their intimate partners to communicate effectively to navigate illness- related challenges. Research indicates that couples who use effective communication strategies have better individual psychological adjustment and higher relationship satisfaction. However, many couples have difficulty communicating about cancer-related issues which can lead to poorer individual, relationship, and patient health outcomes. While dyadic interventions to improve couple communication have proven efficacious, they are often time intensive and have limited reach. The challenges of recruiting couples into dyadic interventions are well-documented, with low recruitment rates especially among underserved couples. Micro-interventions, which consist of brief educational materials and short activities delivered via text message or a mobile application, have significant potential to increase reach and participation in diverse groups of couples by increasing flexibility and reducing barriers to access. Previous studies have found micro-interventions to be effective in promoting health behavior change in a wide range of individuals and in enhancing dyadic functioning in community couples. To date, there are no existing micro-interventions that have been developed and tested in couples coping with cancer. Therefore, the proposed project aims to develop, and pilot test a text-messaging micro-intervention focused on improving communication skills for couples coping with advanced gastrointestinal (GI) cancer. The content of the dyadic micro-intervention will be adapted from existing, empirically validated couple-based interventions developed for cancer and non-cancer couples. Aim 1 focuses on developing the micro-intervention through focus groups and interviews with couples coping with advanced GI cancer and community advisors representative of minority groups, along with user experience testing with the target population. The data collected will be used to increase content relevance, acceptability, feasibility, and cultural sensitivity of the micro-intervention. Aim 2 will be a randomized pilot test of the dyadic micro-intervention to assess feasibility and acceptability. Aim 3 will examine pre-to-post intervention outcomes as measured by improvements in relationship satisfaction and constructive communication. This project is in line with the National Cancer Institute’s priority research area of cancer survivorship and has the potential to increase health equity in cancer by increasing participation of underserved couples. The proposed project will provide training in 5 key areas for the applicant: (1) psycho-oncology and cancer control, (2) theories and didactics related to the development, implementation, and dissemination of behavioral interventions in the context of cancer, (3) mixed research methods specific to development of psychosocial interventions, (4) health equity, diversity, and inclusion training, and (5) professional development. The unique and extensive resources available at Duke University Medical Center, including the Duke Cancer Institute, will aid and enhance training, scientific productivity, and successful completion of the proposed project.

NIH Research Projects · FY 2024 · 2023-09

Project Abstract Older adults with Alzheimer's disease and related dementias (ADRD) have a 2-fold increased risk of clinical bone fracture, and 33% higher rate of morbidity and mortality following fracture. Our prior study showed clinical fractures were reduced by 18% among those prescribed an acetylcholinesterase inhibitor (AchEI). With both cognitive and non-cognitive benefits, AchEIs such as donepezil would be valuable in a fracture prevention program, for older adults with ADRD, with multiple complementary and synergistic components. However, the pathways by which AchEIs reduce fracture risk represent a significant gap in knowledge. Before incorporating AchEIs into a multicomponent program, the specific effects of AchEIs on bone metabolism must be understood. The objective of this application is to measure the effect of ADRD treatment with AchEIs on fracture risk factors including bone mineral density (BMD), bone turnover markers, and bone quality. Our central hypothesis is that AchEIs reduce fracture risk through direct effects on bone metabolism via stimulation of osteoblastic bone formation and reduction in osteoclastic bone resorption. We will recruit adults aged >50 years from the Memory Disorders Clinic with mild to moderate ADRD (N = 45) who will be randomized 2:1 to either the AchEI donepezil 10 mg daily or placebo, respectively. From this biomarker-diagnosed cohort of older adults with mild to moderate ADRD, we will address the following Specific Aims: 1) Determine change over 12-months in Bone Mineral Density measured by dual x-ray absorptiometry associated with the initiation of donepezil; 2) Determine change over 6- and 12-months in Bone Turnover measured by (A) the Bone Resorption Marker C-telopeptide (CTX) and (B) the Bone Formation Marker Procollagen 1 intact N-terminal Pro-peptide (P1NP) associated with the initiation of donepezil; 3) Determine change over 12-months in Bone Quality measured by Trabecular Bone Score associated with the initiation of donepezil. In addressing this significant area, the current application focuses on several NIA priorities including multiple comorbidities and care for adults with ADRD. The proposed study is innovative in its comprehensive, prospective assessment of bone metabolism among adults with biomarker- based diagnosis of ADRD initiating AchE.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Older dialysis patients commonly have geriatric syndromes which contribute to increased healthcare utilization and poor quality of life. Currently, identification and management of geriatric syndromes are not included in dialysis care. Adding geriatric syndrome management into routine care for dialysis patients could result in significant improvements in quality of life and healthcare utilization. Barriers to integration of geriatric evaluation and management into dialysis clinics (e.g., time, personnel, costs) can be minimized through the development of innovative geriatric care models that leverage the existing dialysis unit interprofessional team and workflow. We will pilot a new dialysis care model that includes a centralized geriatric team that uses information from the Geriatric screen for OLder Dialysis patients (GOLD) to develop individualized recommendations for geriatric syndrome management based on the patient’s priorities. The GOLD is a self-administered screening battery of validated instruments to screen for a range of geriatric syndromes (cognitive impairment, depression, falls, mobility disability, social support needs, and malnutrition). The multidisciplinary dialysis team will integrate the recommendations into their care plans with support from the centralized geriatric team. Ultimately, this geriatric care model could be seamlessly integrated into dialysis clinic structure. The objective of this application is to optimize the care model with key stakeholder input and conduct a pilot randomized controlled trial (RCT) to obtain evidence critical to inform a definitive RCT. We propose three aims: 1) iteratively refine GOLD to achieve acceptable agreement with a geriatrician evaluation, 2) iteratively refine the geriatric care model to ensure acceptability and feasibility to key stakeholders, and 3) conduct a pilot RCT (n=100) to evaluate the geriatric care model’s impact on geriatric syndrome management and inform design of a larger RCT. For Aim 1, we will have participants complete the GOLD and undergo geriatric evaluation. We will assess agreement of each GOLD instrument with its corresponding geriatric evaluation, and if target agreement is not achieved, we will iteratively modify and re-test the GOLD instrument. For Aim 2, we will refine the care model using experience-based co- design with a stakeholder advisory board and multiple phases of feedback from study participants. After patients complete the care model, these patients and their dialysis clinicians will assess acceptability and feasibility of the care model through surveys and interviews. The care model will be iteratively refined until specific metrics of success are achieved. For Aim 3, patients will be randomized to receive the geriatric care model or usual care; geriatric syndrome management measured by patient report and chart abstraction will be compared at 3 months. We will also assess patient reported outcomes, physical function, and health care utilization at intervals up to 12 months to assess their adequacy for inclusion as outcomes in a subsequent definitive RCT. Upon completion, we will have key preliminary data for a large RCT testing a novel dialysis geriatric care model. Overall, this application will address the significant problem of geriatric syndromes in the older dialysis population.

