Objectives/Goals: This work aims to identify functional brain networks that differentiate opioid use disorder (OUD) subjects from healthy controls (HC) using machine learning (ML) analysis of resting-state fMRI (rs-fMRI). We investigate the default mode network (DMN), salience network (SN), and executive control network (ECN), as well as demographic features. Methods/Study Population: This work uses high-resolution rs-fMRI data from a National Institute on Drug Abuse study (IRB #HM20023630) with 31 OUD and 45 HC subjects. We extract rs-fMRI blood oxygenation level-dependent (BOLD) features from the DMN, SN, and ECN. The Boruta ML algorithm identifies statistically significant features and brain activity mapping visualizes regions of heightened neural activity for OUD. We conduct fivefold cross-validation classification experiments (OUD vs. HC) to assess the discriminative power of functional network features with and without incorporating demographic features. Demographic features are ranked based on ML classification importance. Follow-up Boruta analysis is performed to study the medial prefrontal cortex (mPFC), posterior cingulate cortex, and temporoparietal junctions in the DMN. Results/Anticipated Results: Boruta ML analysis identifies the DMN as the most salient functional network for differentiating OUD from HC, with 33% of DMN features found significant (p < 0.05), compared to 10% and 0% for the SN and ECN, respectively. The Boruta ML algorithm identifies age and education as the most significant demographic features. Brain activity mapping shows heightened neural activity in the DMN for OUD. The DMN exhibits the greatest discriminative power, with a mean AUC of 69.74%, compared to 47.14% and 54.15% for the SN and ECN, respectively. Fusing DMN BOLD features with the most important demographic features improves the mean AUC to 80.91% and the F1 score to 73.97%. Follow-up Boruta analysis highlights the mPFC as the most important functional hub within the DMN, with 65% significant features. Discussion/Significance of Impact: Our study enhances the understanding of OUD neurobiology, identifying the DMN as the most significant network using ML rs-fMRI BOLD feature analysis. Ethnicity, education, and age rank are the most important demographic features and the mPFC emerges as a key functional hub for OUD. Future research can build on these findings to inform treatment of OUD.

Objectives/Goals: By synergizing our efforts, we believe this to be a fruitful collaboration for UCLA Clinical and Translational Science Institute (CTSI) and California Institute for Regenerative Medicine (CIRM). With multiple levels to stem cell training, focusing on specific educational goals is integral to our pilot event. This was held on July 26th and offered an exciting and valuable day for trainees. Methods/Study Population: Leadership was comprised of leaders at the UCLA Health Alpha Clinic, Broad Stem Cell Research Center (BSCRC), Human Gene and Cell Therapy Facility (HGCTF), UCLA Campus, Clinical and Translational Research Center (CTRC), and the Santa Barbara COMPASS program. Trainees from UCLA, CSUN, and UCSB were represented. The agenda included a didactic overview of the entire translational and clinical research process from discovery in the laboratory to bedside nursing in the patient care areas. Onsite tours were conducted at the HGCTF and the CTRC with a meet and greet with the nurses. The curriculum covered the clinical research process, regulatory requirements, ethics, current clinical trials, manufacturing, quality control, and compliance. A career opportunities discussion and network sessions closed out the day. Results/Anticipated Results: Of the 13 trainees who attended the session, 10 replied to the evaluation survey. All the responding students (100%) rated the event as “excellent” and found it to be “highly valuable” to their current training program. The trainees indicated that they were “very likely” (100%) to recommend a friend to attend this type of event. When asked what they liked most about the event, they indicated that the programming was “insightful, and “inspiring” for seeing beyond their current trainee responsibilities. They valued the responsiveness to questions, sharing experiences, and mentoring for career advancement. They especially liked the tours at the HGCTF and the session with the front-line nurses. Changes for the future will include timing and length, information on graduate programs and more student interactions. Discussion/Significance of Impact: Overall, our first educational session was very well-received by both trainees and staff involved as stakeholders. Due to the success of this inaugural event, we intend to continue to draw on the expertise of this collaboration and use a similar blueprint for future events and scientific sessions.

