Objectives/Goals: This evaluation seeks to demonstrate the feasibility and impact of a new funding program for clinical and translational research launched by U-M Michigan Medicine. ’Research Scouts’ with established research agendas at U-M were identified and trained to invite and fund research proposals from U-M colleagues they had not collaborated with. Methods/Study Population: In 2023, Research Scouts identified and orientated to the program and goals. Each Scouts was given a year to invest $150,000 in innovative research projects. A web-based submission portal was used to anonymize the proposals to remove the potential for bias. All projects were granted a two-year funding period. The sequential mixed methods used for this evaluation includes surveys of ’Research Scouts’ and awardees, followed by the collection on administrative data regarding the awardees scientific production, including both research publications and grants. The survey results and administrative data were analyzed and informed the iterative refinement of an interview protocol designed to investigate the impact of the funding on awardees’ development of novel and collaborative research agendas. Results/Anticipated Results: Surveys of the 2023 Scouts (N=24) and Awardees (N=51) had a 59% response rate. Awardees agreed they were able to pursue new lines of investigation (Mean 4.62, on a 5-point Likert scale); test a new idea that otherwise would go unexplored (Mean 4.62); had more latitude for intellectual creativity and exploration than traditional funding mechanisms permit (Mean 4.46); had new scientific conversations and new connections (Mean 4.54). The results also indicate that Research Scouts agreed with these benefits. Analysis of Awardee’s administrative data yielded evidence that they were actively producing new research; for example, of 146 papers, at least 30 were relevant to their award. Ongoing interviews will yield evidence indicating how the award accelerated their clinical and translational research agendas. Discussion/Significance of Impact: Given the long gestation time for new research ideas to yield new publications or extramural funding, the full impact of the program won’t be known for years. However, preliminary evidence indicates that the Research Scouts program successfully provides a mechanism for pursuing novel ideas with minimal administrative costs and burden.

This case report presents mid- to long-term outcomes of thoracic endovascular aortic repair for high-risk type B aortic dissection in a 15-year-old patient with myosin heavy chain protein 11 genetic mutations. A CT scan showed the primary entry tear located on the lesser curvature near the left subclavian artery, and the dissection originated from the descending aorta to the abdominal aorta, accompanied by 80% narrowing of the true lumen. Following successful endovascular therapy using stent-grafts, the patient recovered well. A four-year follow-up CT scan showed significant thoracic aorta remodelling.

Objectives/Goals: Bardet-Biedl Syndrome (BBS) is a rare, autosomal recessive syndromic ciliopathy. Obesity in BBS has been attributed to melanocortin 4 receptor (MC4R) pathway in the hypothalamus, which plays a major role in appetite control and energy balance. We aim to understand how BBS proteins play a role in hyperphagia and obesity using zebrafish models. Methods/Study Population: We used a published genetically stable bbs4 mutant zebrafish line to investigate metabolic regulation. Mutants harbor a 5 base pair deletion in bbs4 resulting in loss of gene function. We used behavioral assays to quantify feeding activity, lipid accumulation, and BMI. We monitored expression of appetite regulating and glucose homeostasis genes under fasting and feeding conditions using qRT-PCR in biological triplicate pools of larvae with three technical replicates per experimental condition. Comparative analyses between bbs4 mutants and wildtype (WT) controls were conducted to identify transcriptional and physiological alterations; statistical differences were detected with Dunnett’s multiple comparison test, and p<0.05 was considered significant. Results/Anticipated Results: For the behavioral assays, bbs4 homozygous mutants showed no significant differences compared to WT. Preliminary expression data show a significant decrease in mc4r expression in bbs4 mutants versus WT with concomitant decrease in leptin (lepa/lepb) expression versus WT at hunger initiation. Insulin (insa) showed a 6-fold higher activation and elevated glucagon and gluconeogenesis gene activation for mutants versus WT indicating that bbs4 homozygotes have hyperinsulinism even while fasting. However, in feeding conditions, satiety genes showed no activation in mutants, indicating impaired leptin response. Future studies will elucidate further the interplay between ciliary defects in proximal cellular contexts (i.e., hypothalamus, liver, and pancreas) and whole organism impact of impaired BBS protein. Discussion/Significance of Impact: Our findings indicate that bbs4 mutant zebrafish have preserved ciliary control over appetite and glucose homeostasis, as well as defective leptin-MC4R signaling and fasting-induced hyperinsulinism. Our preclinical model will facilitate the development of treatments for obesity associated with BBS.