NIH Research Projects · FY 2024 · 2023-09

PROJECT ABSTRACT Recognizing the significant impact of structural racism on kidney health in minority populations, the NIH is creating a new Consortium focused on Interventions that Address Structural Racism to Reduce Kidney Health Disparities. The Consortium will bring together research teams from six Intervention Sites to conduct community-engaged intervention studies to address structural racism and reduce disparities across the spectrum of kidney health and disease. We propose to serve as the Research Coordinating Center (RCC) for the Consortium. The Duke RCC will leverage extensive institutional resources and expertise at Duke University to provide administrative leadership and research coordination to ensure seamless operations of the Consortium and successful conduct of Consortium studies; support rigorous data collection, data management, and data analysis for Consortium studies; and foster research collaboration, capacity-building, and workforce diversification in kidney health equity research. Three closely integrated cores will lead the core activities of the RCC. The Administrative and Coordination Core will provide administrative, operational, and logistical support to the Consortium, including coordinating meetings, developing a secure web-based communications and collaboration platform, supporting program management and regulatory submissions, administering an opportunity pool for ancillary studies, and continually evaluating and enhancing Consortium operations. The Data Management and Analysis Core will contribute to the final design of Consortium studies, provide data monitoring for multi-site Consortium studies, and support statistical analysis for multi-site Consortium studies and pooled analyses of common data elements across the Consortium. The Collaboration and Community Engagement Core will foster a vibrant and collaborative research community within and beyond the Consortium by facilitating discussion at Consortium meetings and workshops, supporting dissemination of Consortium results to the broader research and lay communities, integrating selected NIH career development awardees in relevant Consortium activities, and exposing early-career trainees from underrepresented groups to kidney- focused structural racism scholarly work in order to successfully implement health equity research and interventions. Together, the multidisciplinary RCC team will ensure the successful completion of multiple intervention trials targeting disparities in kidney health and build a diverse and sustainable community of researchers and community partners focused on improving kidney health in marginalized populations.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT This application is in response to the NOSI of Promoting Fundamental and Applied Research in Inflammation Resolution, in particular to NIEHS’ interest in inflammation resolution related to environmental exposure. It is increasingly recognized that the immune response to an inflammatory stimulus involves specialized pro- resolving mediators (SPMs) that orchestrate the lung’s return to homeostasis by resolving cellular and tissue inflammation. However, little data in humans are available concerning the effects of PM2.5, a ubiquitous air pollutant, on SPMs and inflammation resolution. This is in marked contrast to the large body of literature on the proinflammatory response to PM2.5. Here we hypothesize that PM2.5 impairs cellular biosynthesis and kinetics of SPMs, leading to compromised resolution of inflammation in the airway. As airway inflammation is a hallmark of asthma, it is highly plausible but yet to be confirmed that individuals with asthma are less capable of resolving pollution-induced inflammation. No data are available to support a sex-specific hypothesis on inflammation resolution, despite the known sex-difference in proinflammatory responses to air pollution. Hence, we further hypothesize the effects of PM2.5 on inflammation resolution differ between people with and without asthma and between men and women. We propose to test these hypotheses in a translational study framework by leveraging an existing panel study of air pollution health effects. Our approach comprises of ex vivo cell culture experiments focusing on molecular mechanisms of SPM biosynthesis and resolution kinetics (Aim 1) and a panel study aiming to examine SPM-PM2.5 relationships in vivo (Aim 2) and to examine potential SPMs mediation of the PM2.5 effects on clinical outcomes (Aim 3). To maximize the translatability of the mechanistic findings in Aim 1, we will use primary airway epithelial cells collected from among the panel study participants and will use composition-characterized PM2.5 collected in London, UK, where participants reside. In Aim 2 panel study, 40 participants with and 40 without asthma will be measured 4 times longitudinally for SPMs in nasal fluid and induced sputum, representing the first portal of PM2.5 entry and the lung, respectively. Detailed personal PM2.5 doses and internal doses (biomarkers) of source-specific PM2.5 constituents hours to days prior to SPM measurements will be associated with sputum and nasal SPM concentrations. We anticipate to see differences by asthma and sex, respectively, in the time-concentration profile. In Aim 3, by leveraging the panel study’s rich dataset on health outcomes of clinical relevance, we will examine the mediating effects of SPMs on the exposure- outcome associations at the key time-points of inflammatory and resolution responses identified in Aims 1 and 2. Taking all together, we anticipate to link molecular mechanisms regulating SPM biosynthesis with resolution kinetics and clinically-relevant functional and inflammatory responses to PM2.5. The study will generate real-life data to better understand the role of SPM in resolving pollution-induced inflammation in the airways of asthmatics versus non-asthmatics and those of men versus women. 1

NIH Research Projects · FY 2025 · 2023-08

Metastatic melanoma is the most aggressive type of skin cancer with no treatment. Acquisition of invasive phenotypes is a critical event driving progression from primary to metastatic melanoma which is most detrimental for patient survival in THE case of melanoma brain metastases (MBMs). Invasion of melanoma cells depends on activation of several RHO-GTPases which regulate the biogenesis of cell protrusions. Among RHO-GTPases, RAC1 plays the most prominent role in melanoma progression. Indeed, P29S activating mutation of RAC1 (detected predominately in melanoma) is the 3rd most common mutation in sun-exposed melanomas. In addition, RAC1 and other RHO-GTPases function as effectors and/or regulators of signal transduction programs activated in MBMs, including MAPK, PI3K/AKT or STAT3 pathways. However, mechanisms regulating RHO-GTPase activity in melanoma in general or in MBMs are understudied. Physiological changes of intracellular GTP have not been thought to regulate RHO-GTPase activity because total GTP levels in the cell were considered to be much higher than GTP dissociation constant of RHO proteins (KdRHO•GTP). In our papers (Bianchi-Smiraglia et al. Nature Methods 2017 & Nature Communications 2021; Wolff et al, Cell Chemical Biology 2022), we demonstrated that not only free GTP levels comparable to KdRHO•GTP existed in the cell, but also that RAC1 activation depended on availability of local free GTP. Furthermore, RAC1 interacted with inosine monophosphate dehydrogenase 2 (IMPDH2, a rate limiting enzyme in GTP biosynthesis) and, through this interaction, with other GTP metabolism enzymes (GMEs). Accordingly, IMPDH2 and GMEs were enriched together with RAC1 in cell protrusions of invading cells. Disruption of IMPDH2-RAC1 interaction via sequestration of IMPDH2 away from the plasma membrane did not alter total GTP pools, but decreased local GTP levels in cell protrusions, RAC1 activity, and cell invasion. IMPDH2 and other GTP metabolism enzymes do not contain membrane localization sequences, however, do associate with the plasma membrane. We also demonstrated that high IMPDH2 levels correlate with poor prognosis in melanoma patients. Thus, in the 1st Specific Aim we will establish the mechanisms that recruit IMPDH2 to plasma membrane and cell protrusions. In the 2nd Specific Aim, we will identify and characterize mechanisms maintaining high IMPDH2 levels in metastatic melanoma cells. In the 3rd Specific Aim, we will interrogate the inhibition of IMPDH2 as a novel strategy for treatment of MBMs.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT – Overall Duke University is pleased to respond to RFA-IP-22-075 entitled “Limited Competition: Resources and Workforce Development for the Regional Biocontainment Laboratories”. Duke is one of 12-NIH funded Regional Biocontainment Laboratories (RBLs) in the country and has played an instrument role in the world class translational research being conducted at the DHVI and others throughout Duke and the Research Triangle region, by providing BSL-3 suites for virology, immunology, microbiology, and animal model services. Support from this grant will strengthen the overall infrastructure, ongoing facility and equipment maintenance, establish an enhanced BSL-3 safety and training program, and enhance access to and capabilities of the existing RBL Research Support services that are located in the BSL-3 environment. The significance of this grant is that it will allow the Duke RBL to better respond to emerging infectious diseases and play a leading role in pandemic preparedness. Specific Aims for this proposal are 1) to maintain reliable and uninterrupted facility functionality; 2) to ensure a safe, secure, and compliant work environment; and 3) provide a state-of- the-art biocontainment research environment and related support services that enhance the NIAID Biodefense Facilities Network. This grant will continue to allow Duke to be a crucial resource to a team of internal and external investigators dedicated to studying and countering select agents and BSL-3/ABSL-3 infectious agents of global importance.

