OBJECTIVES/GOALS: We conducted a review of CTSA websites to understand the current landscape for CRP institutional professional development and training revealed in the CTSA hub websites. METHODS/STUDY POPULATION: We accessed and reviewed 59 currently funded CTSA hub websites for evidence of CRP training opportunities. Parameters reviewed included: 1) opportunities were specified for CRPs versus K and T trainees; 2) mandated training; 3) leveling; 4) delivery methods/resources; 5) public accessibility; 6) unique features. The website reviews informed a REDCap survey sent to the CTSA Administrators (n = 149) and the Coordinator Taskforce (n = 105) listservs to gain additional knowledge of CRP training available at the institution. A subsequent repeat review of the CTSA hub websites will be conducted to determine evolving trends. RESULTS/ANTICIPATED RESULTS: A total of 40 responded to the survey from 59 CTSA hubs. Survey results are being analyzed. Website review data are being tabulated and the subsequent review of websites will be collected in February. Those findings are pending and will include a comparison of prior findings. 42% of CRP hubs list CRP training within the CTSA hub website. Required onboarding training (beyond CITI certificates) is revealed for some hubs (15%). DISCUSSION/SIGNIFICANCE OF IMPACT: On our initial website review less than half of the CTSA hub websites list specific CRP training on their website. Many were hidden behind firewalls and could not be reviewed for content. The REDCap Survey will provide more granular descriptions of programs. Data from a second website review will be collected for comparison. Based on a preliminary re-review of sites, there is a suggestion of increasing CRP workforce development information. CTSAs are well-positioned to be a central hub for promoting educational excellence of the institutional workforce, for medical centers and in other venues where clinical research is performed.

OBJECTIVES/GOALS: Novel EEG source localization and functional connectivity will assess frontal and cerebellar activity as predictors of subsequent memory and executive functioning (EF) decline in healthy, asymptomatic older adults who carry the Apolipoprotein-E ε4 allele (ε4+), which conveys up to 12x increased risk for Alzheimers disease (AD). METHODS/STUDY POPULATION: Healthy, cognitively intact ε4+ older adults (n = 23; ages 65-89) completed neuropsychological testing (focus on memory and EF) and EEG at Time 1, returning an average of one year later for neuropsychological retesting. EEG data during successful stop-signal inhibitory control trials will be used for advanced source localization and functional connectivity, with a focus on frontal and cerebellar regions of interest (ROIs). Source analyses will focus on the N200 time window (~200-350ms) to assess conflict processing and P300 (~300-550ms) for performance evaluation. Connectivity analyses are frequency-based, and will focus on theta band connectivity to assess conflict processing and delta to assess performance evaluation. RESULTS/ANTICIPATED RESULTS: Using hierarchical linear regression models, the magnitude of source activation within ROIs and connectivity metrics between ROIs will be used to predict residualized change in memory and executive function metrics between Time 1 and Time 2. We anticipate that 1) greater, compensatory activation in frontal ROIs during the N200 window, and 2) less cerebellar activation during P300, will predict memory and executive decline over the retest interval. Decline will also be predicted by 3) greater inter-hemispheric frontal connectivity in the theta band (conflict processing) and 4) less frontal-cerebellar delta connectivity (performance evaluation). DISCUSSION/SIGNIFICANCE: At most, ~50% of ε4 carriers will develop AD. Thus, identifying which carriers will decline is crucial to enabling successful, early intervention. Cerebellar dysfunction and impaired connectivity may be among the earliest indicators of incipient AD. Cutting-edge cerebellar EEG may enable an accessible option for early discernment of AD risk.