Objectives/Goals: This study aims to uncover immuno-endotypes in sepsis-associated pediatric acute respiratory distress syndrome (SA-PARDS) using single-cell RNA sequencing (scRNA seq) to analyze the immune cell populations of the respiratory tract of intubated pediatric subjects with SA-PARDS. Methods/Study Population: The inclusion criteria are an age of less than 18 years, admission to the PICU, a diagnosis of SA-PARDS, and intubation. Both sepsis and PARDS will be defined using the most recent consensus definitions. Exclusion criteria include an order of limited resuscitation and clinician discretion. A tracheal aspirate and blood sample will be obtained on days 1, 3, and 7. Both samples will be processed for single-cell RNA sequencing via the HIVE platform according to the manufacturer’s protocol. cDNA libraries will be generated and undergo 150 bp sequencing, quality control, and alignment. The Seurat package in R will be used for cell-type annotation and analysis of differential gene expression. Clinical variables, labs, and outcomes will be recorded in REDCap. Results/Anticipated Results: We evaluated five samples from two subjects. After quality control, 31,330 cells remained for analysis. UMAP projections were created, and we annotated cell types. We found that most of the cells were immune cells with very few epithelial cells. There were two groups of neutrophils, which we termed classic and migratory. Classical neutrophils expressed classic neutrophil markers, and migratory neutrophils expressed the classical markers plus increased adhesion markers. We identified a third group of cells that were neutrophil-like, and these were isolated for further analysis. These cells exhibited characteristics of exhausted neutrophils, including downregulation of CXCR1 and CXCR2, and upregulation of PI3 and SLIPI. In each subject, the proportion of exhausted neutrophils increased over time. Discussion/Significance of Impact: We found that in our subjects with SA-PARDS, there were a subset of neutrophils characterized by down-regulation of classical neutrophil markers and up-regulation of neutrophil stress markers, suggested an exhausted phenotype.

Objectives/Goals: The primary objective of this study is to test whether NAD+ modulation lowers mitochondrial oxidative stress to restore endothelial nitric-oxide signaling and limit microvascular remodeling in diabetes in murine and 3D tissue-engineered models. The goal is to define NAD-responsive pathways and candidate blood biomarkers of microvascular injury. Methods/Study Population: Use db/db diabetic mice to measure effects of NAD+ modulation on capillary density, perfusion, endothelial nitric oxide (NO) signaling, and mitochondrial reactive oxygen species (ROS) by histology, immunohistochemistry (IHC), qPCR/immunoblot, and functional assays. Build a 3D microfluidic model of diabetic retinal capillary disease from human endothelial/pericyte co-cultures with a retina-on-a-chip model; induce diabetic stress (hyperglycemia + inflammatory cytokines) and apply an NAD+ modulation to map dose–responses for barrier integrity, ROS, NO, and morphology. Analyze publicly available transcriptomic datasets from people with and without diabetic microvascular disease to identify oxidative-stress signatures and candidate circulating biomarkers. Results/Anticipated Results: Expect increasing NAD+ availability to reduce mitochondrial ROS and inflammatory signaling, increase SIRT3/SOD2 activity, improve NO bioavailability, and prevent capillary rarefaction and leak in db/db mice. In the microfluidic model, anticipate a rescue of barrier function, lumen stability, and branching with NAD+, defining an effective dose window. Cross-platform analyses should converge on NAD+-responsive pathways (redox, mitochondrial metabolism, endothelial junctions) and nominate blood biomarkers that correlate with microvascular status. Together, results will provide actionable preclinical evidence for NAD+ modulation as a therapeutic strategy. Discussion/Significance of Impact: By targeting a shared driver, mitochondrial oxidative stress, this work could deliver a mechanism-based therapy and practical biomarkers for diabetic microvascular disease, accelerating translation from bench models to patient-focused trials and improving cardiovascular outcomes.