NIH Research Projects · FY 2024 · 2023-08

Long-term outcomes for lung transplant recipients remain inadequate. Chronic lung allograft dysfunction (CLAD) is the most important immune-mediated complication of lung transplantation, representing the leading cause of late death among lung recipients. CLAD is diagnosed upon a sustained decline in lung function measures, yet the clinical course after diagnosis is highly variable. Despite the burden of CLAD, there remain no approved therapies. In part, this relates to difficulties in the design of CLAD trials due to a paucity of precise, generalizable information on CLAD progression under standard-of-care (SOC) conditions and lack of established surrogate endpoints for use in CLAD treatment studies. An additional barrier to advancing CLAD trials is the large number of participants needed and ethical concerns regarding enrollment in placebo-controlled studies for a progressive disease with substantial mortality risk. To address these gaps this proposal will create the largest multicenter cohort of lung recipients with CLAD available in the published literature and perform analyses that will inform the rational design of future CLAD trials. Specifically, to be responsive to this Notice of Special Interest (NOSI) this proposal will integrate longitudinal clinical data collected on adult lung recipients through a prior NIAID-funded Clinical Trials in Organ Transplantation (CTOT) study, CTOT-20, and through the NHLBI-funded Lung Transplant Outcomes Group (LTOG) study. Using these rich data, Aim 1 will precisely define the natural history of lung function progression after CLAD and employ latent class analyses of longitudinal lung function measures to discover subphenotypes of CLAD progression, incorporating other clinical variables that may influence disease behavior into the analytical framework. Plausible surrogate lung function endpoints for CLAD trials will be identified by assessing the association between each identified class and graft loss/death using landmarked Cox proportional hazards models. Aim 2 will evaluate the practical use of these real-world lung transplant datasets as a source of external SOC controls to complement future CLAD trials. Key eligibility criteria and endpoints assimilated from ongoing CLAD trials will be applied to evaluate how many patients from CTOT-20 or LTOG could potentially be included as real-world external controls for each trial. Simulations will then be conducted to assess operating characteristics when the external controls are used in hypothetical CLAD trials. This research is innovative because it represents a substantive departure from the existing state of CLAD investigation, namely in the application of modern statistical methods, such as latent class methods, to a large multicenter CLAD dataset and in the examination of innovative concepts, such as the use of external controls from historical data, a model of increasing interest to the FDA and industry. This research is significant because it will establish the natural history of CLAD and resolve the heterogeneity in CLAD progression in a multicenter real-world cohort; informing the design of future interventional CLAD trials in a way that is generalizable across center practices. Thus, aligned with the goals of this NOSI, these data are expected to improve the success of future CLAD trials.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT Computed tomography (CT) imaging for the body can result in thousands of images spanning many organs and myriad possible diseases. With growing patient load as well as increasing resolution and complexity of scans, the task of CT interpretation has become daunting. To improve radiologist performance, many artificial intelligence (AI) algorithms have been produced, but most are limited by their very narrow application to a specific disease in a specific organ or have been trained on limited data due to the high cost and complexity of manual annotation. As a result, there is an unmet need because existing AI solutions have not significantly improved the workflow or performance of radiologists. To meet these needs, we propose to develop a computer-aided diagnosis triage tool for CT of the chest, abdomen, and pelvis (CAP) that would focus radiologists’ attention on regions with a high likelihood of actionable disease while minimizing search efforts in regions of low likelihood. Our hypothesis is that a triage tool will improve radiologist workflow while simultaneously maintaining or improving performance. Our long-term goal is to create a clinical decision support system that will address bottlenecks of radiologist workflow and performance. As key steps toward demonstrating feasibility for that goal, we propose the following three specific aims: 1. Create framework for the assembly, deidentification, annotation, and sharing of over a million chest, abdomen, pelvis (CAP) CT cases from two major health systems. 2. Develop a triage system trained using multi-site CT datasets through collaborative/federated learning. 3. Pilot use of the triage system at multiple sites to allow radiologists to perform efficiently and equivalently for clinical tasks of assessing actionable disease in CAP CT. Key innovations will include the use of weak supervision to label a massive number of cases from two health systems. Labeling will be based on rule-based expert systems as well as natural language processing. Image classification will be based on deep learning models capable of processing an entire 3D CT volume and trained with federated learning to leverage the rich heterogeneity of data from the two health systems. The expected outcome of this project will be evidence to support a new clinical workflow for radiologist interpretation, which is the foundation for all medical imaging. For this project, we will maximize impact by addressing CAP CT because of the large patient load and complex anatomy/disease, and by producing one of the largest medical imaging datasets that can be shared for future research including grand challenges. In addition, by leveraging existing data in patient archives and radiology reports, our approach has the potential to be applicable to other body sites or imaging modalities in the future.

