ABSTRACT IMPACT: This study has uncovered a novel surprising mechanism involving the epithelial adherens junctions and transposon regulation that can deepen our understanding of tumorigenesis. OBJECTIVES/GOALS: Recent studies show that genomic instability in 50% of tumors can be attributed to increased transposon activity, but the the reasons for this activity are unknown. We have evidence of a novel mechanism linking adherens junctions with transposon regulation. We hypothesize that adherens junctions suppress transposons to maintain genomic integrity. METHODS/STUDY POPULATION: We observed co-localization of PIWIL2 with adherens junction components of well differentiated epithelial breast, kidney and colon cell lines MCF10A, MDCK and CACO2, respectively, through immunofluorescence staining, confocal microscopy, and co-immunoprecipitation studies. Breast cancer cell lines MCF7 and MDA-231 were also observed using immunofluorescence to determine the localization of PIWIL2 in cancer cell lines. shRNA knockdown of PIWIL2 in MCF10A cells, followed by western blot, immunofluorescence, and qRT-PCR was performed to confirm the knockdown, observe if transposons were upregulated, and determine the extent of DNA damage to the genome by the marker gamma-H2AX. RNA-seq will be performed to determine piRNA sequences and possible targets of PIWIL2. RESULTS/ANTICIPATED RESULTS: Our data have revealed an interaction of E-cadherin and p120 catenin, core components of adherens junctions, with PIWIL2, a member of the Argonaute family of proteins and a key component of the piRNA processing pathway that is responsible for transposon silencing. piRNAs (PIWI-interacting RNAs) are a distinct class of small RNAs that bind to PIWI proteins, and aid in transposon degradation. We found co-localization of PIWIL2 with E-cadherin and p120 catenin at adherens junctions of well-differentiated epithelial cells, whereas this association was lost in cancer cells. Furthermore, our data show that E-cadherin depletion results in mis-localization of PIWIL2 and TDRD1, another member of the PIWI complex. E-cadherin depletion also results in upregulation of transposons and ?-H2AX, an indicator of DNA damage. DISCUSSION/SIGNIFICANCE OF FINDINGS: Since both loss of junctional integrity and increased transposon activity are universal events in cancer, this study has the potential to further our understanding of the causes of tumorigenesis. Understanding the mechanisms of transposon regulation has the potential to lead to a therapeutic target in the future.

OBJECTIVES/GOALS: While mutant TP53 is an attractive therapeutic target in TNBC, attempts to target the mutant p53 protein directly have failed. Thus, we aim to identify pathways critical for the survival of TP53 mutant cells that can be targeted in TNBC. We have identified Kif11 as one such target and aim to further investigate its function in TP53 mutant TNBC. METHODS/STUDY POPULATION: We conducted a dual in silico/in vivo screen that identified Kif11 inhibition as preferentially inhibiting the growth of TP53 mutant TNBC. We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from TCGA, METABRIC, and CCLE datasets. We treated breast cancer cell lines with the KIF11 inhibitor SB-743921. Cell counts were obtained through staining with DAPI or Hoechst and imaging on the ImageXPress PICO. We detected cell death by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. To investigate mitotic spindle organization, we performed immunofluorescent staining with an anti-tubulin antibody and DAPI co-staining. Cell cycle analysis was performed through flow cytometry. RESULTS/ANTICIPATED RESULTS: KIF11 is highly expressed in TP53 mutant and TNBC clinical samples. High KIF11 expression is associated with poorer clinical outcomes. Kif11 inhibition suppresses growth of both TP53 mutant and wild-type breast cancer cells, but preferentially induces the death of TP53 mutant cells as detected by DRAQ7 and Annexin V/PI staining. Kif11 inhibition induces a G2-M block and growth inhibition in TP53 wild-type cells. On the other hand, following treatment with the Kif11 inhibitor SB-743921, TP53 mutant cells undergo mitotic spindle dysfunction leading to the formation of multinucleated cells and cell death. DISCUSSION/SIGNIFICANCE: These results demonstrate that Kif11 is a promising therapeutic target in aggressive, TP53 mutant TNBCs. Kif11 inhibitors, including SB-743921, have been tested in human trials, and are well tolerated, but it is unclear which patients would most benefit. Our studies show that Kif11 inhibitors may be most useful in patients with TP53 mutant TNBCs.