Objectives/Goals: We aimed to discover treatment candidates for uterine fibroids, a common benign tumor with adverse impacts on quality of life. Repurposing already approved medications for fibroids can expedite treatment option expansion. Using genetic proxies, we identified novel fibroid drug candidates and estimated their effect on risk of fibroid diagnosis. Methods/Study Population: We performed a genetically predicted gene expression (GPGE) analysis using S-PrediXcan and GTEx tissue models with multi-ancestry genome-wide association study (GWAS) summary statistics of fibroids (cases  =  74,294, controls  =  465). There were 81 genes significantly associated with fibroid risk. Querying drug–gene interaction databases identified 56 approved medications that target these genes, including two antihypertensives, hydralazine, and spironolactone. Using independent multi-ancestry GWAS summary statistics (N  =  635,969) for systolic (SBP) and diastolic blood pressure (DBP), we conducted GPGE analyses. Blood pressure (exposure) and fibroids (outcome) GPGE summary statistics in the same tissues were used for two-sample Mendelian randomization (MR) analyses to proxy medication effects. Results/Anticipated Results: GPGE analyses identified hydralazine/tumor protein P53 (TP53) activity and spironolactone/thyroid hormone receptor beta (THRB) activity as drug-gene candidate pairs. Both drugs increase gene activity of their paired gene. Increased TP53 expression was associated with SBP in four tissues (exposure). The MR results indicated hydralazine use, proxied by increased TP53 expression, may reduce fibroid risk by 42% per standard deviation of gene expression (odds ratio [OR]  =  0.58, p  =  1.43E-12). Increased THRB expression was associated with DBP in eight tissues and were included in the MR (exposure). The MR results suggest spironolactone use, proxied by increased THRB expression, may reduce fibroid risk by 23% per standard deviation of gene expression (OR  =  0.77, p  = 5.94E-6). Discussion/Significance of Impact: We provide biologically plausible evidence for repurposing hydralazine and spironolactone for reducing risk of fibroid diagnosis. Repurposing these hypertension medications could provide novel preventative treatments for fibroids, particularly for individuals disproportionately affected by both conditions.

Objectives/Goals: Our goal is to develop a CRM platform to streamline support for investigators, optimize resource use, and enhance service delivery within a CTSA institution. This platform will measure outcomes, improve transparency, and ensure compliance, creating an efficient, supportive research environment. Methods/Study Population: Beginning with the design concepts and collecting technical requirements, ensuring alignment with the needs of investigators, administrators, and service units. Apply project management methodologies to iteratively design, prototype, and test features. Focus on user-centric interfaces and seamless integration with institutional systems. Incorporate stringent data security protocols in compliance with regulatory standards. Conduct pilot studies to refine functionalities and gather constructive feedback. Facilitate extensive user training programs and offer continuous support. Regularly monitor performance metrics, iterating based on empirical data and user feedback to ensure continuous enhancement and adherence to institutional objectives. Results/Anticipated Results: YCCI successfully designed, developed, and implemented a CRM platform for managing the intake, navigation, consultation, service delivery, and outcomes tracking of the clinical and translational science activities that are supported by and beyond the CTSA-supported infrastructure. The CRM platform has been live in production since roughly July 1, 2024, and YCCI is excited to share data, results, and outcomes with the ACTS community. Discussion/Significance of Impact: YCCI’s CRM platform advances the CTS mission by streamlining PI support, enhancing resource utilization, and fostering inter-team collaboration within the research ops and administration teams. Measuring outcomes and service efficiency enables data-driven decisions, ensuring continuous improvement, and impactful translational science.

Objectives/Goals: Research mistrust is well documented. So is community engagement’s potential to foster trust. We share the community engaged development and implementation of informational sessions on research and the protections for participants, aiming at enhancing community members’ knowledge of research. Methods/Study Population: Drawing from a pool of community members who had participated in research projects with a community engaged (CE) research center, we established a four-member Research Partner Committee (RPC) to work with the Center’s research team (CE Team) in the co-creation of materials for an informational session covering two main topics: the fundamentals of biomedical research and of protections for research participants.. The RPC and CE Team also co-developed two sets of pre- and post-tests to evaluate knowledge acquisition at session implementation. Community partners assisted in recruiting their constituents to participate in the sessions and provided convenient sites for the presentations. Results/Anticipated Results: The CE Team and RPC met 7 times to co-create the research and protections presentations (e.g., research definition, importance, inclusion of diverse populations, reasons we have protections, Institutional Review Boards, and informed consent). They also co-developed a 7- and a 9- item pre-post test, respectively. Five informational sessions, implemented by the CE Team with RPC members as observers drew 49 participants (96% African American, 73% female). 55.1% and 75.5% of participants showed a positive change in knowledge after the research and the protections presentations, respectively. 91% agreed to be notified about future research studies at the center. Discussion/Significance of Impact: Engaging community members in dialogue around research and the reasons for mistrust in research encourages relationship development. This engagement equips community members with knowledge to join in conversations around medical studies and make informed decisions about participation in research.