OBJECTIVES/GOALS: The Translational Science Benefits Model offers an approach for evaluating the downstream health and social impact of research. Using the new Translating for Impact Toolkit of nine web-based tools, researchers can create free, secure accounts to plan, track, and demonstrate the impact of their work. METHODS/STUDY POPULATION: Development of the online toolkit includes 6 phases: 1) Review of existing tools, 2) Development of fillable PDF tool prototypes, 3) Pilot testing, 4) Development of web-based tools, 5) Usability testing, and 6) Refinement of web-based tools. First, we reviewed existing tools for measuring research impact. We then created prototypes of nine tools, published on the TSBM website, and pilot tested with researchers. Based on feedback and testing, we developed and launched web-based versions of the tools. We are currently conducting usability testing with researchers, which we will use to evaluate the ease-of-use and quality of the tools, identify areas for improvement, and refine the tools. RESULTS/ANTICIPATED RESULTS: Researchers can sign up for user accounts, create projects, invite collaborators and program administrators, and save progress as the complete the nine tools in the Translating for Impact Toolkit. The tools are divided into three steps: Plan (Roadmap to Impact, Benefits 2x2, Partner Mapper, and Team Manager), Track (Impact Tracker), and Demonstrate (Product Navigator, Case Study Builder, Impact Profile, and Dissemination Planner). The toolkit also includes a dashboard that provides a quick snapshot of translational impact for each project. The toolkit will help both individual translational scientists demonstrate the impact of their work and CTSA hubs evaluate impact of their projects. DISCUSSION/SIGNIFICANCE: The TSBM online toolkit is a free, secure, easy-to-use platform researchers can use to plan for, track, and demonstrate the impacts of their work. The toolkit provides a structured process that will help the next generation of scientists prioritize and promote translational impact in their work.

OBJECTIVES/GOALS: Research emphasizes the importance of play in early childhood to support social, emotional, and physical development. This study explores how the Prescription for Play (P4P) is executed in clinical contexts by analyzing implementation fidelity and contextualizing the adaptations, challenges, and facilitators to the program’s functionality. METHODS/STUDY POPULATION: This project is an ongoing multi-site case study. At the time of study completion in December of 2023, there will be over 40 clinical observations of pediatric well-child check (WCC) visits completed across 7 Federally Qualified Health Centers (FQHC) participating P4P, a play promotion program wherein providers discuss the importance of play in WCC visits and provide a free play kit. All visits are with children 18-36 months old, with a broad demographic spread across sites. Observations are recorded through a guided observation protocol informed by a standard implementation fidelity framework, conducted by 5 researchers. Through inductive thematic analysis, this study will analyze observations of WCC visits to understand the ways providers engage with P4P across sociocultural contexts within FQHCs. RESULTS/ANTICIPATED RESULTS: Preliminary analysis of clinic observations (N = 30) indicates the degree of implementation fidelity varies across sites, with particular variances between WCC visits conducted in English versus non-English languages (NEL). In NEL visits, there were discrepancies among indicators of quality of delivery and participant responsiveness. NEL visits were less likely to have the provider model play with the caregiver and far less likely to open the play kit given to the family. Providers in NEL visits were also less likely to discuss certain benefits of play like brain development and reduced screen time. Across all observations, providers “prescribed play” approximately half the time. As more observations are conducted, researchers anticipate seeing continued differences between English and NEL visits. DISCUSSION/SIGNIFICANCE: From preliminary analysis, discrepancies in implementation fidelity indicate the P4P intervention may require adaptation and additional training related to how to prescribe and discuss play in WCC visits conducted in NEL visits. Additionally, this study elucidates the impact language can have on the fidelity of clinical interventions.

OBJECTIVES/GOALS: This project will design, develop and pilot-test a Spanish-mobile application for breast cancer patients. It is intended that patients will improve treatment adherence, have a personal information storage system for their follow-up healthcare and can have greater control in the processes of diagnosis, treatment, education, and recovery. METHODS/STUDY POPULATION: In the first phase, a team of undergraduate engineering students was recruited to work on the areas of: Front End, Database Development, Security Protocols, and Integration. Team Venus will provide the cancer-related content. In the second phase, the engineering team will identify bugs, usability, satisfaction, and workability of the application among a pilot convenience sample of 20 participants from four cancer clinics and the community. The inclusion criteria are being > 21 years old and having a cell phone. The exclusion criterion is not knowing how/being able to use a mobile application. Participants will complete a questionnaire on sociodemographic data, mobile phone and application use, efficacy, usability, and security. Univariate and bivariate statistical analysis will be performed using SPSS. RESULTS/ANTICIPATED RESULTS: The researchers are defining specific visual and content features they would like the application to have. They are also searching and collecting reliable information about cancer from primary sources to incorporate into the tools available to the application’s users. In addition, they have also begun to identify potential cancer treatment facilities and medical personnel to assist in the recruitment of patients for pilot-testing the application. The researchers have divided them into geographical areas in Puerto Rico and each Team Venus member will approach and orient patients and medical personnel about the project. DISCUSSION/SIGNIFICANCE: It is intended that patients will improve treatment adherence, have a storage system for their follow-up healthcare and greater control in their processes of diagnosis, treatment, education, and recovery. Future goals include use of the database feature by researchers and expanding the testing of the application with a larger patient-base.