OBJECTIVES/GOALS: Stress is amplified in parents of children with Down syndrome (DS). However, little is known about caregivers’use of coping strategies in relation to stress-related eating and their child with DS. The purpose of this study is to explore stressors and coping resources that increase or decrease stress-related eating among caregivers of children with DS. METHODS/STUDY POPULATION: This descriptive qualitative study sought to understand and describe the lived experiences of caregivers of children with DS. Fifteen caregivers of children, ages 2-6-years with DS, completed an online survey and individual interview. The Transactional Model of Stress and Coping was used to develop the interview guide and to assess related constructs using the Mindful Eating Questionnaire (MEQ), Perceived Stress Scale (PSS), Salzburg Stress Eating Scale (SSES), and a subscale of the Eating and Appraisal Due to Emotions and Stress Questionnaire (EADES). Interviews were audio-recorded, transcribed verbatim, coded using NVivo 12, and analyzed using deductive thematic analysis based on theoretical constructs and content analysis approaches. RESULTS/ANTICIPATED RESULTS: Thirteen of the 15 participants reported moderate to high perceived stress on the PSS. Stressors identified by caregivers included caring for a child with DS, feeding difficulties, physical and mental health, family and social interactions, and finances. Caregivers utilized various interpersonal resources to cope, including family, friends, and social media groups. In the interviews, stress-related eating was revealed as a coping mechanism for 9 caregivers. Also, 13 caregivers reported eating more or eating less when stressed on the SSES and 7 caregivers reported increased emotional eating as measured on the sub-scale of the MEQ. Only two caregivers described using mindfulness strategies throughout the interview. DISCUSSION/SIGNIFICANCE: Overall, caregivers of children with DS report increased perceived stress related to their role as a caregiver of a child with DS, which may be related to changes in caregivers’dietary intake. Dietary interventions developed for these families should include mindful eating and stress management strategies.

OBJECTIVES/GOALS: Team science competencies are not well-defined for nonfaculty staff of Clinical Research Professionals (CRPs) who conduct research. Using an existing framework, our work has determined skills associated with team science competencies as related to CRPs. Our team also outlined examples of those skills on a fundamental, skilled, and advanced level. METHODS/STUDY POPULATION: The team consists of both CRPs and those working in the Team Science space. This team used a modified Delphi approach to determine the skills and leveling examples of each team science competency. The team broke into four groups and was assigned 3-4 competencies each. Each group determined skills needed to support (exhibit, promote) each competency and then described an example of this skill at the fundamental, skilled, and advanced levels. Once each group was finished with their assigned competencies, they were re-assigned to a different group for review and changes. Finally, team science and CRP experts reviewed the skills and levels. RESULTS/ANTICIPATED RESULTS: Our results are a rubric that defines 3-5 practical skills per described competency. These skills are needed to support and promote each competency as a CRP. An additional outcome from this work includes examples of each skill at the fundamental, skilled, and advanced levels in a CRP’s career. Each leveled example is described in a concise, actionable way using Bloom’s taxonomy. This rubric is meant to be easily understood, very useable and able to be used in conjunction with existing CRP competency frameworks. By using Bloom’s taxonomy, we set the stage for future educational programming in Team Science skill-building for clinical research professionals. DISCUSSION/SIGNIFICANCE: Team science concepts and competencies have been increasingly integrated into translational science teams. However, team science competencies related to CRPs have remained largely undefined. Our work helps to define these competencies for CRPs in a practical way. Our rubric fills gaps in, and builds on, existing CRP competency frameworks.