Objectives/Goals: The Research Participant Advisory Group (RPAG) was born out of the concept that, by creating a structured way for researchers to collaborate with research participants and/or community, we can improve, grow, and support clinical/research-based research, from design to dissemination. Three distinct groups exist across our Academic Health Center (AHC). Methods/Study Population: The RPAG has 3 groups: The Cincinnati Children’s Hospital Research Participant Advisory Council (CCHMC RPAC) addresses clinical research needs, such as research design, recruitment, and consenting. Adult/youth research participants and family members (n = 21) provide feedback about how to improve a research process, form or other research tool. In Cincinnati’s underserved West End, the West End Community Research Advisory Board (WE C-RAB: n = 18) focuses on the needs of underserved minority groups and supports researchers wishing to do community-based research. The newly formed University of Cincinnati RPAC (UC RPAC; n = 17) addresses the UC research needs. All groups meet monthly and are offered a meal and incentive. All members have received training in the responsible conduct of research. Results/Anticipated Results: Over 70 researchers have brought research challenges to the RPAG groups over the last 8 years, leading to improved consent forms, more targeted recruitment, clearer language and a more streamlined screening process. RPAG member surveys indicate a greater understanding of clinical research challenges, critical health issues, and how research can benefit them. The WE C-RAB has improved community recruitment efforts, as well as survey and study design. Researchers highlight the importance of feedback in creating greater study participant engagement, indicating deeper understanding of the community/participant perspective and how to work “with” community. Ongoing WE C-RAB-faculty partnerships have led to at least 3 federally funded grants. Discussion/Significance of Impact: The 3 RPAGs provide the versatility to meet the needs of the diverse research spectrum across the AHC. This includes the type of research as well as the level or degree of participant/community engagement needed. RPAGs create greater connection and understanding leading to better participant experiences and the promise of better health outcomes.

Objectives/Goals: Transgender women who have sex with men (TGWSM) have higher HIV risk. The rectal mucosal (RM) immune environment of TGWSM who choose feminizing hormone therapy (FHT) has been shown to be distinct from the RM of cisgender men who have sex with men (MSM). We studied the impact of FHT on the adaptive immune cellular composition of the RM. Methods/Study Population: We sampled cross-sectional and longitudinal cohorts of TGWSM and cisgender MSM from The Silom Clinic in Bangkok, Thailand from December 2020 to December 2023. We included participants aged >18 years, all cisgender MSM and TGWSM with FHT levels in the therapeutic range for cisgender women. We performed RM biopsies and analyzed the adaptive immune cell characteristics via flow cytometry. We will perform binary linear regression to assess the association between systemic FHT levels and the percentage of CD4+ T cells expressing key biomarkers. Primary outcomes include the percentage of CD4+ T cells that express CCR5, with a secondary outcome of the percentage of CD4+ T cells that express Ki67. Results/Anticipated Results: The cross-sectional cohort included 100 TGWSM on FHT and 50 cisgender MSM. The longitudinal cohort included 25 TGWSM who were initiating FHT. Similar primary and secondary outcomes are to be elucidated in both cohorts. We anticipate the RM environment of TGWSM using FHT in both cohorts compared to the RM environment of cisgender MSM in the cross-sectional cohort will be associated with greater percentages of activation/co-receptor expression of CD4+ T cells that express biomarkers of interest. In the longitudinal cohort, we similarly anticipate increased percentages and activation/co-receptor expression of CD4+ T cells expressing biomarkers of interest in TGWSM after in comparison to before initiating FHT. Discussion/Significance of Impact: This is the largest study of its kind to compare HIV target cells in RM of TGSWM, which challenges prevailing perspectives suggesting to group cisgender MSM with TGWSM. Anticipated results will inform HIV prevention strategies and future vaccine studies in this high-risk population.