NIH Research Projects · FY 2025 · 2023-08

Abstract Treatment tailored to specific patient characteristics (i.e. precision medicine) has the potential to offer more effective options for post-operative pain management. Chronic pain conditions are a heavy burden to society due their economic impact and individual suffering they cause; and developing precision medicine approaches are a high priority for the Federal Pain Research Strategy. Accordingly, in this proposal, we will initiate a precision medicine approach for total shoulder arthroplasty by identifying a risk phenotype that is predictive of chronic post-surgical pain. Biologic, psychologic, and social factors (alone and in combination) will be considered as candidates for the risk phenotype. Additionally, changes in biochemical biomarkers and psychologic factors will be considered as post-operative time-varying factors to enhance predictive accuracy of the risk phenotype. This proposal will support a prospective cohort study of 461 individuals undergoing primary total shoulder arthroplasty (anatomic or reverse) at Duke Health. The primary outcome is chronic post- surgical pain 12-months after total shoulder arthroplasty. Risk phenotype measures will be collected pre- operatively and time-varying factors will be collected pre-operatively and post-operatively. We will investigate novel combinations of biologic, psychologic, and social factors to determine a chronic post-surgical pain risk phenotype for total shoulder arthroplasty. Additionally, time-varying factors will be tested for improving predictive accuracy of the risk phenotype. This proposal will advance the pain research field by addressing important knowledge gaps in chronic post-surgical pain development for adults with osteoarthritis that are undergoing a surgical procedure that is a) rapidly increasing in volume and b) has a high risk of post-operative pain. Successful completion of the study has the potential to alter standard of care for surgical selection and provide new avenues of precision medicine for post-operative pain management. Specifically, completion of this study may allow for risk stratification approaches that improve surgical decision making to be implemented in health care systems and/or identify novel treatment targets that could be tested for effectiveness in preventing chronic post-surgical pain.

NIH Research Projects · FY 2026 · 2023-08

Abstract Genetic diagnosis of complex disease is an important challenge in modern medicine. As a majority of GWAS hits implicate noncoding regions of the human genome, regulatory elements such as enhancers have become a major focus in the search for causal mechanisms. This proposal focuses on the development of computational methods for analyzing experimental data relevant to gene regulatory mechanisms and the variants that can perturb them, leading to disease. My lab is well positioned to have a sizeable impact on the experimental science in gene regulation ongoing at Duke and elsewhere, via existing collaborations and memberships in multiple consortia. In particular, my lab has been developing statistical models for detecting allele-specific gene expression in individuals and trios, to identify genes that may be under dysregulation. My lab also continues to develop methods for analyzing genetic variant data from massively parallel reporter assays, which was a focus of my Ph.D. thesis. And my lab has begun developing statistical methods for analyzing CRISPRi perturbations in single-cell data to identify gene-enhancer relationships. We expect these synergistic projects to result in more effective identification of causal variants and a higher diagnosis rate for currently undiagnosed patients of a wide variety of diseases, as well as potential leads toward the design of therapeutics.

NIH Research Projects · FY 2025 · 2023-08

Alcohol misuse is strongly associated with suicide crises (i.e., acute suicidal ideation or attempts) and death. The standard care for a suicide crisis, including for persons who misuse alcohol, is acute psychiatric hospitalization. Acute psychiatric hospitalization focuses on stabilization and crisis resolution prior to quickly discharging at-risk patients back into their stressful environments with a referral for outpatient care. Outpatient- based interventions focused on emotion regulation training have been shown to simultaneously reduce alcohol misuse and suicidal behavior. Yet, less than 50% of psychiatric inpatients follow through with outpatient treatment, which creates a dangerous gap in care; risk for suicide is the highest among recently discharging patients who misuse alcohol. This Mentored Patient-Oriented Research Career Development Award (K23) involves the development of a novel adjunctive intervention to (1) enhance standard care for at-risk psychiatric inpatients who misuse alcohol, and (2) create an opportunity for sustained recovery and reduced risk for a subsequent suicide crisis during the post-discharge period. This intervention, entitled mHealth-supported Skills Training for Alcohol and Related Suicidality (mSTARS), combines emotion regulation skills training implemented in the acute setting with a mHealth app designed to encourage utilization of these skills during the risky post-discharge period. The research plan for this K23 has two phases: development (Phase 1: AIMS 1 and 2) and evaluation of feasibility and acceptability of mSTARS (Phase 2: AIM 3). To inform mHealth app development, we will conduct a 6-week ecological momentary assessment (EMA) study on suicidal psychiatric inpatients who misuse alcohol (N = 35) to elucidate time-varying predictors for alcohol consumption and suicidal ideation, and examine the role of specific emotion regulation deficits. Analyses will facilitate adjustments to the app to make empirically-based recommendations for emotion regulation skills in real time (AIM 1). mSTARS, including the inpatient skills training component and mHealth app, will be iteratively refined per patient-driven modifications over two successive cohorts (n = 5 in each) of suicidal psychiatric inpatients who misuse alcohol (AIM 2). The finalized version of mSTARS, while incorporating AIM 1 findings, will be evaluated in AIM 3 in a three-arm feasibility/acceptability randomized control trial comparing mSTARS (n = 15) to inpatient skills training (n = 10) and treatment as usual (TAU) only (n = 10). The research plan for this K23 is closely tied to the PI’s training goals, which are to gain experience with (1) advanced longitudinal modeling of EMA data, (2) mHealth-supported treatment development, and (3) clinical trials design and management. Over the 5-year K23 award period, these training goals will facilitate the PI’s overarching career goal of becoming an independent clinical researcher. Beginning with this K23, the broader aim of the PI’s research program is to develop integrated, scalable, and cost-effective mHealth-supported interventions to treat concurrent alcohol misuse and suicidal behavior across treatment settings, reducing risk for this underserved population.

NIH Research Projects · FY 2024 · 2023-08

Severe, disabling pain is the hallmark of sickle cell disease (SCD). SCD pain is associated with poor quality of life, early mortality, and high healthcare costs. Clinicians face great challenges in managing SCD pain because of the poor understanding of the etiology of chronic/persistent SCD pain and the absence of validated clinical prognostic tools that can accurately identify individuals with SCD who are at risk of developing severe, persistent pain with associated physical and/or psychological disability. The overall objective of this project is to identify predictors of pain severity and pain-related outcomes in SCD using a prospective, longitudinal study design informed by the biopsychosocial model of pain. This proposal is supported by the hypothesis that painspecific psychological and sensory factors are strong, modifiable predictors of SCD pain severity and painrelated outcomes. The understanding of pain-specific psychological and sensory predictors of SCD pain outcomes is anticipated to have important implications for (1) identifying SCD patients who are at risk for severe pain outcomes, (2) informing preventive and therapeutic management of SCD pain, and (3) selecting patients for clinical trials of non-opioid interventions for SCD pain. The hypothesis will be tested by pursuing two specific aims: Aim 1) Determine psychological predictors of pain outcomes; and Aim 2) Ascertain the strength of pain distribution and sensitivity as predictors of pain outcomes. The candidate will use reliable and well validated pain-specific patient-reported outcome (PRO) questionnaires to evaluate the strength of psychological factors for predicting pain severity and other pain-related outcomes in the study cohort (Aim 1) and will use body mapping and quantitative sensory testing (QST) to examine sensory predictors of pain outcomes (Aim 2). The prospective, longitudinal design of this study and the use of biopsychosocial model of pain are innovative. The candidate's long-term goal is to become an interdisciplinary SCD pain expert who is a leading contributor to the treatment and understanding of the etiology and prognosis of chronic pain in adolescents and young adults with SCD. Her interdisciplinary training background in pediatrics and anesthesiology uniquely positions her for pursuing this research agenda. Through a detailed and specific career development plan developed in conjunction with her mentoring panel, the candidate will acquire formal training and expertise in SCD management, QST, assessment of the conceptual and psychometric properties of PROs, and advanced statistical modeling (risk prediction and prognostic modeling). Her mentoring and advisory committee includes an outstanding group of experts in psychosocial influencers of chronic pain (Francis J. Keefe PhD, Duke), QST (Claudia M. Campbell PhD, Johns Hopkins), SCD (Nirmish Shah MD and Paula Tanabe PhD, Duke; and Wally Smith MD, Virginia Commonwealth University), predictive modeling (Yi-Ju Li, PhD, Duke) and prognostic research in pain (Andrey Bortsov MD/PhD, Duke). Successful completion of this project is anticipated to improve the quality of life of individuals living with SCD.