OBJECTIVES/GOALS: Illuminate processes and findings of the Environmental Scan of Adaptive Capacity and Preparedness of CTSAs done by the CTSA Working Group. Share challenges, strategies, and lessons learned for CTSAs to build Capacity to address clinical and translational barriers while responding to emergencies. METHODS/STUDY POPULATION: An Environmental Scan approach for searching, collecting, analyzing and using information from diverse sources regarding CTSA hubs experiences during emergency as related to research implementation, translation, support, adaptation, and preparedness: - Triangulating multiple data sources and mixed methods (e.g., literature review, document/RPPR analysis, and expert review); - Secondary analysis of the JCTS COVID-19 Survey of the CTSAs: challenges, lessons learned, and practices that work in various program components/areas; - Using feedback of CTSA professionals from multiple disciplines to enhance our knowledge of emergency preparedness and efficient adaptation to public health and other crises. RESULTS/ANTICIPATED RESULTS: The E-scan approach helped identify challenges, successful practices, and evidence-based strategies for building adaptive capacity and preparedness of CTSAs across various scientific sectors of the translational science spectrum. Some of the findings include: - Roadmaps for the creation of new collaborative research resources (biobanks; data repositories, etc.); - Rapid clinical and research decision making during public health crises; - New community-based research strategies to facilitate communication, research dissemination, and participant recruitment based on existing trust-based networks; - Innovative resource allocation to guarantee continuity of training and research opportunities for trainees. DISCUSSION/SIGNIFICANCE: The Environmental Scan of the Adaptive Capacity of CTSA hubs provides useful knowledge and tools to diverse clinical research stakeholders for mitigating the impact of a disaster via adjusting programs, practices, and processes, and building capacity for effective, emergency-ready and responsive research, training, and community engagement.

Attacks on minoritized communities and increasing awareness of the societal causes of health disparities have combined to highlight deep systemic inequities. In response, academic health centers have prioritized justice, equity, diversity, and inclusion (JEDI) in their strategic goals. To have a sustained impact, JEDI efforts cannot be siloed; rather, they must be woven into the fabric of our work and systematically assessed to promote meaningful outcomes and accountability. To this end, the University of Pittsburgh’s Institute for Clinical Research Education assembled a task force to create and apply a rubric to identify short and long-term JEDI goals, assess the current state of JEDI at our Institute, and make recommendations for immediate action. To ensure deep buy-in, we gathered input from diverse members of our academic community, who served on targeted subcommittees. We then applied a three-step process to ensure rapid forward progress. We emerged with concrete actions for priority focus and a plan for ongoing assessment of JEDI institutionalization. We believe our process and rubric offer a scalable and adaptable model for other institutions and departments to follow as we work together across academic medical institutions to put our justice, equity, diversity, and inclusion goals into meaningful action.

The Community Research Liaison Model (CRLM) is a novel model to facilitate community-engaged research (CEnR) and community–academic research partnerships focused on health priorities identified by the community. This model, informed by the Principles of Community Engagement, builds trust among rural communities and expands capacity for community and investigator-initiated research. We describe the CRLM development process and how it is operationalized today. We followed a multi-phase process to design and implement a community engagement model that could be replicated. The resulting CRLM moves community–academic research collaborations from objectives to outputs using a conceptual framework that specifies our guiding principles, objectives, and actions to facilitate the objectives (i.e., capacity, motivations, and partners), and outputs. The CRLM has been fully implemented across Oregon. Six Community Research Liaisons collectively support 18 predominantly rural Oregon counties. Since 2017, the liaison team has engaged with communities on nearly 300 community projects. The CRLM has been successful in facilitating CEnR and community–academic research partnerships. The model has always existed on a dynamic foundation and continues to be responsive to the lessons learned by the community and researchers. The model is expanding across Oregon as an equitable approach to addressing health disparities across the state.