Objectives/Goals: Nutrition status is an overlooked, modifiable driver of total joint arthroplasty (TJA) recovery that can reduce complication rates. Therefore, we will profile preoperative nutrition status in TJA patients using integrated clinical measures, identify metrics that predict postoperative complications, and validate their clinical utility. Methods/Study Population: We will enroll 800 elective hip (THA), knee (TKA), and shoulder (TSA) arthroplasty patients. Assessments will include blood biomarkers (albumin, lymphocytes, vitamin D/A, zinc, HbA1c, lipids), bioelectrical impedance (BIA) for body composition metrics (phase angle, body fat%, skeletal muscle index: SMI, visceral adipose area: VFA), and handgrip strength (muscle quality index: MQI). Biomarkers will inform on deficiencies, immunity, and metabolic health, reflecting surgical readiness. We will compute Onodera’s Prognostic Nutritional Index (OPNI), SMI, and MQI and will evaluate infections, hospitalizations, revisions, and prolonged stay via records and encounters at 3 months and 1 year postop. Analyses will link nutrition metrics to complications to identify the strongest, actionable predictors. Results/Anticipated Results: Among the first 100 arthroplasty participants assessed (THA: 43%, TKA: 49%, TSA: 8%), 56% were male (age 66.3±10.4 yrs). Mean lymphocytes was 1.9±0.6×109/L (normal: 1.0-4.8×109/L). Zinc averaged 59.9±11.6 µg/dL (normal: 60-120 µg/dL) with 47% deficient (50.9±6.6 µg/dL) versus 53% in normal range (66.8±5.0 µg/dL). Average vitamin D was 36.3 ng/mL (sufficient: >30 ng/mL), with 42% of patients low (20.8±5.6 ng/mL) versus 48% sufficient (47.8±15.1 ng/mL). Body composition measures: BMI 30.2±5.9 kg/m2 (2% underweight, 18% normal, 27% overweight, 27% obese 1, 14% obese 2, 6% obese 3); body fat 35.1±9.4%, VFA 155.8±55.3 cm2 (healthy: <100 cm2), SMI 7.9±1.5 kg/m2, phase angle 5.0±0.8°; and MQI 1.4±0.4. Comorbidities: T2DM 18%, HTN 67%, hypercholesterolemia 28%, CVD 37%; and 8% used GLP-1 medications. Discussion/Significance of Impact: These early data show high zinc and vitamin D deficiency (47% and 42%, respectively), obesity (47% class I–III), and substantial comorbidities, underscoring the need for standardized nutrition screening in orthopedics. These findings justify targeted preop interventions and support an NIH R01 focused on reducing complications.

Objectives/Goals: Common mamographic findings, breast arterial calcifications (BAC), are a sex-specific biomarker for cardiovascular disease in women. While less common, BAC is frequently encountered on mammograms in males. This study aims to investigate BAC and its association with cardiovascular risk factors in men. Methods/Study Population: A retrospective single-institution study of male patients with mammography studies was performed between September 1, 2018, and September 3, 2025. Diagnostic mammographic images of male patients from a high-volume breast imaging facility were evaluated for the presence of BAC. BAC severity will be assigned a score based on the Canadian Society of Breast Imaging’s grading system. Unique ICD-10 diagnosis codes from the electronic medical record that have been established as independent risk factors for cardiovascular disease will also be reviewed. Male patients who have a prior history of myocardial infarction, coronary revascularization, stroke, heart failure, peripheral vascular disease, breast cancer, dementia, or chronic dialysis/renal transplant were excluded. Results/Anticipated Results: The study is in process of pulling male mammograms from our institution. Current volume of male mammograms at the institution is about 160 male mammograms performed per month. Therefore, we anticipate having around 13,600 male mammograms to analyze the BAC presence. Anticipated results include that patients with independent cardiovascular risk factors such as increased systolic blood pressure, increased LDL levels, and current smokers will have a higher prevalence of BAC presence. Additionally, those with a presence of BAC will be older compared to those that have an absence of BAC on mammography. If BAC is shown to be a significant risk factor for the development of cardiovascular disease, perhaps a mammogram might be recommended in men purely for cardiovascular risk assessment. Discussion/Significance of Impact: A significant association between the presence of BAC and CVD risk factors may provide a supplementary assessment of CVD risk among men, offering a novel tool for risk stratification. Implementing a standardized BAC reporting model may allow for earlier identification of asymptomatic patients at high risk for adverse cardiac events.