NIH Research Projects · FY 2025 · 2023-08

Limb girdle muscular dystrophy 2B (LGMD2B) is a late-onset progressive muscular dystrophy resulting from mutations in the dysferlin gene. Dysferlin is a membrane-associated protein, highly expressed in skeletal and cardiac muscle fibers where it orchestrates membrane repair in response to various injuries. Currently, there are no ongoing clinical trials or therapies to slow disease progression or cure LGMD2B. While useful for in vivo mechanistic studies, dysferlin-deficient (BLAJ) mice, a model of LGDM2B, exhibit a mild disease phenotype compared to humans, limiting mouse utility for translational studies. Developing a high-fidelity in vitro model of human LGMD2B muscle would complement mouse studies and allow patient-specific disease modeling and drug discovery. Thus, the overarching goal of this project is to engineer a novel 3D human skeletal muscle tissue model (“myobundle”) that replicates the main structural, functional, and metabolic features of LGMD2B. Specifically, we will utilize human iPSC lines from three healthy and three LGMD2B donors to engineer LGMD2B myobundles that exhibit reproducible deficits in muscle contractile function, calcium homeostasis, and lipid handling, while showing drug responses consistent with studies in BLAJ mice and LGMD2B patients. Importantly, a defining feature of LGMD2B muscle is the ectopic fat formation suggested to occur due to adipogenic differentiation of muscle interstitial cells (MICs). We will thus develop a novel tissue-engineered model of intramuscular adipose tissue (IMAT) accumulation in LGMD2B muscle by co-culturing MICs isolated from LGMD2B human muscle biopsies and iPSC-derived muscle progenitor cells. In this novel co-culture system, we will identify pro-adipogenic factors secreted from LGMD2B muscle and study their ability to induce ectopic fat formation. Since immune cell infiltration and biased macrophage polarization are additional defining features of LGMD2B muscle, we will engineer co- and tri-cultured muscle-macrophage myobundles to further characterize roles of heterocellular interactions and inflammatory milieu in injury response and fat accumulation in LGMD2B. Finally, our preliminary studies suggest that the cholesterol metabolism in LGMD2B muscle is impaired and contributes to the disease, which we will further study pharmacologically, biochemically, and histologically in LGMD2B myobundles and BLAJ mice. Overall, we expect that the novel tissue-engineered model of human LGMD2B muscle developed in this project will enable new mechanistic and pharmacological studies, eventually leading to first clinical trials for LGMD2B.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Black Americans face nearly double the risk of Alzheimer’s disease (AD) and related dementias (ADRD) compared to Whites. Black American men may be at increased risk for late-life neurological disorders associated with exposure to repetitive head impacts (RHI) and traumatic brain injury (TBI) from contact sports participation and other risk factors. Exposure to RHI/TBI is linked with late-life cognitive impairment, neuropsychiatric disturbances, and structural brain changes. Black racial identity and exposure to RHI/TBI have been shown to have an additive effect on MRI metrics of atrophy and cerebrospinal fluid neurodegenerative disease proteins. These neurological disparities might be explained by contextual risk factors (e.g., educational access and quality, environmental and personal circumstances, neighborhood environment, and economic conditions), which may affect resistance and resilience to other neurological disorders arising from RHI/TBI. Contextual factors might indeed contribute to neurological outcomes in adult Black men irrespective of RHI/TBI. Yet, there has been persistent under-representation of Blacks in biomedical and health research, including among studies of the late effects of RHI from American football play. The overarching goal of this study is to examine the impact of RHI/TBI from American football and the contribution of early-life contextual risk factors to later-life cognitive function, neuropsychiatric symptoms, structural MRI features, and plasma biomarkers in Black male former American football athletes and non-RHI/TBI-exposed Black men. We will recruit 100 Black male former American football athletes (across all levels of play and cognitive continuum, 50+ years) and 100 age-matched Black males without RHI/TBI. Participants will enroll to complete harmonized cognitive and neuropsychiatric tests, MRI, and blood draw to assess neurodegenerative disease proteins. A battery of questionnaires (e.g., Adverse Childhood Experiences, Childhood-Experiences Survey, SHARELIFE survey) will be administered to assess Early-Life Contextual Risk Factors (ELCRF). In Aim 1, we will investigate the association between RHI/TBI and later-life cognitive and neuropsychiatric symptoms in Black male former American football athletes. Aim 2 will investigate the association between RHI/TBI and later-life biomarkers of Aß, p-tau, and neurodegeneration outcomes in Black male former American football athletes. Aim 3 will examine the contribution of ELCRF to later-life cognitive and neuropsychiatric symptoms and later-life biomarkers of Aß, p-tau, and neurodegeneration outcomes. Our hypotheses are that (1) RHI/TBI will be associated with worse cognitive and neuropsychiatric function and compromised structural gray/white matter in Black former football players; (2) impoverished ELCRF will increase risk and reduce resilience to the late-life effects of RHI/TBI; and (3) impoverished ELCRF will mediate the effects of RHI on late-life neurological outcomes. This R01 will discover the role of RHI/TBI and ELCRF in older Black men. We will address racial disparities present in data on late-life neurological outcomes from RHI/TBI to facilitate accurate disease detection and diagnosis at the individual level.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Cell fates are decided as an organism develops. In human development, pluripotent stem cells differentiate into the three layers of ectoderm, mesoderm, and endoderm. These classes of tissue further differentiate into specific cell types with specific functions including neurons, immune cells, and skin cells. These identities are stable; once a cell differentiates into its final state, it will not revert back to a stem cell state, nor will it transform into another cell type. A skin cell will not spontaneously become a neuron, even if the neuron is damaged. However, Takahashi and Yamanaka demonstrated that cells have the potential to revert back to a stem cell fate when they reprogrammed mouse fibroblasts into induced pluripotent stem cells (iPSCs) by forced overexpression of stem cell-specifying transcription factors. In 2010, Vierbuchen and colleagues demonstrated that fibroblasts could be reprogrammed directly to neurons using neuron-specific transcription factors, bypassing the need for an iPSC-intermediate. However, reprogramming efficiencies in each of these systems was low; very few cells are actually capable of changing their cellular identity. In 2019, Babos and Galloway greatly improve reprogramming efficiencies in direct motor neuron reprogramming, demonstrating improved reprogramming yields 100 times greater than the original process. They drew upon factors that enhanced another cell fate transition: cancer. Genes that promote a healthy cell’s transition to cancer also improved the ability of a cell to change its cell type. Thus, reprogramming can serve as a model of cancer initiation. By understanding the molecular mechanisms by which these oncogenes promote reprogramming, we can understand how oncogenes evade cellular barriers to cancer and establish tumors. In the F99-phase of the proposed research, I will investigate the role of the tumor suppressor protein p53 in oncogene-mediated reprogramming. p53 is the most frequently mutated gene in cancer. Rather than p53 expression being lost in cancer, it is most often mutated to create a protein unable to perform its designated functions and accumulates to abnormally high levels. As a synthetic biologist, I will design synthetic gene circuits that track and report p53 levels during reprogramming. I will isolate cells that accumulate p53 and investigate their ability to reprogram. In the K00-phase of the proposed research, I will extend my investigations of p53 to three-dimensional models of ovarian cancer. Ovarian cancer is often diagnosed at late stages, after the cancer has metastasized, leading to poor patient outcomes. 3D models of tumor initiation can shed light on the early stages of ovarian cancer and enable clinicians to catch the cancer early, when the disease is most easily treated. By inducing cancer initiation in 3D models of ovarian cancer and tracking cancer progression using p53-sensors, I will identify the drivers of tumor establishment and factors associated with early-stage disease.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Oxidative DNA damage is frequently generated by radiation, chemicals, and endogenous oxygen radicals, contributing to genomic instability during both aging and tumorigenesis. Oxidative damage at telomeres can lead to telomere loss or attrition, which triggers cellular senescence and limits the lifespan of dividing cells. While it is clear that oxygen radicals can inflict multiple types of DNA lesions, how these lesions are repaired at telomeres is still largely unknown. By inducibly and locally generating reactive oxygen species (ROS) at telomeres, we discovered a novel DNA repair pathway critical for the protection of cells against telomeric oxidative damage. This pathway is activated by ROS-induced R-loops, and is mediated by break-induced replication (BIR), a process that “jumpstarts” DNA synthesis at collapsed replication forks. In parallel with our studies on the oxidative damage response at telomeres, we also investigated how cancer cells maintain telomeres to bypass senescence. In particular, we have molecularly dissected the alternative lengthening of telomere (ALT) pathway, which is used by ~10-15% of human cancers to extend telomeres. Interestingly, we found that ALT is also an R-loop-triggered and BIR-mediated repair pathway. The unexpected similarities between the repair pathway dealing with telomeric oxidative damage and the ALT pathway lead us to hypothesize that these telomere repair pathways are mechanistically linked. Furthermore, cancer cells hijack the R-loop and BIR-mediated repair pathway to extend telomeres and bypass senescence. In Aim 1, we will systematically delineate the R-loop and BIR-mediated pathway that repairs telomeric oxidative damage, and investigate if this pathway contributes to ALT activation in cancer cells. In Aim 2, we will develop strategies to exploit the cellular dependency on the R-loop and BIR-mediated ALT pathway, which may allow us to selectively kill ALT+ cancer cells and aged cells harboring high telomeric oxidative damage. Our studies may establish a new link between cellular aging and tumorigenesis, and provide new opportunities to eliminate cancer cells by targeting a hijacked DNA repair pathway. These studies may have transformative impacts at the interface between aging and cancer research, opening a new avenue to future preclinical and clinical investigations.

