Objectives/Goals: Engaging interest holders in research is increasingly common, and guidelines include creating engagement plans. A detailed plan may be especially helpful when researchers perceive engagement as difficult or less relevant. We tested whether a study’s translational stage or an investigator’s years of research experience affect their perceptions. Methods/Study Population: Since 2019, the Tufts Clinical and Translational Science Institute Pilot Studies Program required applicants to submit plans to engage interest holders. Applicants in three cohorts responded to a survey about this requirement, including perceived difficulty developing an engagement plan, perceived relevance of engagement, and self-reported years of research experience (≤5, 6–10, and ≥10 years). Two raters assigned translational stage(s) of proposed studies: T0 (basic science), T.5 (pre-clinical to initial human studies), and T1 through T4. Separate analyses were conducted when multistage studies were coded as the earliest vs. latest stage and for individual stage vs. groups of stages (T0/T.5/T1 vs. T2/T3/T4). The Fisher’s exact statistical test was used to assess associations between variables. Results/Anticipated Results: Analyses included 67 participants. Developing an engagement plan was perceived as more difficult for studies at earlier translational stages when those studies were coded as the earliest applicable stage. This significant association held both when stages were grouped as T0/T.5/T1 and T2/T3/T4 (P  =  .03) and when analyzed as a single stage (P  =  .01); however, when studies were coded as the latest applicable stage, there were no significant associations. Similarly, when multistage studies were coded as the earliest applicable stage, engagement was perceived as less relevant for early-stage studies when grouped (P  =  .04), but not for individual stages or when studies were coded as the latest applicable stage. No significant association between years of research experience and perceived difficulty was identified. Discussion/Significance of Impact: Results show that investigators conducting early-stage research perceive more difficulty engaging interest holders, aligning with prior qualitative studies. These investigators may need more evidence of the value added to early-stage studies, targeted and practical training, and funder requirements to establish a culture of engagement.

Objectives/Goals: Clinical trial success requires recruiting and retaining diverse participants. The ER&R Certificate Program trains clinical research professionals (CRPs) in equity, diversity, and inclusion (EDI), addressing biases, and integrating regulatory knowledge with practical skills to foster inclusive research practices. Methods/Study Population: An interdisciplinary Steering Committee, supported by Duke CTSI and DOCR, developed and implemented an engagement, recruitment, and retention certificate program (ER&R) for CRPs. With expert-led instruction, including e-learning, group sessions, and hands-on activities, ER&R integrates EDI into participant engagement practices. Participants complete 7 core courses and at least 3 elective courses, reflecting their unique responsibilities. Program evaluation uses the Kirkpatrick model to assess participant learning, competency, and EDI integration into clinical research. Since launch, the program has expanded to include clinical research trainees from Durham Technical Community College. All elements of the program were designed to allow for sharing across academic medical institutions. Results/Anticipated Results: A total of 202 CRPs and trainees have participated since launch (2020), including 17 trainee participants from Durham Technical Community College (2022–2024). Post-program evaluations showed significant growth in recruitment and retention self-efficacy. An early evaluation of the first 2 cohorts (n = 59) included a self-assessment across defined competencies showing marked increases in comfort across all learning objectives, with notable gains in: Community and Stakeholder Engagement, Recruitment on a Shoestring Budget, Community-Engaged Research Initiatives, and Social Marketing. Participants valued the program’s focus on EDI and sought more practical strategies and peer collaboration. 50 additional institutions have engaged with our implementation consultations and program repository. Discussion/Significance of Impact: Barriers to equitable ER&R exist at the individual, study, and system levels. Addressing these requires more intentional engagement practices. The ER&R certificate program is an innovative model for integrating equity principles with practical and required knowledge and skills training for participant-facing research professionals.