Objectives/Goals: Incorporating community voice ensures that research meets real world needs and enhances community trust and engagement. The objective of this program is to train community members on research to become community scientists and strengthen their role as partners in research, advancing the translation of discoveries into community practice. Methods/Study Population: Based on the University of Florida Citizen Scientist model, Mayo Clinic launched a Community Scientist (CS) program in 2021 as a one-day event. It expanded to a 4-week program in 2022, an 8-week program with a 6-week experiential assignment in 2023−2024 and added asynchronous training in 2025. The CS curriculum integrates didactic and experiential assignments with pre/post evaluations implemented in 2023 and expanded in 2024 to include post evaluations for the 6-week experiential assignment. Participants represented three Mayo Clinic regions (Arizona, Florida, Midwest), reflecting different geographies and backgrounds. Evaluations assessed participants’ knowledge, confidence, and engagement in multiple topics such as genomics, ethics, biobanking, and researcher–community collaboration. Results/Anticipated Results: 30 CS participants completed training once evaluations were implemented, and the CS participants’ familiarity with the role of a community scientist increased from 20% to 90%. Understanding of genomics and biobanking increased from <20% to 80% and knowledge of research ethics and IRB processes increased from 30% to 75%. Participants agreed that the experiential assignment deepened their understanding (82%), confidence (80%), and ability (70%) to partner with researchers. High satisfaction scores (89%) from the CS participants supported the program’s value and 100% noted readiness to engage as a CS in research. Overall results demonstrate the program’s scalability and effectiveness while also noting the need for consistent evaluation for program improvement. Discussion/Significance of Impact: The CS program empowers communities with knowledge and confidence to engage with research teams, advocate locally and partner with investigators. Strong gains and consistent growth across domains highlight the CS program’s potential as a scalable model for meaningful community engagement in research.

Objectives/Goals: Given the close proximity of two academic institutions with CTSAs, we piloted distinct training topics – psychological safety and evaluation of opportunities – with K and T scholars at both academic hubs. Each topic was originally developed and delivered by one of the partnering hubs, allowing for cross-institutional sharing of expertise. Methods/Study Population: The team science educators at both institutions had a series of conference calls to share information about the types of professional development activities offered to early-career investigators at respective institutions. Each group then agreed to offer a new workshop at the partner institution. Team 1 presented “Should I Join This Research Team? Strategically Evaluating a New Teaming Opportunity,” while Team 2 presented “Promoting a Positive Culture in Teams.” Our instructional goal was to deliver skills-based training that addressed opportunities and challenges unique to research teams. Each session incorporated interactive methods, including scenario analysis and discussion, think-pair-share activities, and short video clips. Results/Anticipated Results: Likert-scale items and open responses will be completed by workshop participants before and after participating in the workshops. Attendees will evaluate their confidence in their perceived abilities related to workshop objectives and their perceptions of the utility and application of the content. In addition to the trainee evaluations, the two institutional teams completed a joint qualitative interview to provide feedback, discuss andragogical strategies to improve educational delivery and impact, and assess how to leverage the expertise of each team to provide future team science programming across the two institutions. Given that the workshops are scheduled to be completed by November 2025, the results will be collated and shared at the Translational Science 2026 conference. Discussion/Significance of Impact: We believe partnerships can work in a variety of settings and with multiple topics to enrich the experiences of both early-career investigators and team science educators at partnering hubs. The collaboration also promotes sharing of expertise, content, and resources across institutions which expands educational content across CTSAs.