NIH Research Projects · FY 2025 · 2023-08

Abstract Individuals with chronic MSK pain conditions who experience disability-related limitations in physical activity participation are at higher risk for poor physical and mental health outcomes. Chronic patellar tendinopathy is a highly prevalent chronic MSK condition that severely limits performance, physical activity, self-perceived function, and quality of life. Whole person health (WPH) is a comprehensive framework that considers interconnected domains (biological, behavioral, social, environmental) that foster health, and is designed to facilitate multicomponent therapeutic interventions. The applicant’s long-term career objective is to establish a funded research program that develops and evaluates a WPH model to decrease disability and improve quality of life for individuals with for chronic musculoskeletal pain conditions. Current standard of clinical care for patients with chronic patellar tendinopathy take a unifocal approach, emphasizing local tendon pain and capacity, does not incorporate other factors (psychological, social, environmental) that may contribute to the patient’s overall health. The overall objective of this study for this career development award is to develop a WHP framework that is responsive to the critical gaps in the literature and will inform future intervention approaches for patellar tendinopathy. The approach will expand the standard of care progressive loading exercise treatment (PLE) utilizing individualized biofeedback augmented movement retraining and behavioral graded exposure techniques to develop and refine a novel patellar tendon WPH intervention (WPH), and evaluate feasibility, acceptability, and responsiveness of key biopsychosocial outcomes (landing mechanics, psychological distress, tendon stiffness). The central hypothesis of this study is that individuals with chronic patellar tendinopathy who utilize novel WPH for patellar tendinopathy rehabilitation will demonstrate improvements in self-reported function, landing biomechanics, and tissue capacity. The hypothesis will be tested through three specific aims: 1) refine WPH intervention protocols through a small case series study using mixed-methods approach, 2) establish feasibility and acceptability benchmarks, and 3) determine safety and responsiveness of WPH compared to PLE on primary outcome of (a) patient self-reported function and psychological distress, and secondary outcomes of (b) landing biomechanics and (c) patellar tendon stiffness through a pilot randomized controlled study. The proposed work is innovative, in that: 1) it will be the first study to utilize a WPH framework compared to standard of care and evaluate a novel combination of outcomes for the treatment of chronic tendinopathy. The study is significant as it addresses an important gap in knowledge, moving beyond outdated traditional care models to create novel clinical care model designed to mitigate the debilitating effects of tendon pain on quality of life. Doing so takes a substantial step forward to enhance patient-oriented long-term outcomes for individuals with chronic musculoskeletal pain conditions.