Objectives/Goals: Osteosarcoma (OS) is the most common primary bone malignancy in humans and dogs. >40% of children and >90% of dogs succumb to metastatic disease. We hypothesize MYC overexpression in metastatic canine and human OS contributes to an immunosuppressive tumor environment by driving tumor-associated macrophage influx and T lymphocyte exclusion. Methods/Study Population: To characterize the role of oncogenic MYC signaling in the canine metastatic tumor immune microenvironment (TIME), 42 archived FFPE lung metastatic canine OS samples were evaluated for MYC copy number variation (CNV), mRNA, and protein expression via ddPCR, nanostring analysis, and immunohistochemistry (IHC). Seven samples also underwent GeoMX spatial profiling to more specifically evaluate T cell and macrophage transcriptional profiles based on MYC status. To determine the role of MYC target modulation as a potential therapeutic option, canine and human OS cell lines were treated with a novel MYC inhibitor (MYCi975) and assessed for effects on survival, proliferation, and cytokine profiles. Results/Anticipated Results: We demonstrate that copy number gains are not a key driver of MYC hyperactivity in canine metastatic OS. However, stratification based on MYC protein expression demonstrates that “MYC-high” tumors are associated with downregulation of cytotoxic effector T-cell associated transcripts and upregulation of tumor-associated macrophage (TAM) and extracellular matrix remodeling transcripts. We also report that MYCi975 treatment of canine and human OS cell lines results in significant inhibition of OS cell survival and proliferation at concentrations that are pharmacologically achievable in mice. Furthermore, we demonstrate MYC inhibition by MYCi975 is associated with reduced pro-inflammatory cytokine secretion in OS cell culture models. Discussion/Significance of Impact: While MYC overactivity in metastatic canine OS may not be genomically driven, other mechanisms that lead to increased MYC protein expression are associated with transcriptomic profiles supportive of local immunosuppression. Pharmacologic targeting of MYC may serve as a strategy to bolster immunotherapeutic options in metastatic OS treatment.

Objectives/Goals: To develop and deploy an academic learning health system (aLHS) Bridge Program to capitalize on our unique organizational strengths in Implementation Science (IS) and to overcome the gap between science and practice that threatens the success of an aLHS. The aLHS Bridges includes an IS Shared Resource, intended to advance IS to practice. Methods/Study Population: The new IS Shared Resource is built on our expertise in dissemination and implementation science, pragmatic, and adaptive trials and the CTSI’s prior success in integrating academic and clinical missions. We also leveraged our existing experts to co-lead the aLHS Bridge including Kristie Foley, PhD, inaugural Chair of the Department of Implementation Science, and Gary Rosenthal, MD, Chair of the Department of Internal Medicine. Specifically, the new IS Shared Resource builds on the capacity of the Department of IS, comprised of 36 faculty members (19 primary and 17 secondary/adjunct appointments) with expertise in qualitative and mixed-methods research, stakeholder engagement, participatory research, digital health, and organizational theory. Results/Anticipated Results: The IS Shared Resource is primed to aid faculty with dissemination and implementation needs, including shortening the time of intervention adoption and using implementation science to inform sustainable and effective implementation practice. The IS Shared Resource is equipped to provide consultation services to faculty members to understand their specific request and match IS faculty members who are expertly trained in specific strategies or contexts. Discussion/Significance of Impact: Leveraging current resources and our first-of-its-kind Department of Implementation Science, our CTSI was able to stand up the IS Shared Resource to support the goals of the CTSA and our greater institution mission. Using a multidisciplinary approach was essential to the success of the IS Shared Resource.

Objectives/Goals: Neuroendocrine malignancies are heterogeneous cancers with varied clinical outcomes, yet the molecular landscape driving this heterogeneity has not been fully characterized. Here, we investigate the gene expression and mutational profiles of neuroendocrine malignancies to better understand the underlying biology and therapeutic targets. Methods/Study Population: Patients with neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) treated at Cleveland Clinic (2000–2022) with molecular profiling (n = 66) were identified. Mutational and gene expression profiles were abstracted from electronic health records (EHR). Clinico-pathological characteristics and overall survival (OS) were obtained from EHR. Statistical analyses were performed by R v.4.0.5 and R package Limma for differential gene expression, as well as Chi-square, Fisher’s exact, and Wilcoxon rank sum tests. Results/Anticipated Results: The cohort consisted of 38 cases with NEC, 18 NET g3, and 10 NET g1/2. EZH2 and cyclin E1 were differentially over-expressed in NEC vs. NET (p < 0.05), while PTEN and MSLN were differentially under-expressed in NEC vs. NET (p < 0.005). Several recurrent alterations co-segregated with aggressive histology (NEC vs. NET): TP53 (p 60. Also, there was no difference in gene expression profiles between the two age groups among NETs or NECs. Discussion/Significance of Impact: This study explores the molecular landscape of NETs and NECs, revealing distinct gene expression and mutation profiles related to clinical outcomes. High expressions of cyclin D1 and EGFR were significantly associated with improved 2-year OS in NECs, highlighting potential therapeutic targets. Future studies are needed to validate these findings.