Objectives/Goals: Scientific writing is vital for TL1 trainees, yet evaluating writing programs effectiveness is challenging. Current evaluations gauge writing confidence, not skill competency. Our study aimed to develop a quantitative writing evaluation tool to assess technique and improvement longitudinally across the TL1 program. Methods/Study Population: The writing evaluation tool uses 19 typical grammar concepts embedded within three paragraphs. TL1 trainees received a list of these concepts without definitions and were instructed not to research them. They were asked to identify each concept, its location, and explain the reasoning behind of their choice (e.g., Qualifier: “very” and “significant” are redundant, thus “very” can be removed). Points were awarded when concepts were identified (0.5 point) and reasoned appropriately (0.5 point). The participants included 12 predoctoral and 3 postdoctoral trainees. Evaluations were administered at the start of Fall 2025 and again in Spring 2026. A paired t-test was used to assess longitudinal growth, with significance determined at p ≥ 0.05. Results/Anticipated Results: We separated our participants into two groups by experience level: 1Y) 8 trainees (1-year predoctoral) and 2Y) 4 (2-year predoctoral) and 3 (1-year postdoctoral) trainees. After the Fall 2025 evaluation, the highest score was 9.5, with the lowest score being 0.5. On average, group 1Y scored 3.8 and group 2Y scored 3.1, indicating that although the trainees were separated by experience level, their writing abilities did not differ (p = 0.57). We anticipate that the Spring 2026 evaluation will show positive longitudinal growth for both groups as they learn to identify grammar concepts. Discussion/Significance of Impact: Developing an effective quantitative writing evaluation tool allows facilitators to refine writing programs for trainees. This study is the first step in testing the tool’s impact. Results will guide improvements in the evaluation tool and support ongoing evaluations of writing program effectiveness within TL1 training.

Objectives/Goals: Methicillin-resistant Staphylococcus aureus is a nefarious bacterial pathogen and is classified as a serious threat. MRSA is resistant to most B-lactam antibiotics (penicillins, cephalosporins, etc.). The goal of this study is to identify an antibiotic adjuvant capable of resurrecting B-lactam antibiotics for treatment of MRSA infections. Methods/Study Population: A fluorescence-reporter assay was used to screen a compound library. Minimum-inhibitory concentrations were assessed for the compounds against various MSSA and MRSA strains. A common resistance mechanism to B-lactams by MRSA is by the function of the bla operon. One gene in this operon encodes for a B-lactam sensor/signal transducer protein BlaR, the primary target of this study. Inhibition of BlaR by compound 1 (best potentiator of oxacillin) was studied by nano-differential scanning fluorimetry (nanoDSF), surface plasmon resonance (SPR), scanning electron microscopy (SEM), and in vivo assays. Results/Anticipated Results: We identified 80 compound hits from a 1,974-compound NCI library. Twenty-four compounds showed potentiating ability (2- to 4,096-fold decrease in MIC for oxacillin). Seven compounds exhibited melting temperature shifts by nanoDSF of BlaR, indicating binding. SPR determined compound 1 has a binding affinity of 31 mM to BlaR-SD. SEM images showed disruption in the S. aureuscell wall on exposure to compound 1and oxacillin. S. aureusN315 showed 3-log reduction in bacterial count treated with a mixture of compound 1 and oxacillin. Discussion/Significance of Impact: Compound 1 targets BlaR-SD, which restores S. aureussusceptibility to treatment by oxacillin. There are currently few antibiotics available in the clinic capable of treating MRSA infections. The combination hold promise of a treatment option for MRSA.

Objectives/Goals: Artificial Intelligence(AI)and automation are rapidly transforming healthcare, yet their integration into clinical workflows often falls short due to technical, ethical, and organizational challenges. A standardized, quality-driven approach can aid the AI implementation process and ensure value delivery. Methods/Study Population: Trust emerges as the central hurdle, encompassing both patient and provider confidence in AI systems. Patients raise concerns over safety, transparency, and the physician-patient relationship, while providers express apprehension towards algorithmic opacity, data quality, and legal ambiguity. To address these concerns, the Understand, Transform, Sustain (UTS) framework offers a behavior-based, systems-level approach to AI deployment. Developed by Mayo Clinic’s Quality Academy, UTS integrates process improvement (PI) principles across three phases, emphasizing stakeholder engagement, transparency, and patient safety throughout the AI lifecycle. Results/Anticipated Results: The Understand phase identifies inefficiencies by mapping workflows, collecting data, and recognizing areas for improvement, ensuring appropriate priorities are addressed. In the Transform phase, interventions are designed, implemented, and tested through improvement cycles and feedback loops. Data to build algorithms are carefully evaluated to avoid biases, and AI output is assessed for opacity risk to maintain transparency and explain ability. The Sustain phase monitors outcomes and standardizes practices for long-term value. Data audits and automated extraction tools are applied for fidelity and harmonization, promoting scalability and collaboration among organizations. Discussion/Significance of Impact: While keeping human intelligence central to AI integration, UTS ensures sustained efficiency and trust – critical for successful healthcare transformation. This framework positions itself as a catalyst for responsible innovation, aligning technological advancement with clinical priorities and patient protection.