OBJECTIVES/GOALS: Metabolic syndrome (MetSyn) is a risk for World Trade Center-Lung Injury (WTC-LI; defined as developing FEV1<lower limit of normal [LLN]). Metabolic health is a modifiable disease risk factor. We propose to characterize how time-dependent covariates of MetSyn are longitudinally associated with WTC-LI. METHODS/STUDY POPULATION: WTC-particulate exposed firefighters, consented, with pre-9/11 FEV1 LLN (N = 5,746). Data assessed from last pre-9/11 till August 1, 2017. Longitudinal MetSyn characteristics were assessed using 3 models: i. A linear mixed effect model to assess the effect size of longitudinal MetSyn and its components on longitudinal FEV1% predicted as an outcome; ii. a time-dependent Cox regression to assess the associations of MetSyn to time of onset of WTC-LI; iii. a novel, partially linear single index regression model with repeatedly measured MetSyn to assess their joint effects and delineate their relative contribution on the longitudinal lung function in the WTC-FDNY cohort. RESULTS/ANTICIPATED RESULTS: In Model I, BMI 30 kg/m2 had the largest effect size compared to ever-smoking, with −2.524 (95%CI: −2.708,−2.340) compared to −1.681(−2.325,−1.038) respectively. Having MetSyn, defined as 3/5 risk factors, had an effect size of −2.319(−2.526,−2.112). In Model II, hazards of triglycerides 150mg/dL were highest at 1.497(1.336, 1.677), followed by BMI 30 kg/m2 at 1.406(1.256, 1.575), and HDL<40mg/dL 1.355(1.176-1.561), compared to ever-smoking (1.201, p = 0.002). Having high exposure to PM by being present in the morning of 9/11 was a significant covariate only in Model II investigating HDL<40mg/dL or triglycerides 150mg/dL. Model III The proposed methods will be applied to our cohort study. DISCUSSION/SIGNIFICANCE OF IMPACT: MetSyn is both a predictor and concurrent marker of WTC-LI. The single index model can not only reduce dimensionality of the covariates, but also provides efficient estimates of the joint MetSyn effects, allowing linear or nonlinear effects. Future studies will investigate dietary intervention as a potential disease-modifying factor. CONFLICT OF INTEREST DESCRIPTION: NA, nothing to disclose.

Identifying and disseminating actionable intelligence is a challenging task that requires thoughtful planning. The National Center for Advancing Translational Sciences instituted the Common Metrics Initiative with the goal of evaluating the Clinical and Translational Science Awards (CTSA) Programs using a standard set of metrics. Initially managed by Tufts University, the Center for Leading Innovation and Collaboration (CLIC) at the University of Rochester began leading this initiative in 2017. In directing this work, CLIC created a framework for communicating and disseminating data insights. Insights to Inspire emerged from the need to share strategies and lessons learned to improve metric performance at the local level to a network of 60+ academic research institutions. Insights to Inspire employs a mixed methods approach for translating data into actionable intelligence. A series of blogs, webinars, and webcasts were designed to communicate metric-specific strategies used by individual sites to the broader CTSA consortium. A dissemination plan to expand the reach beyond metric stakeholders utilized focused communications including social media channels, network newsletters, and presentations at national meetings. This framework serves as a blueprint for other national evaluation programs interested in a systematic approach to using data insights for continuous improvement.

Health systems currently underutilize systematic reviews. Here, we describe a proof of concept project designed to augment the standard systematic review process by presenting qualitative information as a companion to a review on deprescribing interventions. We conducted a thematic analysis of semi-structured interviews with Veterans Health Administration clinicians and Veterans to describe first-hand experiences of engaging in the deprescribing process. Qualitative findings were incorporated into an interactive, web-based product designed to supplement the systematic review report. Preliminary evaluation suggests that integration of narratives as a companion to systematic reviews is of interest to frontline clinicians, researchers, and health system administrators.

OBJECTIVES/GOALS: High cholesterol is among the major causes of cardiometabolic complications. People with high cholesterol have about twice the risk of heart disease as people with lower levels. Approximately every 40 seconds, an American will have a heart attack. Costs related to Heart Attack exceed 60 Billion USA dollars per year. Renin-Angiotensin-Aldosteron System (RAAS) is implicated in the genesis of coronary heart disease and in the perpetuation of heart failure. Angiotensin-Converting Enzyme inhibitors (ACE-I) have emerged as the treatment of choice for patients with all degrees of heart failure. Many clinical trials (Consensus, 1987; Save 1990) provide the evidence that ACE-I preserves cardiac function, prevents cardiovascular death, myocardial infarction & stroke and limit remodeling after myocardial infarction. However, there are still controversies in cardiology and a debate over cardioprotection is continuing:

• Do ACE Inhibitors have unique properties, beyond their antihypertensive effect?

• Can we protect the heart during hypercholesterolemia?

• In which way hypercholesterolemia affects mitochondria bioenergetics?

• How does ramipril affect mitochondrial bioenergetics during the course of experimental hypercholesterolemia?