Objectives/Goals: Pharmacogenomic (PGx) testing identifies genetic variations affecting medication response but is not yet in routine clinical whole-genome sequencing (WGS) workflows. We aimed to establish a streamlined bioinformatics pipeline for incorporating PGx reporting into clinical WGS and to determine clinical implications for medication treatment. Methods/Study Population: A PGx profiling pipeline based on existing WGS data was developed, integrating three WGS-based PGx calling tools: Aldy, PyPGx, and Cyrius (CYP2D6 only), to provide genotype calls for 17 key pharmacogenes. The pipeline was validated using WGS data from 70 individuals with diverse backgrounds (36% European, 27% African, 27% Asian, and 10% admixed) from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results were manually reviewed against published data. The validated pipeline was then applied to 144 clinical patients previously screened for neurodevelopmental disorders or suspected hereditary diseases, followed by diplotype-to-phenotype translation and preemptive PGx-guided medication recommendations based on consensus guidelines and FDA labeling for commonly used medications. Results/Anticipated Results: Congruent phenotype call rates for GeT-RM samples were 100% for 13 genes (CFTR, CYP2B6, CYP2C19, CYP2C9, CYP3A4, CYP4F2, DPYD, G6PD, IFNL3, NAT2, NUDT15, TPMT, and VKORC1), 99% for three genes (CYP3A5, SLCO1B1, UGT1A1), and 97% for CYP2D6, indicating strong pipeline performance. Among 144 clinical patients, 99.3% had at least one clinically actionable PGx results relevant to 36 of top 300 medications in the USA across psychotropic, cardiovascular, musculoskeletal, gastrointestinal, and other therapeutic areas. The most prevalent drug–gene interactions involved sertraline and CYP2B6, affecting 49% patients: 41% were intermediate metabolizers who may require slower titration and lower maintenance doses, while 8% poor metabolizers may benefit from a lower starting dose or alternative antidepressants. Discussion/Significance of Impact: Our validated WGS-based PGx profiling pipeline successfully extracted actionable PGx data from clinical WGS. By aligning PGx profiles with guideline-recommended clinical actions, we demonstrated the clinical value of integrating PGx reporting in WGS workflows, improving personalized medication management.

Objectives/Goals: To facilitate engagement between university researchers and Appalachian Kentucky communities, the UK Rural Research Hub (RRH) promotes Community Engaged Research (CEnR) and academic–community partnerships that have the greatest potential to conduct impactful research to improve health and reduce regional health disparities. Methods/Study Population: Through the UK RRH, a wealth of expertise and a range of services sustain successful CEnR. Hub coordinators provide research consultations, accelerate researchers’ engagement with the community, and facilitate the success of studies through study coordination, assistance with participant recruitment, data collection and interventions, and through dissemination back to the community. Results/Anticipated Results: UK RRH coordinators have supported numerous studies across the region. For example, RRH staff facilitated recruitment of and collected data from 40 Appalachian caregivers of patients with Alzheimer’s disease and related dementias (ADRD) in a study to improve home environments for patient well-being. The study provided pilot data for a successful K23 application. Other examples of supported research include studies to improve cancer screening uptake, self-management of diabetes, and cardiovascular disease risk reduction, resulting in improved care in the community and often providing pilot data leading to larger national grants. Discussion/Significance of Impact: Research addressing the complex health issues that burden Appalachian Kentucky requires community engagement to be successful. The UK RRH is at the heart of successful CEnR that benefits researchers and communities alike.

Objectives/Goals: Currently, a lack of screening markers and targeted therapies prevent clinicians from successfully treating PDAC. Precision medicine may allow oncologists to better combat this disease. To personalize care, knowledge of tumor protein posttranslational modifications, extracellular matrix makeup, and infiltrating immune cells is imperative. Methods/Study Population: Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was employed to characterize the N glycosylation state, the ECM composition, and immune cell populations present within 10 formalin fixed paraffin embedded PDAC patient samples. Molecular dry spray of PNGase F and Collagenase III followed by enzymatic digestion allowed for the release of N glycans and ECM peptides from the tissue. Multiplex immunohistochemistry with photocleavable, mass-tagged probes was also performed on each tissue. This analysis produced a spatial map of N glycans, ECM peptides and immune cells with their distribution and abundance color-coded as a heat map of each tissue. Results/Anticipated Results: This analysis produced a unique N-glycan signature associated with specific tumor regions (necrosis, invasive margin, etc.) and immune cell clusters. Additionally, immune cells within the PDAC tumor microenvironment were found to be organized into immature tertiary lymphoid structures composed primarily of CD20+ B cells. Finally, a distinct distribution of ECM peptides within and surrounding tumor tissue was visualized, and putative identifications have been assigned to these stromal elements. Discussion/Significance of Impact: In the future, insights from this hypothesis-generating study may be leveraged to identify diagnostic and prognostic biomarkers for PDAC to improve early diagnosis and treatment response rates. The N glycan signature, ECM composition, and immune activation state in liquid biopsies including serum and PBMCs will be compared to data from this study.