Objectives/Goals: Prenatal exposure to phthalates, parabens, and phenols (EDCs) may be risk factors for adverse birth outcomes, but the biological mechanisms driving these relationships are not well understood. This study aims to explore the associations between prenatal EDC exposure – individually and as a mixture – and the neonatal metabolome. Methods/Study Population: This study included 180 pregnant people from a Columbia Center for Children’s Environmental Health cohort in New York City (1998-2002). Eleven phthalates, 1 phthalate replacement, 5 phenols, and 3 parabens were quantified in umbilical cord plasma samples using gas chromatography-tandem mass spectrometry. Metabolomic features in cord plasma were characterized via liquid chromatography coupled with high-resolution mass spectrometry. Metabolome-wide association studies using linear regression and quantile g-computation were used to identify features associated with exposure to the individual and mixture of EDCs, respectively. Correlated metabolites were grouped into modules using weighted gene co-expression network analysis (WGCNA) to identify associations with EDC exposure at a systemic resolution. Results/Anticipated Results: We observed metabolome-wide associations between 18 individual EDCs and 64 unique features in the umbilical cord plasma (p-value <0.05). For example, 9 different phthalates were significantly negatively associated with methionine, creatinine, and 2-methylcitrate. The overall EDC mixture was negatively associated with 2-methylcitrate, arginine, and creatinine (p-value <0.05). In the WGCNA analyses, 13 individual EDCs and the overall EDC mixture were significantly associated with at least 1 of the modules. Pathway enrichment analyses revealed that these metabolites were related to energy, amino acid, and lipid metabolism. Discussion/Significance of Impact: Prenatal EDC exposure, individually and as a mixture, is associated with levels of metabolites measured in umbilical cord plasma at delivery. The identified EDC-responsive metabolites and associated pathways offer insight into the biological processes that could underpin the relationship between prenatal EDC exposure and adverse birth outcomes.

The use of restraint in hospital settings is divisive, and internationally there are calls for its elimination. However, this is at odds with the experience of many hospital staff, who consider restraint, at times, a “necessary evil”. In this paper, we explore the definition of restraint and potential ethical justifications for its use. We argue that the current ethical literature employs two definitions of restraint — outcome-oriented and intent-oriented — neither of which successfully captures all ethically relevant features of the practice. We propose a new conceptualization of restraint which centers on the number of individuals impacted by an act of patient restraint — a continuum between therapeutic restraint and public-safety restraint. Understood in this way, neither the principlist nor human rights frameworks that dominate the current literature are appropriate for assessing the ethical legitimacy of restraint. We suggest that, given the similarities between restraint and public health interventions, the use of public health ethics principles to consider the ethical justifiability of restraint in hospitals is a potentially productive way forward in this controversial area.

Objectives/Goals: The specialized riboflavin (vitamin B2) biosynthetic enzymes are targets for novel antibiotics. This project provides direct evidence for the chemical mechanism of Mycobacterium tuberculosis lumazine synthase, the penultimate enzyme of the riboflavin production and target for therapeutic intervention. Methods/Study Population: Lumazine synthase catalyzes the condensation of 3,4-dihydroxy-2-butanone-4-phosphate and 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione forming 6,7-dimethyl-8-ribityllumazine. To determine the chemical mechanism of Mycobacterium tuberculosis lumazine synthase, we performed transient state kinetics to monitor the formation and disappearance of intermediates and products over time. These traces indicated time points to flash-freeze the reaction to isolate intermediates that are analyzed with 13C-NMR to determine the chemical structure. Time resolved X-ray crystallography is used as a complementary method to determine the structures of the reaction intermediates in the context of the active site. Results/Anticipated Results: The NMR data provide direct evidence of the chemical mechanism of lumazine synthase. Complementary crystal structures of lumazine synthase with intermediates bound to the active site will corroborate the chemical mechanism determined by NMR and model key residues in the active site that facilitate catalysis. These data will inform steps of the chemical mechanism that can be targeted for therapeutic intervention. Discussion/Significance of Impact: This work informs drug design to address the growing threat of antibiotic resistance. The riboflavin biosynthetic enzymes do not have human homologs, so drugs designed to specifically target these enzymes will minimize off target effects.