OBJECTIVES/GOALS: This project examines the initial feasibility, acceptability, and appropriateness of using population-health data and geospatial information systems (GIS) technology to guide opioid overdose prevention in a Black community facing increasing racial inequities in fatal overdose since 2016. METHODS/STUDY POPULATION: We applied a mixed-methods approach, using GIS to examine hot spots of fatal opioid overdose among Black, adult decedents in St. Louis City and County, Missouri, derived from cause-of-death data from the Medical Examiner’s Offices from 2016-2021 (N = 1,612). Hot spot analysis was conducting using the Getis-Ord Gi* statistic. We then conducted a focus group with community-based providers in Black neighborhoods (N = 8) to assess the acceptability and appropriateness of mapping overdose data and to determine the utility of GIS for guiding decision-making in outreach activities aimed at overdose prevention. The focus group was recorded and transcribed for analysis to extract themes. RESULTS/ANTICIPATED RESULTS: Among 340 census tracts in the St. Louis region, 41 were statistically significant hot spots, indicating high clustering of fatal overdose. All hot spots were clustered together in North St. Louis, an area characterized by high racial and income segregation. Focus group participants reported potential pitfalls of GIS, including data inaccuracy for Black individuals and unwanted attention in hot spot areas (e.g., increased policing). Potential benefits included improved care coordination between organizations, assessing effectiveness of outreach interventions, and improving community-level awareness. Hyper-local neighborhood rather than regional maps were perceived as useful for outreach. DISCUSSION/SIGNIFICANCE: Artifacts of structural racism that drive Black opioid overdose deaths limit the utility of GIS to guide outreach in communities facing this systemic disinvestment. However, neighborhood-level GIS may serve as a tool for communities to build awareness and collaboration toward overdose prevention.

OBJECTIVES/SPECIFIC AIMS: N/A. METHODS/STUDY POPULATION: N/A. RESULTS/ANTICIPATED RESULTS: N/A. DISCUSSION/SIGNIFICANCE OF IMPACT: There is an increasing need to foster cross-disciplinary research to address complex problems within healthcare. The Sinai Team-based Translational Education Program: the URM Propeller (STTEP-UP) is a NCATS funded program through the Icahn School of Medicine at Mount Sinai. Its goal is to facilitate URM post-doctoral trainees becoming innovative leaders in clinical and translational research. The program includes a team-based research component, where fellows collaborate on a project. This year, disciplines represented by the four fellows include Cardiology, Psychiatry, Neurology, and Pediatrics. Identifying a clinical question and designing an investigation was facilitated by group brainstorming meetings with program mentors. Fellows designed a project to identify medical testing and prescribing that were not clinically indicated throughout the healthcare system, with the goal of exploring whether an intervention, including provider education, could reduce ordering practices. In addition to regular in-person meetings, a licensed virtual learning environment and free web-based sharing platform were used to foster collaboration. Challenges faced throughout this process, included fellows struggling to find protected time, difficulties accessing broad sets of data across the healthcare system, and overcoming administrative barriers between departments. Strengths of this approach, included fellows learning new research strategies and feeling a deeper sense of commonality with their peers. Overall, this experience supports the idea that cross-disciplinary research improves the collaboration and education of emerging researchers. However, addressing logistical and systems-based barriers may better facilitate this education and research.