Objectives/Goals: Ischemic stroke treatments assist in restoring blood flow, but do not guarantee good outcomes. Since extracellular vesicles (EVs) able to cross the blood brain barrier, total (nonspecific) and astrocyte enriched EVs (TEVs, AEVs, respectively) from plasma may emerge as plasma biomarkers for prognostication and targeted therapeutics. Methods/Study Population: “Blood and Clot Thrombectomy Registry and Collaboration” (BACTRAC; NCT03153683) is a human stroke biobank at the University of Kentucky that collects samples at the time of mechanical thrombectomy during emergent large vessel occlusions (ELVO; ischemic stroke). EVs were isolated, via size exclusion chromatography, from unbanked plasma and concentrated resulting in TEVs. AEVs were immunoprecipitated with anti-EAAT1 (GLAST), an astrocyte-specific transmembrane glycoprotein. Isolated protein was sent to Olink and ran on their metabolic panel. Demographics and medical histories of the subjects were exported from REDcap and investigators were blinded during EV analysis. Results/Anticipated Results: ELVO subjects (8 females/ 5 males) were an average age of 71.1 ± 11.7 years. Lower TEV enolase 2, a neuronal glycolysis enzyme, associated with increased stroke severity (NIHSS; rs =  -0.7819, p = 0.0476). Higher systemically TEV quinoid dihydropteridine reductase (QDPR), essential co-factor enzyme, was associated with more severe strokes (NIHSS; rs =  0.8486, p = 0.0123) and lower cognition (MoCA; r2 =  0.7515, p = 0.0254). Interestingly, higher intracranial AEVs QDPR was associated with lower infarct volumes (rs =  -0.7333, p = 0.0202), less severe strokes (NIHSS; rs =  -0.6095, p = 0.0388), and better cognition (MoCA; r2 =  0.6095, p = 0.0388). Increased AEV nicotinamide adenine dinucleotide kinase another essential co-factor enzyme, intracranially also correlated to higher cognition (MoCA; rs =  0.8356, p = 0.0298). Discussion/Significance of Impact: Plasma TEV and AEV metabolic proteins correlate with the progression of stroke outcomes and should be investigated as target therapies during MT to improve outcomes.

Objectives/Goals: To support the growing number of clinical research professionals (CRP) working in behavioral and social science contexts, relevant formative assessments of research skills are needed. This study examines the development of an objective formative assessment designed to assess skills relevant to conducting social and behavioral research. Methods/Study Population: A multidisciplinary group at the University of Michigan was convened to develop the assessment. Case studies depicting clinical and translational research conducted in behavioral and social science contexts were used to measure proficiency in seven of the eight ECRPTQ competencies: Scientific Concepts & Research Design, Ethical & Participant Safety Concerns, Clinical Trial Operations (Good Clinical Practice), Study & Site Management, Data Management & Informatics, Leadership & Professionalism, Communication, and Teamwork & Team Science. Three difficulty levels of questions were developed: basic, intermediate, and advanced. To reinforce knowledge and skill development, the assessment was designed to give respondents formative feedback after responding to each question. Results/Anticipated Results: A preliminary “pre-pilot” test was conducted with three postdoctoral scholars to ensure that the assessment items were understandable. The assessment was then pilot tested with a larger group of 40 clinical research professionals (CRP) to test for the clarity and difficulty level of the items. A smaller group of 20 of these CRPs agreed to participate in focus groups to obtain feedback on their user experience. Data regarding years of experience as a CRP, types of studies engaged with, and information regarding professional certification were collected and used in the analyses. Demographic data collected were not connected to user responses. Results of the pilot test and focus groups were used to revise the questions on the final version of the assessment. Discussion/Significance of Impact: To our knowledge, this is the first objective assessment of research skills for CRP working in behavioral and social science contexts. We will discuss how other institutions can use this instrument to evaluate the training needs of their social and behavioral research workforce.