Objectives/Goals: To probe microbe and bacterial extracellular vesicle (bEV) mobility through biological barriers, we use novel multiple-particle tracking technology. The goal is to evaluate changes caused by extracellular vesicles relevant to placental function and neonatal development. Methods/Study Population: We conducted multiple particle tracking to assess whole bacterial and bEV mobility in cervicovaginal mucus. To accomplish this, cervicovaginal mucus was self-collected from 10 women. Mucus samples were characterized via wet mount, Nugent score, and pH measurements. In parallel, we cultured commercially available vaginal bacteria strains in anaerobic conditions. We isolated bEVs via ultracentrifugation, and subsequently characterized them via nanoparticle tracking analysis to measure size, ζ-potential, and concentration. We investigated reproductive tract tissues response to bEVs. We dosed vaginal, endometrial, myometrial, and placental cells lines with bEVs over a 24 h period and determined uptake, viability, and cytokine production. One-way analysis of variance was used for statistical analysis. Results/Anticipated Results: Based on our previous work, size and ζ-potential greatly affect particle mobility in mucus. G. vaginalis and M. mulieris were smaller than L. crispatus and L. iners. G. vaginalis had a more net-neutral ζ-potential compared to other bEVs. During multiple-particle tracking analysis, whole bacteria were unable to diffuse through vaginal mucus, while bEVs showed increased mobility. Through fluorescence levels, we determined M. mulieris bEVs reach >90% uptake at 24 h. Uptake was verified via microscopy. Across all strains, bEVs were not detrimental to placental viability. When investigating cytokine production in placental cells, an increase in IL-6 was seen after treatment with L. iners bEVs, while TNFα was increased after treatment with G. vaginalis bEVs. Discussion/Significance of Impact: Vaginal microbiome dysbiosis increases adverse obstetric indications. We demonstrate that bacteria are unable to ascend to reproductive tissues. We propose that bEVs travel through vaginal mucus, facilitating microbe–host communication. This impacts obstetric disease pathology and is relevant for diagnostic criteria during pregnancy.

Objectives/Goals: Large-scale tumor sequencing efforts have led to annotations of novel cancer hotspot mutations that may underlie driver or cooperative function. We have sought to define the molecular consequences of such hotspots associated with pediatric DICER1 syndrome cancers, with the ultimate goal of revealing novel targets that may inform new standards of care. Methods/Study Population: We have performed genomic analysis to identify tumor types (in TCGA and MSK-IMPACT patient data) for which mutations in the Dicer1 gene (encoding Dicer protein) emerge as the dominant signature of driver function. As Dicer is a critical RNA processing factor responsible for the generation of microRNAs, which are posttranscriptional gene regulatory molecules, we have modeled these mutations in human embryonic stem cells in order to study the direct effects on miRNAs and their target genes in an isogenic background. In addition to providing the required setting for unambiguous attribution of function to specific mutations, clonal human ES cells offer an opportunity for modeling of both developmental and cancer requirements associated with altered Dicer function. Results/Anticipated Results: Through generation of genomics and functional datasets from matched genotypes in Dicer mutated human ES cells, we have identified specific alterations in miRNAs and their effects on target genes. Unexpectedly, we found direct evidence for both loss of function and gain of function attributable to Dicer mutations. In addition, through integrated analysis of genomic data from tumor sequencing datasets and our human ES cell models, we have identified potential miRNA and target gene alterations that underlie tumorigenic potential, nominating gene candidates for targeted therapy in DICER1 syndrome. Direct mouse modeling of such candidate gene targets has revealed evidence for driver function of identified miRNA and their targets. Discussion/Significance of Impact: DICER1 syndrome cancers comprise a wide variety of rare pediatric tumor types. Presently, we still lack an effective standard of care. Furthermore, the previous lack of molecular profiling precluded targeted therapy opportunities. Our precise knock-in modeling of Dicer hotspots and deep profiling of relevant tumors now provide candidate targets.