OBJECTIVES/GOALS: By establishing a youth-centric web-app as a central hub of information and inspiration in an attempt to engage a young demographic, this project aims to increase community awareness and reduce misconceptions surrounding clinical trials, in hopes of fairly representing marginalized communities among future clinical trial participants. METHODS/STUDY POPULATION: We designed a children’s web-app to host a collection of child-friendly educational materials (such as picture books, games, and age-appropriate articles about advances in clinical research) explaining clinical research and its process. An emphasis was put on ensuring the web-app and its contents were understandable and appealing to children. The effectiveness of this tool will be tested through a focus group study. Children ages 7-10 will be given a preliminary survey measuring their knowledge and opinions about clinical research, and then given time to explore the web-app. Afterwards, they will be given a secondary survey to gauge their acquired knowledge from the website and asked about their opinion on the design and usability of the web-app and its materials, as well as how likely they were to revisit the site. RESULTS/ANTICIPATED RESULTS: We anticipate a very positive response from the children regarding the design and usability of the web-app and its materials. By using an adolescent-focused design methodology at every step of the design process, we will ensure that all materials are attractive and engaging to our younger target audience. Exposing children to accessible information about clinical trials at a young age allows us to build their trust in the research process prior to the possible internalization and acceptance of cultural misconceptions. Over time, we hope to see a change in attitudes toward clinical research as well as increased participation, whether from under-represented groups or a younger demographic, and positively contribute to T3 and onwards in the translational continuum. DISCUSSION/SIGNIFICANCE: In a rapidly changing world, the best approach to making change is through targeting the younger demographic, the leaders of tomorrow. Our project will allow adolescents to foster a more well-rounded opinion of clinical research, increasing their participation and better paving a more positively received future for translational science as a whole.

OBJECTIVES/SPECIFIC AIMS: Endogenous RT (eRT) is necessary for the function of retrotransposons, elements that replicate via an RNA intermediate. One source of eRT activity is long interspersed elements (LINE). LINEs, of which there are several subgroups (L1, L2, L3), are retrotransposons that regulate cellular growth and gene expression. Given their diverse and important roles, we hypothesized that L1 elements regulate functional responses in megakaryocytes and platelets; a concept not yet examined in the field. METHODS/STUDY POPULATION: To study eRT in human platelets we used RT activity assays, PCR, and Western blot approaches. Furthermore, we used an RT-inhibitor to dissect the function of eRT, analyzed RT-dependent protein synthetic capacity, and immunoprecipitated RNA-DNA hybrids. RNA-DNA hybrids were also detected by means of ICC and automated analysis using CellProfiler software. RNA-DNA hybrids were validated by PCR and eRT regulated synthesis of target proteins was analyzed using autoradiography and Western blot techniques. Platelets from patients with HIV+ were examined in parallel. RESULTS/ANTICIPATED RESULTS: We identified that highly purified, isolated platelets from healthy subjects possess eRT activity. eRT activity was blocked with the non-nucleoside RT inhibitor nevirapine at concentrations within the therapeutic drug range. L1 elements are bicistronic, containing 2 open reading frames (ORFs), ORF1 and ORF2. Thus, we next identified that human platelets express full-length L1 mRNA containing ORF1 and ORF2. In human platelets, eRT activity was localized to L1 protein containing ribonucleo particles. Platelet eRT reverse transcribed exogenous RNAs, a process inhibited by nevirapine, acting in trans using the 3′-UTR of exogenous mRNAs as a template. To dissect the function of eRT in platelets, we next examined cytoskeletal and protein synthetic events in the presence or absence of nevirapine. Inhibition of eRT in isolated platelets led to characteristically beaded platelets in appearance, strongly resembling bone marrow proplatelets. Parallel increases in platelet reactivity were also observed. As these changes occurred over hours, not minutes, we hypothesized that inhibition of eRT would affect platelet protein synthetic events. Consistent with this, RT inhibition resulted in upregulation of global platelet protein synthesis. We validated upregulation of the synthesis of specific proteins (mitofilin, p-selectin, and L26—a component of the 60S ribosomal subunit essential for mRNA translation). RNA-DNA hybrids, noncanonical nucleic acid structures that regulate gene expression, are enriched in regions where L1 is abundant. RNA-DNA hybrids were present in platelets and expression confirmed via differential digestion of RNAs (eg, with RNase A and RNAse I). Next-generation sequencing of pulled down (eg, immunoprecipitated) platelet RNA-DNA hybrids identified numerous differentially expressed transcripts and we focused on MAP1LC3B (LC3B), a primary regulator of autophagy. Hybrid sequencing results for LC3B were validated using qPCR and we confirmed that LC3B RNA binds to L1-encoded RNA binding protein. Platelets treated with nevirapine had increased total LC3B protein expression. As RT inhibition is an important mechanism to control HIV infection, we examined platelet morphology, activation, and LC3B expression in platelets from HIV+ subjects treated with nevirapine. HIV+ patients treated with RT inhibitors had higher numbers of platelets that were beaded in appearance at baseline, increased platelet reactivity, and differential LC3B expression compared with healthy controls. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, these results demonstrate that platelets possess eRT activity that regulates platelet morphology, platelet hyperreactivity, and protein synthetic events. We postulate that eRT activity in platelets may be a new post-transcriptional regulatory checkpoint. Moreover, our findings have implications in HIV+ patients treated with RT inhibitors, where off-target effects may contribute to platelet activation and an increased risk of thrombosis.