Objectives/Goals: Despite the acknowledgment of post-Ebola syndrome (PES), young EVD survivors have received little attention. The mechanistic drivers and long-term consequences of PES and EVD early in life are unknown. We aim to define PES presentations in pediatric EVD survivors and propose potential mechanistic factors contributing to PES in young people. Methods/Study Population: Here we focus on physical health outcomes in an ongoing cohort study assessing mental and physical health in pediatric EVD survivors (age Results/Anticipated Results: 671 participants were enrolled between 2021 and 2022 (Infected: n = 226, Affected: n = 207, and Control: n = 238). Groups were similar in sex distribution (52.7%, 54.0%, and 53.8% female, respectively) and mean age, although the Infected group was slightly older (14.6 y) than the Affected (13.5 y) and Control groups (14.1 y), a difference unlikely to be clinically significant. Notably, the EVD Infected group exhibited a higher burden of symptoms, with significant findings in cardiac, MSK, ophthalmologic, and “ear, nose, and throat” systems. Principal component analysis showed differential patterns of sequelae across the groups, primarily defined by MSK. Discussion/Significance of Impact: PES is heterogeneous in pediatric EVD survivors. EVD Affected children exhibit a similar yet distinct pattern of clinical sequelae indicating ecological factors impact sequelae and raising questions about the mechanistic drivers of PES in children. Potential mechanisms include inflammation or accelerated aging and immune dysfunction.

Objectives/Goals: Nucleoside transport by ENT2 facilitates transit of the lupus anti-DNA autoantibody Deoxymab into cells and across the blood–brain barrier into brain tumors. This work examines the Deoxymab-nucleoside interactions that contribute to membrane crossing and apply them in brain tumor therapeutics. Methods/Study Population: Deoxymab interactions with individual nucleosides, nucleobases, and pentose sugars are examined by surface plasma resonance (SPR) and cell penetration assays in a panel of cell lines including glioblastoma, breast cancer, and normal breast epithelial cells. Deoxymab-conjugated gold nanoparticles are generated and tested for binding to normal human astrocytes and glioma cells, and the impact of supplemental nucleosides on this binding is determined. Deoxymab-gold nanoparticles are tested for brain tumor localization by systemic and local administration in mice bearing orthotopic glioblastoma brain tumors and enhancement of laser interstitial thermal therapy (LITT) examined. Results/Anticipated Results: Individual nucleosides significantly increase the efficiency of cell penetration by Deoxymab in all cell lines tested. In contrast, component nucleobases and pentose sugars significantly suppress the uptake of the autoantibody into cells. Deoxymab-conjugated gold nanoparticles bind DNA in vitro and to astrocytes in culture and are anticipated will enhance the efficacy if LITT in vivo by associating with DNA released by necrotic tumors and/or by locally administered nucleosides in brain tumor environments and subsequently act as heat sink to amplify LITT impact. Discussion/Significance of Impact: Deoxymab is a DNA-targeting, cell-penetrating autoantibody. These findings establish nucleosides as the components of DNA that promote autoantibody membrane crossing through ENT2 activity and indicate potential for use of Deoxymab-gold nanoparticles in combination with LITT for brain tumor therapy.

Objectives/Goals: Diabetic kidney disease (DKD) affects 40% of diabetic patients, leading to renal failure, yet the molecular drivers remain elusive. MicroRNAs, noncoding regulators of gene expression, may hold the key. This study aims to identify key miRNAs in DKD, providing crucial insights for early intervention. Methods/Study Population: miRNA sequencing was conducted on kidneys from 8-week old male BTBR wild type and BTBR ob/ob mice. BTBR ob/ob mice lack the hormone leptin and spontaneously develop type 2 diabetes, with morphological renal lesions characteristic of human DKD. Total RNA was extracted from whole kidney sections and processed using the QIAseq miRNA library kit. Sequencing was performed on an Illumina NextSeq 550 platform. GeneGlobe analysis was used to identify differentially expressed miRNA functional pathways, while ingenuity pathway analysis (IPA) was employed to predict master regulators and causal networks involved in DKD. Results/Anticipated Results: miRNA sequencing identified significantly differentially expressed miRNAs (p < 0.05) between 8-week-old BTBR WT and BTBR ob/ob male mice, including miR-34a (-6.86 fold), miR-122 (-5.01 fold), miR-129 (-2.23 fold), miR-142a (+2.78 fold), miR-346 (+4.66 fold), miR-547 (-2.49 fold), miR-592 (+11.81 fold), miR-802 (-6.95 fold), and miR-6539 (-7.93 fold). Qiagen GeneGlobe analysis revealed biological processes potentially targeted by these miRNAs, including endocytosis, phagocytosis, hyperglycemia (p = 7.59e-3), and insulin-dependent diabetes (p = 4.32e-4). IPA predicted activation of RRAS, a small GTPase regulating cell growth and signaling (Z-score +2), with miR-34a and miR-122 targeting MYC, PI3K, and TGF-β in DKD progression in BTBR ob/ob mice. Discussion/Significance of Impact: We identified kidney miRNA expression in BTBR ob/ob mice at a pivotal disease stage. miR-34a, miR-122, and RRAS emerged as key drivers in DKD progression, showing remarkable early biomarker potential. These findings lay the groundwork for early detection and innovative therapies to halt DKD and improve patient outcomes.