Objectives/Goals: Primary sclerosing cholangitis (PSC) manifests with an inflammatory milieu that leads to fibrotic scarring of the liver. Human PSC liver bile ducts are enriched with neutrophils; however, their infiltration and functional role is unexplored. Our goal is to investigate the mechanism and impact of peribiliary neutrophil infiltration observed in PSC. Methods/Study Population: Primary cholangiocytes (bile duct cells) isolated from WT and mouse models of PSC (3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice and Mdr2-/- mice) were analyzed by RNA-sequencing. Immunofluorescence (IF) was performed on liver tissues from PSC patients and mouse models of PSC for markers of bile ducts and neutrophils (KRT19 and MPO). Intrahepatic leukocytes (IHL) isolated from mice livers were evaluated for neutrophil abundance and activation state. Anti-Ly6G antibody-mediated neutrophil depletion in Mdr2-/- mice was analyzed by IF, histology, and cytometry by time-of-flight (CyTOF). Cholangiocytes stimulated with TNFα (to induce an inflammatory phenotype) were analyzed for neutrophil chemoattractants with genetic and pharmacological interventions. Results/Anticipated Results: RNA-seq analysis of primary cholangiocytes from PSC mouse models revealed enrichment in inflammatory and neutrophil degranulation pathways. Flow cytometry and RT-PCR analysis revealed an increase in the neutrophil population in PSC mice with activated phenotype. Peripheral depletion of neutrophils in Mdr2-/- mice alleviated liver injury and inflammation, along with a reduction in peribiliary neutrophil infiltration and attenuated bridging fibrosis. CyTOF analysis showed a significant reduction in CD8+ T cells upon neutrophil depletion, implying neutrophils sustain CD8+ T cells in PSC liver. Mechanistically in cholangiocytes, TNFα mediates expression of neutrophil chemoattractants, CXCL1 and CXCL8, through the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway. Discussion/Significance of Impact: Our findings suggest that activation of the STING pathway in cholangiocytes in cholestatic liver disease triggers an immune response resulting in peri-portal neutrophil infiltration via CXC chemokines. The sustained presence of these activated neutrophils engages the adaptive immune system to perpetuate the inflammation and fibrosis seen in PSC.

Objectives/Goals: Depression is common among people living with HIV (PLWH). This study explored the link between reduced metacognitive awareness and depression in PLWH. It utilized a positive emotion regulation task to compare brain activation during viewing versus upregulating positive emotions. Methods/Study Population: Depressed PLWH (N = 24; mean age  =  53; HAM-D mean  =  19) participated in an emotion regulation task while blood oxygen-level-dependent (BOLD) responses were recorded. In the emotional regulation task, participants were shown the International Affective Picture System (IAPS) a series of positive, negative, and neutral images. Participants were asked to view these images and given instructions to either negatively reappraise (RN) or positively reappraise (RP). In the RP condition, participants were no longer shown the image and asked to upregulate their positive emotional responses associated with it. Ten onset times were included for each trial. Results/Anticipated Results: A one-sample t-test was conducted to analyze contrasts between reappraisal of positive images and viewing positive images (RP > VP). Results showed significantly greater activation in the posterior cingulate and angular gyrus during the RP condition (peak MNI: 18, -52, 34; p < 0.001, uncorrected, k > 10 voxels). In comparing the reappraisal of negative images to viewing negative images (RN > VN), there was increased activation in the right supramarginal gyrus (peak MNI: 50, -28, 22; p < 0.001, uncorrected, k > 10 voxels). When contrasting the reappraisal of positive to negative images (RP > RN), BOLD signals were higher in the left dorsolateral prefrontal cortex (peak MNI: 40, -38, 32; p < 0.001, uncorrected, k > 10 voxels). Discussion/Significance of Impact: Findings underscore that depressed PLWH demonstrates BOLD responses in brain regions linked to appetitive motivation and meta-cognitive awareness during the RP condition which demands more executive resources among those with depression, highlighting the complexity of emotional regulation in this population.