OBJECTIVES/GOALS: Young women (18 – 45 years of age) with breast cancer have complex medical and psychosocial needs. Educational materials are often used as tools in patient-centered communication. However, these materials disseminate complex health information in print-heavy formats and can be difficult to understand for women with varying health literacy levels. METHODS/STUDY POPULATION: In the first phase of this study, the principal investigator (PI) will recruit 40 diverse women to participate in four focus groups (FG) to explore the perceived usefulness of the most frequently used cancer educational materials. The PI will also obtain demographics and heath literacy levels of the FG participants using the Newest Vital Sign. In the second phase, the PI will assess the literacy demands of the ten most frequently used cancer educational print materials and five most frequently used websites described by the FG participants. The perceptions of the usefulness of materials and the literacy demands will then be used to appraise the effectiveness of materials within patient-centered cancer communication. RESULTS/ANTICIPATED RESULTS: Results from this study will provide a patient-centered blueprint that will be used to design more effective educational materials that treatment centers can incorporate into their patient-centered cancer communication process. The next step of this research will be to determine providers’ perceptions of cancer education materials used to exchange information within the patient-centered communication process. This will complement the patient findings and inform the development of the provider aspect of a communication intervention centered on designing educational materials for women with various health literacy levels within the patient-centered cancer communication process. DISCUSSION/SIGNIFICANCE OF IMPACT: Detecting the usefulness of cancer educational materials, as perceived by young women with breast cancer, is foundational to developing communication interventions that improve cancer outcomes. This study will identify how materials can be improved in the critical informational-exchange component of the patient-communication process.