Objectives/Goals: Aim 1: Assess the correlation between Tuft cell T2R functionality and Th2 sinonasal inflammation, disease burden, and post-surgical outcomes in AERD patients. Aim 2: Determine if hyperfunctional Tuft cell T2Rs enhance denatonium-stimulated inflammatory responses in nasal epithelial air–liquid interface (ALI) cultures from AERD patients. Methods/Study Population: Aim 1: We will conduct a prospective cohort study with 82 AERD patients. Taste sensitivity to denatonium (DB), serving as a proxy for tuft cell T2R functionality, will be assessed using a validated 13-point scale, and correlations with clinical outcomes – SNOT-22, histopathologic, CT, and endoscopic scores – will be analyzed using linear regression. Aim 2: Sinonasal epithelial cells will be collected from AERD patients either hyper- or hyposensitive to DB and from healthy controls. We will establish ALI cultures and expose them to varying DB concentrations. Secretions will be analyzed for antimicrobial peptide release via bacterial kill assays and for IL-25 and β-defensin 2 via ELISA. Tuft cell frequency and baseline IL-25 mRNA expression will be assessed using immunofluorescence and quantitative real-time polymerase chain reaction, respectively. Results/Anticipated Results: We expect that higher DB taste sensitivity in AERD patients will correlate with worse clinical outcomes, reflected by elevated 6-month postoperative SNOT-22 scores, indicating increased symptoms. Additionally, we anticipate that preoperative Lund-MacKay and Lund-Kennedy scores, along with histopathological metrics, will be worse in DB-hypersensitive patients, establishing a link between taste sensitivity and disease burden. In vitro, we predict that AERD patients with DB hypersensitivity will demonstrate significantly higher IL-25 and β-defensin 2 secretion and reduced bacterial colonies in kill assays. We also expect increased tuft-cell frequency and baseline IL-25 mRNA in AERD-derived cultures compared to healthy controls, highlighting T2R functionality’s role in AERD pathogenesis. Discussion/Significance of Impact: This project aims to investigate a putative new role for Tuft cells in AERD pathogenesis by correlating Tuft cell T2R functionality with outcomes in AERD patients and with inflammatory response in vitro. Findings could lead to predictive clinical taste tests and future genotyping studies to identify T2R polymorphisms correlated with AERD severity.

Objectives/Goals: This study examines associations between childhood violence exposure, accelerated biological aging, and adolescent depression using DNA methylation-based epigenetic clocks. Findings aim to identify biomarkers for early detection, guide interventions, and address youth mental health disparities. Methods/Study Population:Data from the Future of Families and Child Wellbeing Study (N = 4,898), a diverse urban U.S. cohort, were analyzed. Childhood violence exposure, assessed using the Parent-Child Conflict Tactics Scale, included measures of physical, emotional, and psychological aggression and neglect. Biological aging at age 15 was evaluated using second-generation epigenetic clocks derived from saliva DNA methylation patterns, while depressive symptoms were measured with the CES-D scale. Multiple linear regression models tested associations between early violence exposure, epigenetic aging, and depressive symptoms, adjusting for socioeconomic status, caregiver mental health, and other key covariates. Results/Anticipated Results: Preliminary results suggest that early violence exposure may be linked to accelerated biological aging and depressive symptoms during adolescence, a critical developmental period. Epigenetic clocks offer an objective method for identifying high-risk youth, complementing mental health evaluations. With further validation and participatory action research, these findings could guide the development of biomarkers for longitudinal testing in school-based screenings and community health programs. These tools aim to be accessible, culturally relevant, and tailored to diverse populations, enhancing early detection, informing personalized interventions, and supporting scalable clinical applications. Discussion/Significance of Impact: This study explores links between early adversity, biological aging, and mental health, advancing understanding of adolescent depression. Epigenetic biomarkers could improve risk detection and guide tailored interventions in schools and community settings, enhancing access and reducing disparities.