OBJECTIVES/GOALS: Patients frequently need or desire fat grafting to improve common issues such as implant visibility and contour deformity, often done as a second, staged procedure following immediate reconstruction. This study aimed to identify which patient factors and reconstructive techniques predict the need for revision with AFG after IBBR METHODS/STUDY POPULATION: Patients who underwent IBBR with either tissue expanders or implants following mastectomy from 2017 to 2021 were identified. Demographics, comorbidities, and the postoperative course were reviewed. The primary outcome variable was AFG after the initial reconstruction. Univariate and regression analyses were performed to identify factors predictive of AFG. RESULTS/ANTICIPATED RESULTS: Five-hundred twenty-nine patients were included in our analysis, with 43% having AFG. Univariate regression displayed single-stage reconstruction (OR=0.53, 95% 0.37-0.75) and previous radiation (OR 0.59, 95% 0.35-0.99) negatively predicted the need for AFG, while bilateral breast reconstruction (BBR) was a predictor (OR 2.32, 95% 1.58-3.4). On multivariate analysis, decreasing age and BBR remained predictive of AFG. The odds of AFG decreased by 3% for every one-unit increase in age (95% CI [0.96, 0.99]). Interestingly, neither pre-pectoral breast reconstruction nor specimen weight:implant ratio was associated with increased need for AFG on univariate/multivariate analysis. DISCUSSION/SIGNIFICANCE: Patients requiring AFG were likely younger and had undergone BBR with tissue expanders. Plane of implant did not appear to affect need for AFG. Knowledge of these predictive factors may help plastic surgeons in preoperative counseling before implant-based breast reconstruction.

ABSTRACT IMPACT: Our work might lead to a new treatment for patients with acute myeloid leukemia OBJECTIVES/GOALS: Acute myeloid leukemia (AML) is a devastating hematologic malignancy, with dismal 5-year survival. Chimeric antigen receptor (CAR) T cells have been approved for B cell malignancies but not for AML. The goal of this study is to explore the safety and efficacy of CAR T cells targeting CD105 (endoglin) to treat AML. METHODS/STUDY POPULATION: We have constructed human and murine CAR T cells targeting CD105. The CARs were created by sequencing the V(D)J regions of hybridomas and designing single chain variable fragments that target CD105 which were subsequently introduced in a CAR backbone via Gibson assembly. The CAR T cells were produced via transduction using retrovirus or lentivirus. Leukemia cell lines were assessed for CD105 expression with flow cytometry. Killing assays were performed via measurement of luminescence of target cells after co-culture with CAR T cells. Activation assays were performed with co-culture of CAR T cells and target cells and measurement of activation markers with flow cytometry. To assess in vivo efficacy and safety, murine CAR T cells were infused into C57BL/6J mice carrying B16 melanoma after lymphodepletion. RESULTS/ANTICIPATED RESULTS: All human leukemia cell lines assessed (Nalm6, MOLM-14, MV4-11, Kasumi-1, THP-1) expressed some degree of endoglin apart from the T cell leukemia Jurkat. Human CD105 CAR T cells were activated by co-culture with leukemia cell lines and effectively killed leukemia cells in vitro in a CD105-specific manner. Murine CAR T cells killed efficiently both murine solid tumors (B16 melanoma) and murine leukemias (C1498) in vitro. Murine CAR T cells did not exhibit any toxicity when infused after low-dose lymphodepletion (cyclophosphamide 100mg/kg) but caused significant morbidity after higher doses (cyclophosphamide 200mg/kg). Murine CAR T cells delayed the growth of B16 melanoma in immunocompetent mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have constructed human and murine CD105 CAR T cells with excellent activity in vitro. The activity of human CD105 CAR T cells in xenografts and the biologic relevance of the toxicity of murine CD105 CAR T cells in humans needs to be further investigated. CD105 CAR T cells might prove an important therapeutic option for patients with AML.