Objectives/Goals: KSHV is well documented to infect and establish latency in endothelial cells; however, infected cell types in the Kaposi Sarcoma (KS) tumor microenvironment are not well defined. Here, we present the first application of the Lunaphore COMETTM multiplexed immunofluorescence platform for spatial proteomic analyses of KS lesions. Methods/Study Population: Custom antibody panels were validated and optimized for characterization of the TME components, as compared to marginal structures. The Ab panels supported the detection, quantification, and comparison of >15 cell types in regions of interest in the KS tissues in a single automated run. For data analysis, we developed a rule-based decision tree for accurate cell classification and used the resulting cell populations to perform a nearest-neighbor analysis. We assessed spatial proximity among cell types located within, adjacent to, or distal from the areas with a large number of cells expressing KSHV LANA. Results/Anticipated Results: The highest KSHV load was detected in CD34+ cells with or without CD31, consistent with an endothelial progenitor phenotype (EPCs). We also defined 12 cell types with KSHV-LANA detected in <1% of each cell type. Among them, tumor-infiltrating CTLs were proximal to or overlapping with infected EPCs, suggesting they have cytolytic activities. In addition, we observed areas with T-cells detected at high frequency near normal vascular endothelial cells (VECs), indicating immune cell trafficking toward the lesion in these marginal areas. While supporting previous findings that T-cells are not infected by KSHV, our data also revealed areas having high viral Ag load and abnormal vasculature, versus surrounding areas with much lower Ag load, normal vasculature, and robust immune infiltration. Discussion/Significance of Impact: Our approach not only demonstrated its ability to characterize the KS TME at a previously unapproachable level of complexity but has also revealed gaps in lineage assignments in substantial proportions of the tumor, which will be subjected to further characterization.

Objectives/Goals: To date, no study has jointly profiled RNA isoforms and modifications associated with Alzheimer’s disease neuropathological changes (ADNC) in a human cohort. Therefore, the overall objective of this study is to identify and characterize RNA isoforms and modifications that associate with ADNC by leveraging long-read direct RNA sequencing. Methods/Study Population: This translational study will analyze human prefrontal cortex tissue from a well-defined cohort of 12 individuals. The cohort consists of six individuals with severe Alzheimer’s disease neuropathological changes and six age- and sex-matched non-demented controls. Leveraging long-read direct RNA sequencing, which uniquely quantifies both RNA isoform and modifications simultaneously, can provide novel insights into how severe Alzheimer’s disease neuropathological changes drive changes in RNA isoform expression patterns and alter RNA modifications contributing to isoform dysregulation. Results/Anticipated Results: My preliminary data from human prefrontal cortex (n=2) revealed pseudouridine sites, 1 of ~170 known RNA modifications, in Alzheimer’s disease-risk genes (APP, CLU, SORT1, and BIN1), supporting the need to map RNA modifications across Alzheimer’s disease-relevant isoforms. My central hypothesis is that severe Alzheimer’s disease neuropathological changes (ADNCs) drive transcriptomic and epitranscriptomic dysregulation by altering RNA isoforms. Thus, my two working hypotheses are 1) severe ADNC drives transcriptomic dysregulation of RNA isoforms and 2) severe ADNC drives epitranscriptomic dysregulation of RNA modifications. Discussion/Significance of Impact: This project will be the first to jointly profile RNA isoforms and modifications accounting for Alzheimer’s disease neuropathological changes, offering candidate regulatory targets for therapeutic development. Importantly, this research will equip me with an expertise in RNA biology, neurodegenerative diseases, and bioinformatics.