NIH Research Projects · FY 2024 · 2023-08

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen. C. trachomatis infections can ascend to the female upper genital tract (UGT) leading to acute and chronic infections than cause pelvic inflammatory disease and fallopian tube scarring. These inflammatory pathologies have a large negative impact on women’s reproductive health. We know remarkably little as to the cellular and molecular processes underlying acute and chronic infections of the UGT, and how they promote permanent tissue damage even long after the pathogen has been cleared. Similarly, what role Chlamydia virulence factors play in this process is largely unknown. In this application we propose to apply emerging transcriptional profiling technologies to catalogue the cellular and gene expression events from initial infection, to resolution, to fibrotic damage post bacterial clearance at single cell resolution in the female UGT following a challenge with Chlamydia muridarum, a mouse adapted Chlamydia sp. In addition, to understand the mechanisms underlying the emergence of fibrosis and how long-lasting tissue damage that can lead to infertility, we will compare cellular responses in the UGT and their corresponding single-cell gene expression profiles upon challenges with Chlamydia mutants that cause altered pathologies. These longitudinal studies will define the types of cells that drive Chlamydia-dependent inflammatory damage and provide new insight as to how the spatial distribution of these cells correlate with pathogen clearance and pathology. Furthermore, the proposed studies will provide a unique resource for the characterization of inflammatory processes and fibrotic damage in an organ of significant importance for reproductive health.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Spending time outdoors increases children’s physical activity and promotes engagement with nature, which has been shown to have positive benefits on children’s mental, cognitive, and behavioral health. However, outdoor time is difficult to quantify, with most studies relying on self- or caregiver-reported outdoor time. One solution is to use accelerometers equipped with ambient light sensor technology. The light sensor delivers an estimate of light exposure (i.e., lux values) that can be used to objectively quantify children’s outdoor time. Despite the ubiquitous use of accelerometers, most researchers have not taken advantage of this tool. Of the studies that have used ambient light data, analytic approaches have varied. Specifically, studies have used different lux cut points to distinguish between indoor and outdoor time, and have not considered placement of the accelerometer (i.e., hip vs. wrist worn) or the potential error due to seasonality or weather conditions. Therefore, the objective of this project is to develop and disseminate a set of best practices for analyzing accelerometer-derived ambient light data in order to provide an objective assessment of outdoor time in children. The proposed project will analyze secondary data from two NHLBI funded intervention trials targeting physical activity promotion in the child care setting. Both trials included accelerometer data with the ambient light sensor enabled as well as data from the Environment and Policy Assessment and Observation (EPAO) tool, an observational measure that contains detailed time stamped data of the child care day including indoor and outdoor time. In Aim 1, accelerometer data will be harmonized to facilitate comparisons between hip- and wrist-worn devices and then combined with the corresponding EPAO data. In Aim 2, receiver operator characteristic curve analysis will be used to identify the optimal lux cut point to distinguish between indoor and outdoor time, comparing accelerometer lux values to the documented indoor and outdoor time from the EPAO. Then, we will compare how well the identified lux cut point performs in distinguishing outdoor time in wrist vs. hip worn accelerometers. Finally, we will compare how seasonality and different weather conditions (i.e., cloudy vs. clear days) affect lux readings. In Aim 3, we will examine the association between objectively measured outdoor time and children’s physical activity while at child care using the newly developed analytic guidelines. In order to promote consistency in the field, we will disseminate the best practices by developing open source code for other researchers that can be applied to existing datasets and repositories of accelerometer data. Overall, this project will address key measurement gaps in assessing young children’s physical activity and outdoor time. Improving measurement of outdoor time will further add to research into the health impact of spending time outdoors, aid in the identification of disparities regarding access to the outdoors, and provide support for policies and regulations promoting youth outdoor time.

NIH Research Projects · FY 2026 · 2023-08

The design of drug formulations is an essential part of pharmaceutical development to enable the safe and effective delivery of medications. Unfortunately, formulation optimization is currently done using a trial-and-error approach or by adhering to already established formulation strategies following a one-size-fits-all mindset. This has resulted in formulations that are simple and only ensure appropriate physical properties such as shelf life and liberation. Complex, targeted formulations can increase the safety and efficacy of medications, but such systems are expensive to design, manufacture, and administer. Here, we describe our goals to expand and augment our efforts in developing innovative machine learning methods and integrate them with experimental workflows for the design of novel, targeted drug formulations. We will specifically focus on the machine learning-guided design of (1) functional excipients that prevent microbiome metabolism, (2) targeted self-assembling nanoparticles, and (3) tissue-selective prodrugs. Our machine learning models will enable us to circumvent billions of otherwise necessary trial-and-error experiments by predicting the most promising candidates for experimental validation. This allows us to systematically explore novel drug delivery systems to identify better solutions. Our in vitro and in vivo experiments will validate our predictions and provide pre-clinical data for innovative drug delivery solutions positioned for further translation. We expect that our platform will enable the rapid and effective design of advanced drug delivery solutions to create safer and more efficacious therapeutics.