OBJECTIVES/SPECIFIC AIMS: The overall goal of this project is to determine whether timing of seasonal influenza vaccination affects the severity of illness in vaccinated individuals who are hospitalized with influenza. This will be assessed with the following aims: 1. To determine whether differences in demographic and clinical characteristics exist among patients with short duration between seasonal influenza vaccination and influenza-related hospitalization and those with longer duration. 2. To determine whether time between seasonal influenza vaccination and hospitalization is associated with the duration of influenza-related hospitalization. 3. To determine whether time between seasonal influenza vaccination and hospitalization is associated with the rate of influenza-related ICU admission among patients hospitalized with influenza. 4. To determine whether time between seasonal influenza vaccination and hospitalization is associated with the rate of influenza-related death among adults hospitalized with influenza. METHODS/STUDY POPULATION: The Influenza Hospitalization Surveillance Network (FluSurv-NET) is a surveillance platform of influenza-related hospitalizations through the CDC Emerging Infections Program (EIP). FluSurv-NET conducts active surveillance for influenza-related hospitalizations of both children and adults in selected counties in California, Colorado, Connecticut, Georgia, Maryland, Michigan, Minnesota, Ohio, Oregon, New Mexico, New York, Tennessee, and Utah with a total catchment population of over 27 million people (~9% of the US population). Using this platform, we will retrospectively evaluate four influenza seasons using FluSurv-NET data to look at the timing of influenza vaccination and severity of illness among patients with influenza-related hospitalization. We will conduct a multivariate analysis to assess for differences in severe outcomes including duration of hospitalization, ICU admission, and death among patients with varying lengths of time between influenza vaccination and influenza-related hospitalization. Separate analyses will be performed among different age groups and influenza type/subtypes, as well as specific seasons as a surrogate for most common circulating strain. RESULTS/ANTICIPATED RESULTS: We hypothesize that patients with chronic medical conditions and those at the extremes of age will have a longer duration between vaccination and hospitalization as they are more likely to get vaccinated earlier. We also hypothesize that patients with longer duration between seasonal influenza vaccination and hospitalization will have a longer duration of hospitalization and a higher rate of other severe outcomes (e.g., ICU admission, death). Such data would suggest that immune protection wanes during the influenza season. DISCUSSION/SIGNIFICANCE OF IMPACT: Limited data suggest that vaccine-induced influenza immunity may wane during the influenza season. It is not known whether the impact of influenza vaccination upon severity of disease might wane with increasing time between vaccination and influenza infection. In contrast to many previous studies evaluating vaccine effectiveness which have assessed medically-attended influenza illness as a primary outcome, our dataset is a large cohort of hospitalized patients which allows us to assess rare yet critical outcomes such as ICU admission and death. This study will also have a substantially larger amount of pediatric data than previous studies, which will provide the opportunity to determine whether timing of vaccination affects children and adults differently. Improving our understanding of whether influenza vaccine-induced protection might wane over time could ultimately impact U.S. influenza vaccination policy resulting in decreased morbidity and mortality attributed to influenza each season.

ABSTRACT IMPACT: This work will accelerate the translation of post stroke rehabilitation devices from the research lab to clinic use. OBJECTIVES/GOALS: Rehabilitation device efficacy alone does not lead to adoption into clinical practice. The objective of this work was to increase understanding of the landscape for clinical adoption of post-stroke physical rehabilitation devices. METHODS/STUDY POPULATION: We conducted interviews with 107 stakeholders including patients who have had strokes, rehab directors, and physical/occupational therapists to understand their viewpoints for adopting new rehabilitation devices. To contribute to previous literature, interviews were analyzed qualitatively using direct content analysis to provide more specific details about the most appropriate adoption settings, specific roles for stakeholders, and drivers for all stakeholders involved in the adoption process. RESULTS/ANTICIPATED RESULTS: Unique to this work, care settings in which therapy goals are best aligned for restorative devices were found to be outpatient rehabilitation, followed by inpatient rehabilitation. Therapists are the major influencers for adoption because they typically introduce new rehabilitation devices to patients for both clinic and home use. We also learned therapists’ utilization rate of a rehabilitation device influences a rehabilitation director’s decision to acquire the device for facility use. Additionally, device setup in <7 minutes will allow for increased use without reducing therapist productivity. DISCUSSION/SIGNIFICANCE OF FINDINGS: Rehabilitation device development should consider the best settings to first introduce the device, roles of each stakeholder, and drivers that influence each stakeholder to accelerate successful adoption of the developed device.

OBJECTIVES/SPECIFIC AIMS: To review the multiple differences between traditional research design and on the ground pragmatic trials. To review two pragmatic projects, identify core assumptions and to contrast assumptions with the reality of conducting T3 and T4 research. METHODS/STUDY POPULATION: Observational mixed methods multi trial review of large multi site implementations. RESULTS/ANTICIPATED RESULTS: The complexities of implementation on the ground were consistently greater than anticipated and required changing assumptions and research design elements. DISCUSSION/SIGNIFICANCE OF IMPACT: Research findings are tremendously influenced by design and design implementation decisions. Anticipating the scope and breadth of the challenges will assist potential of successful implementation.