Objectives/Goals: Current techniques for immunosuppressant therapeutic drug monitoring (TDM) are not patient-centric which is prohibitive for those in remote areas. We conducted stakeholder engagement meetings to identify context, attitudes, design requirements, and potential unintended consequences for developing an at-home test for immunosuppressant TDM. Methods/Study Population: We conducted two virtual focus group discussions with 10 organ transplant recipients, some of whom were located in rural and remote locations, including members of the community engagement committee of the Center for Innovations in Cancer & Transplant and the Transplant Recipient Advisory Council. We also conducted a virtual focus group discussion with transplant providers and had individual conversations with one provider and one patient who were unable to attend the focus groups. Results/Anticipated Results: Patients had strong interest in at-home testing to reduce financial and time burdens. Providers wanted faster test turnaround times than available with current centralized labs, especially for managing patients in remote settings. Both patients and providers agreed that at-home tests could enhance patient engagement and empowerment in healthcare, and they preferred a device that would provide highly quantitative and accurate results available on the same day. Finally, both patients and providers also expressed concerns about potential increased work created for caregivers and healthcare workers. Discussion/Significance of Impact: We will incorporate these findings into our design engineering and continue iterative stakeholder engagement to co-design a device that can transform TDM and transplant care.

Objectives/Goals: To evaluate an undergraduate nursing externship revised to integrate the updated International Association for Clinical Research Nursing (IACRN) curriculum, designed to strengthen knowledge, skills, and career readiness for the specialty of clinical research nursing (CRN). Methods/Study Population: A 192-hour, 8-week summer externship was conducted at the Rockefeller University (2022–2025), accepting two rising junior nursing students annually. The curriculum included lectures on clinical research principles and processes, IRB observation, informed consent simulation, a mock IRB, and rotations in bionutrition, pharmacy, bioinformatics, and laboratory processing. Externs also completed the Collaborative Institutional Training Initiative on Good Laboratory Practice and lab safety modules. Program evaluations included a pre- and post-test, a 30-item knowledge exam to assess understanding of clinical research concepts, as well as ratings by the mentors across professional competencies, and open-ended questions to obtain feedback from the externs. Results/Anticipated Results: Externs’ knowledge scores rose from pretest averages of 58–70% to post-test scores of 73–86%, a gain of ~20 points. Surveys revealed an improved understanding of research processes, particularly in areas such as informed consent and protocol design. Hands-on activities, lab processing, dietary simulation, and pharmacy were most valued. Mentors consistently rated externs as “Good” to “Excellent” in terms of professionalism, adaptability, and teamwork. Externs reported a greater interest in CRN careers and a deeper appreciation for the specialty’s breadth. Discussion/Significance of Impact: This externship, which embedded updated IACRN competencies at the undergraduate level, successfully enhanced knowledge and understanding of the field. Its innovative, replicable model demonstrates how early exposure can expand the clinical research nursing pipeline and prepare future CRN leaders.

Objectives/Goals: We sought to describe perspectives among Black nursing professionals and community leaders regarding the return of genetic test results and place perspectives into context with aggregated findings in the All of Us Research Program’s Data Browser. Methods/Study Population: Semi-structured, virtual interviews were held with adults (≥18 years of age) self-identifying as Black. A two-step thematic analysis process was used to assess interviewee perspectives with (sub)themes identified in the literature across two topics: drug/medication response and hereditary disease risk. Themes were placed into context with Data Browser content, focusing on genes and their respective alleles with frequencies ≥0.10 in African ancestry populations in All of Us. Results/Anticipated Results: Interviewee perspectives aligned with two known themes (research engagement and care), with five emerging subthemes. In the Data Browser, seven alleles were observed with frequencies ≥0.10 for three pharmacogenomic (PGx) biomarkers for African ancestry populations:CYP2C19 (SNV, 10-94761900-C-T; SNV,10-94775367-A-G; SNV 10-94781859-G-A), DPYD (SNV, 1-97883329-A-G; SNV, 1-97515839-T-C), and UGT1A1 (insertion, 2-233760233-C-CAT; SNV, 2-233757136-G-A). Also, four alleles were observed with frequencies ≥0.10 for three genes implicated in hereditary disease risk, two of which hold PGx implications: CACNA1S (PGx, SNV, 1-201112815-C-T; SNV, 1-201110107-C-T), SCN5A (no PGx, SNV, 3-38603929-T-C), and TP53 (PGx, SNV, 17-7676154-G-C). Discussion/Significance of Impact: Our findings convey important clinical and translational science considerations for individuals and community leaders of African ancestry and researchers seeking reputable, publicly available information to understand, communicate, and act on genomic findings.