NIH Research Projects · FY 2025 · 2023-08

Abstract Aggressive malignant progression and spreading has limited a clear understanding of the pathophysiology of SCLC. The overarching goal of this “bedside to bench” project is to elucidate the role of inter-metastatic divergence in therapeutic resistance in small cell lung cancer (SCLC), with a focus on how antigenic heterogeneity and dominant immunosuppressive butyrophilins thwart protective adaptive immune responses in this disease, to develop novel interventions to overcome these hurdles. We will leverage a Rapid Tissue Donation (RTD) program established at Moofitt Cancer Center, which provides timely access to the entire repertoire of metastatic lesions in terminal patients, who generously donate their tissues for this research. Using this resource, plus CDX models routinely generated for each SCLC patient, we will define barriers that impair the effectiveness of immunotherapies. During the 5-year tenure of this grant we will generate a rationale for novel immunotherapeutic trials to overcome overcome these elusive hurdles, with a focus on antigenic heterogeneity and the role of immunosuppressive butyrophilins. Based on our expertise on tumor immunology (Dr. Conejo-Garcia) and clinical immunotherapy (Drs. Perez), as well as access to a Rapid Tissue Donation program, we postulate that the effectiveness of immunotherapies against small cell lung cancer is thwarted by heterogeneous immunogenicity across different tumor masses, along with high expression of immunosuppressive CD277+ butyrophilins. Based on our preliminary results, our central hypothesis is that pleural effusions and trogocytic tumor-infiltrating T cells could provide a source of effector lymphocytes for cell therapies that would target multiple tumor masses, in combination with targeting immunosuppressive BTN3A butyrophilins and/or tumor-derived antibodies. We propose the following Specific Aims: Aim 1. Define the role of inter-metastatic heterogeneity in therapeutic resistance in human SCLC. Aim 2. Design novel T cell immunotherapies that overcome metastatic heterogeneity in SCLC. Aim 3. Determine the potential of antibodies produced in SCLC to overcome inter-metastatic heterogeneity. These studies will, first, define the role and heterogeneity of neoantigens and immune cells in SCLC; a poorly characterized disease, due in part to its aggressiveness. Most importantly, we will provide a mechanistic rationale for more effective immunotherapies that target the diversity and dominant immunosuppressive drivers of this human disease, which will target in novel clinical trials at Moffitt.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Permanent smell loss is a common morbidity associated with esthesioneuroblastoma (ENB), a tumor thought to arise from the olfactory epithelium in the nose. This is due to necessity for bulk surgical resection, regardless of tumor grade. As such, the development of novel medical treatment strategies is necessary to preserve olfaction in this patient population. The olfactory epithelium is a neurogenic niche that produces neurons and supporting epithelial cells throughout life, and various undifferentiated stages of normal epithelial proliferation and development have been observed in ENB. The epigenetic regulator polycomb repressive complex 2 (PRC2) has been implicated in driving proliferative cell states in stem cell niches throughout the body. We have shown that PRC2 is crucial for basal cell proliferation in the olfactory epithelium, and we have identified that it is expressed in proliferating cells in ENB. Because ENB is a rare tumor, mechanistic biological studies are sparse, and no cell lines or mouse models exist. Furthermore, the majority of ENBs do not share common driver mutations, emphasizing the importance of epigenetic regulation. Here I propose to utilize newly-developed multi-omic studies to investigate how PRC2 expression in human ENB specifies cellular states and contributes to tumor growth. I will perform single cell chromatin and transcriptomic assays on multiple tumors to identify areas of chromosomal accessibility. Furthermore, I will use pharmacologic assays in human ENB samples to assess the downstream effects on transcription. Finally, spatial transcriptomics will be used to define ENB cellular and molecular composition, including PRC-associated expression, and how this compares to normal olfactory epithelium, to elucidate cellular signaling dynamics among tumor and immune cells within the ENB microenvironment. Completion of the proposed studies will define epigenetic drivers co-opted by ENB with the goal of identifying new druggable targets, allowing for improved survival and preservation of smell. Altogether, the research and training plan outlined in this proposal combined with expert mentorship from Dr. Bradley Goldstein will prepare me with the rigorous training necessary for a successful career as a surgeon-scientist in rhinology and anterior skull base surgery.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Society faces increasing burden from respiratory immune challenge including respiratory viral infection. Respiratory viral infection, including with influenza A virus, can cause changes in brain function; understanding the link between lung and brain health is critical to anticipating the shifting health needs of our society. The lung and brain engage in bidirectional communication through several signaling mechanisms, allowing these organs to influence each other. It is unknown how lung-brain communication impacts the progression of respiratory viral infection, or how different communication mechanisms are prioritized as inflammatory response progresses. Using a mouse model of infection with influenza A strain PR8, this proposal explores immune signaling in the bidirectional lung-brain axis. The overarching goal of this proposal is to test the hypothesis that the lung and brain exert bidirectional influence during respiratory infection, altering each other’s immune states. My preliminary data shows that during PR8 infection, changes in central nervous system (CNS) occur prior to inflammatory gene upregulation in lung tissue. The signaling mechanisms influencing these rapid CNS changes in neuronal activity, as well as the role of this CNS response on infection progression, remain unknown. Pulmonary neuroendocrine cells (PNECs) are sensory cells which mount an immune response during respiratory inflammatory challenge. These cells are also the only cells in the lung epithelium directly innervated by the vagus nerve. Despite the known immune function of both PNECs and the vagus nerve, the role of PNECs signaling to the CNS during a respiratory infection remains unstudied. In Aim 1, I will explore the role of PNEC signaling in infection by characterizing molecular and vagal signaling from these cells during infection. I will then ablate these cells prior to PR8 infection using a cre-dependent AAV delivery mechanism in calcacre mice to determine whether their signaling contributes to the peripheral or CNS response to infection. Regardless of signaling mechanisms initiating the rapid CNS response to PR8 infection, the ultimate consequence of this response is unknown. Neuronal ensembles can encode immune memory, impacting the immune states of peripheral organs. In Aim 2, I will determine how neuronal activity influences the microglial and peripheral immune response throughout PR8 infection onset. Using high-throughput imaging and a machine learning analysis pipeline, I will map the neuronal and microglial response to PR8 infection in TRAP2::TdTom mice. I will then explore the role of neuronal activity by capturing and later re-activating the neuronal population active during PR8 infection using TRAP2::hM3Dq mice. I will characterize microglial response in CNS tissue and peripheral immune response to determine if activity of the PR8-responsive neuronal population is sufficient to drive immune outcomes. Altogether, this proposal will explore the connection between lung and brain health, focusing on these organs’ influence on each other’s immune states in the onset of respiratory infection.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The visual cortex can process identical stimuli differently depending on context; behavioral states such as locomotion or arousal can alter the magnitude and the specificity of visual responses. The neuromodulator acetylcholine (ACh) is implicated in state-dependent processing and acts on diverse inhibitory interneurons in cortical circuits, but it remains uncertain how interneuron classes contribute to state-dependence. Particular controversy surrounds the role of the somatostatin-positive (SOM) cells, which shape circuit output by directly inhibiting pyramidal cells. One model suggests that ACh action on upstream interneurons triggers suppression of SOM cells via release of the inhibitory neurotransmitter γ-Aminobutyric acid (GABA). This disinhibits pyramidal cells to increase gain in visual circuits during locomotion and potentially other states. However, contradictory findings reveal that SOM cells, which can be directly facilitated by ACh through muscarinic receptors, are actually more active during locomotion, indicating the disinhibitory model is not sufficient to explain context dependence. This proposal tests the hypothesis that muscarinic and GABAergic action on SOM cells have complementary effects on modulating visual cortex circuits and shaping in visual discrimination. I hypothesize that muscarinic action on SOM cells contributes to tuning of the pyramidal population, while GABAergic action on SOM cells contributes to pyramidal cell gain. I will dissect this utilizing unprecedented intersectional control of specific receptors on specific cell types via the Drugs Acutely Restricted by Tethering (DART) methodology coupled with 2-photon calcium imaging of mouse primary visual cortex. In Aim 1, I will selectively antagonize muscarinic receptors on SOM cells and record activity of SOM cells and nearby pyramidal cells as mice passively view visual stimuli. I will assess visual responses and how responses are altered by locomotion and arousal, to reveal the direct impact of ACh on SOM cells in basal visual processing and modulation by behavioral state. In Aim 2, I will selectively block GABA receptors on SOM cells, again recording SOM and pyramidal cell activity during passive viewing. This will allow me to clarify how inhibition onto SOM cells contributes to basal visual process and circuit modulation during locomotion and arousal. If, as hypothesized, muscarinic and GABAergic control impact tuning and gain of pyramidal cells, this could meaningfully impact visual discrimination. To assess how these two pathways act on animals' ability to perceive and use visual information, in Aim 3 I will selectively antagonize muscarinic or GABAergic receptors on SOM cells, and record activity of SOM and pyramidal cells, while mice perform an orientation change detection task. Together these data will resolve longstanding questions around how neuromodulators imbue visual circuits with context specificity.