OBJECTIVES/GOALS: This study examined youth tobacco use, disaggregated by sexual and gender minority (SGM) identity and race, in Massachusetts before and after the state implemented a flavored tobacco restriction. We assessed if the policy differentially impacted groups that have had higher rates of flavored tobacco use (i.e., SGM and African Americans [AAs]). METHODS/STUDY POPULATION: Data for this analysis came from the 2019 and 2021 Massachusetts Youth Risk Behavior Survey (YRBS), a biennial, national survey conducted among high school students, provided by the Massachusetts Department of Elementary and Secondary Education. Changes in current use of cigarettes and e-cigarettes between 2019 and 2021 were examined for the entire sample and by SGM identity and race/ethnicity. Current cigarette use and current e-cigarette use were defined as reporting any use of the product in the past 30 days. We received confirmation from the IRB that because the data are de-identified and available to the public, this research is considered Not Human Subjects Research. RESULTS/ANTICIPATED RESULTS: Between 2019 and 2021, current cigarette use and current e-cigarette use decreased for the entire sample (3.78% to 2.79% and 27.69% to 15.74%, respectively). Decreases were also observed after disaggregating results, but smaller changes were observed among minoritized groups (i.e., SGM and AAs), particularly for e-cigarettes. Current e-cigarette use decreased 25.56% among individuals identifying as SGM (28.14% to 20.95%) compared to a 49.33% decrease among non-SGM individuals (27.63% to 14.0%). Among all races, AAs had the lowest prevalence of current e-cigarette use in 2019 (15.10%), but also saw the lowest percentage decrease (17.68%). Among whites, current e-cigarette use decreased 45.75% from 32.33% in 2019 to 17.54% in 2021. DISCUSSION/SIGNIFICANCE: After implementation of Massachusetts’ flavored tobacco restriction, current cigarette and e-cigarette use declined among Massachusetts youth overall and among groups that have been most affected by flavored tobacco. However, minoritized groups (i.e., SGMs, AAs) had lower percentage decreases compared to non-minoritized groups.

OBJECTIVES/SPECIFIC AIMS: (1) Correlate PBX1 mRNA expression as measured by RNAScope in situ hybridization, at an RNA number/cell measurement, Versus by RT-qPCR by the ddCt method. (2) Validate PBX1 mRNA expression in a second independent cohort of neuroblastoma tumor samples, and correlate with patient outcomes. We expect that PBX1 expression will correlate whether detected by RNAScope or by RT-qPCR. This work has the promise of validating a novel biomarker of disease severity, and for clinical translation as the RNAscope technology has been CLIA-certified for clinical use for other genes. METHODS/STUDY POPULATION: Primary neuroblastoma tumor samples were acquired through the Children’s Oncology Group Tumor Bank, The Cooperative Human Tissue Network Tumor bank, and the Westmeade Tumor Bank (Westmeade, Australia), with patient outcomes annotated but sequestered until experiments are completed. RT-qPCR is performed using 1 μg total RNA isolated from each sample by Nucleospin RNA kit (Clontech), reverse transcribed by SuperScript VILO (ThermoFisher Scientific) and amplified using KiCqSTART SYBR Green qPCR mix (Sigma Aldrich). RNAScope was performed on sections of fresh frozen tumor, in triplicate, per manufacturer protocol (ACDBio) using company-designed probes. Statistical analyses performed using GraphPad Prism5. RESULTS/ANTICIPATED RESULTS: PBX1 mRNA expression as measured RNAScope correlated well with matched RT-qPCR values, with most PBX1 transcripts identified within the malignant cells and not in tumor stroma. Correlation with patient outcomes is ongoing (expected to be available at the time of presentation), but as the RNAScope values correlate with R>0.9 with RT-qPCR values, we expect good correlation with outcomes in our primary data set and matching validation set. DISCUSSION/SIGNIFICANCE OF IMPACT: PBX1 mRNA expression is an accurate prognostic biomarker of outcome in low and intermediate-risk neuroblastoma, and testing on an additional validation set is planned based on thresholds established by RNAScope. RNAScope is a method readily translatable to clinical use and its inclusion in future clinical trials will be further studied. It provides an additional benefit that concomitant immunohistochemistry can also be performed. Analysis of high-risk neuroblastomas for responsiveness to retinoic acid based on PBX1 expression is planned.