Objectives/Goals were: To evaluate the mitochondrial actions of chronically administered ramipril (non-SH-containing ACE inhibitor) in cholesterol-fed rabbits by determining the influence of ramipril on:

• myocardial oxygen consumption (State 4, State 3), Respiratory Control Ratio (RCR), and adenosine diphosphate - oxygen index (ADP/O) and

• oxidative stress biomarkers

METHODS/STUDY POPULATION: Animal treatments. In the course of twelve-week the male Chinchilla rabbits (n = 10/group) received once a day a single dose, in group: A - sunflower oil (control animals); B – atherogenic 2% hypercholesterolemic diet; C - atherogenic diet and ramipril (1 mg/kg) and D - ramipril (1 mg/kg) only. Animals were terminated in accordance with the U.K. “Animals (Scientific Procedures) Act.” Isolation of mitochondria - Mitochondria from rabbit heart were isolated by tissue digestion (trypsin), fractionation and differential centrifugation. Mitochondrial respiratory functional measures (State 4 - Basal; State 3 - ADP-stimulated respiration, RCR and ADP/O) and biochemical markers of oxidative damage (the nitrite level) were measured polarographically (Clark electrode, YSI, USA) and spectrophotometrically, respectively, in isolated heart mitochondrial suspensions. Statistics - All results are reported as means ± SD. Comparisons between ramipril treated and control animals were performed by unpaired t-test or one-way ANOVA with a Tukey adjustment for multiple comparisons. A P value < 0.05 was considered significant for all tests. RESULTS/ANTICIPATED RESULTS: Plasma cholesterol levels: After a period of 12 weeks

• Plasma cholesterol levels in control rabbits (A) were low (1.36 ± 0.23 mmol/l).

• Cholesterol-fed rabbits became hypercholesterolemic and their plasma total cholesterol level was higher even than 10 mmol/l. The level of total cholesterol in the high-cholesterol-diet group was significantly increased compared with the level in the normal-diet group (p < 0.01).

• In the high-cholesterol-diet group treated with ramipril (C), the plasma cholesterol level was not affected by the drug ramipril (10.54 ± 1.31 mmol/l). ACE-I ramipril did not infuence the concentration of total cholesterol.

• Plasma cholesterol levels in group D were low (1.46 ± 0.29 mmol/l).

• Atherogenic 2% cholesterol diet (B) caused a decline in mitochondrial function (in both, State 3 and 4) (−25%).

• Mitochondria from group C animals (treatment with ramipril along with 2% cholesterol diet) exhibited higher State 3 respiratory rates compared with group B.

• Mild inhibition of mitochondrial respiration was recorded in group D, in both respiratory states (V4&V3).

• In cholestrerol-supplemented hearts myocardial oxygen consumption was markedly reduced (State 4 and State 3) compared to controls. Administration of high-cholesterol diet decreased not only the respiratory activity of rabbit heart mitochondria (RHM), but also the sensitivity of respiratory chain to ADP (ADP/O), while concomitantly caused an increase in nitrite production. Possible explanation: high-fat diet affects the fluidity of mitochondrial membrane – Electron transport chain (ETC) may be damaged, and unable to support high rates of respiration (e.g. substantial cytochrome c could be lost).

• Administration of ACE-I ramipril along with cholesterol diet partially abolish reduction in MQO2 and improved coupling efficiency (ADP/O). Possible explanation: reduced coupling efficiency means the coupling mechanism itself is altered (e.g. the respiratory complexes slip and pump fewer protons than normal and less ATP is produced per oxygen consumed. Ramipril partially improved coupling efficiency and increased the amount of ATP per oxygen consumed.

• RCR - No significant difference between the groups were found. High RCR indicates good function (a high capacity for substrate oxidation and ATP turnover). Low RCR usually indicates dysfunction. However, there is no absolute RCR value that is diagnostic of dysfunctional mitochondria, because values are substrate- and tissue-dependent.

• NO• exerts metabolic control over mitochondrial respiration. Group B: The lowered state 3-respiration in heart mitochondria seems to contribute to the increased NO production, and elevated nitrite level.

• In a system as complex as OXPHOS, conclusions about overall efficiency must involve measurements of: mito membrane potential, proton transport, ATP synthesis and modular kinetic analisys.