This abstract is based on unpublished data. OBJECTIVES/GOALS: The estimates of unbiased first-degree relatives (FDRs) risk of cancers would enhance genetic counseling of at-risk FDRs in families where the pancreatic cancer (PC) proband carrying a germline variant. This study aims at quantifying gene-specific risks of six cancers among FDRs of PC patients with germline variants in cancer-associated genes. METHODS/STUDY POPULATION: In the prospective, clinic-based Mayo Clinic Biospecimen Resource for Pancreas Research registry, 4,562 PC patients had previously undergone germline genetic testing for pancreatic cancer-associated genes through either research studies or clinical testing. Of these, 234 PC probands were found to carry germline pathogenic/likely pathogenic variants (PLPV) among 9 genes of interest and had provided detailed demographic and cancer data on their FDRs by questionnaire. We focused on six cancer types (ovary, breast, uterus, pancreas, colon, and malignant melanoma) in FDRs as reported by the probands. Standardized incidence ratios were calculated to estimate risk of six cancers among FDRs of PC patients carrying PLPV by gene. RESULTS/ANTICIPATED RESULTS: 1,670 FDRs (mean age 58.1+17.8SD; 48.9% female) were included in the study. We found significantly increased risk of ovarian cancer in female FDRs of PC probands who carry PLPV in BRCA1 (SIR 9.49, 95%CI:3.06-22.14) or BRCA2 (3.72, 95%CI:1.36-8.11), and breast cancer risks were higher with BRCA2 (2.62, 95%CI:1.89-3.54). Uterine cancer risk was increased in FDRs of PC probands who carry PLPV for Lynch Syndrome mismatch repair (MMR) (6.53, 95%CI:2.81-12.86). PC risk was also increased (ATM 4.53, 95% CI:2.69-7.16; BRCA2 3.45, 95%CI:1.72-6.17; CDKN2A 7.38, 95%CI:3.18-14.54; PALB2 5.39, 95%CI:1.45-13.79). Increased colon cancer risk was observed in FDRs of probands who carried MMR PLPV (5.83, 95%CI:3.70-8.75), while melanoma risk was elevated for FDRs of probands with CDKN2A PLPV (7.47, 95%CI:3.97-12.77). DISCUSSION/SIGNIFICANCE: PLPV in nine syndrome-associated genes in PC probands are associated with increased risk of six cancers in FDRs. The findings underscore the importance of risk estimation of various other cancers in PC families for screening, early detection, intervention, and cascade genetic testing.

OBJECTIVES/SPECIFIC AIMS: This study aims to estimate patient attitudes and receptiveness towards stem cell-based cell replacement devices to management Type 1 Diabetes. The primary outcomes of this study are mean response values to questions interrogating patient attitudes, knowledge, and receptiveness. METHODS/STUDY POPULATION: A RedCap survey was generated for this study. 100 participants will be drawn from Mayo Clinic Rochester, MN patients living with Type 1 Diabetes. RESULTS/ANTICIPATED RESULTS: Response values will be used to estimate broader patient attitudes. DISCUSSION/SIGNIFICANCE OF IMPACT: The response values of this survey will help inform future directions of cell replacement device development. Additionally, understanding patient attitudes may be useful in crafting more effective education strategies.

OBJECTIVES/GOALS: Chronic or new symptoms after infection with severe-acute-respiratory-coronavirus-2 (SARS-CoV-2) has been termed post-acute sequelae of Covid-19 (PASC) or Long Covid. Our objective is to present results from COVID-OUT, a phase 3 double-blind, randomized controlled trial of early outpatient treatment of Covid-19 with repurposed medications. METHODS/STUDY POPULATION: COVID-OUT enrolled adults age 30 to 85 with overweight or obesity who had proof of SARS-CoV-2 infection and fewer than 7 days of symptoms. In this 2 by 3 factorial design trial of metformin, ivermectin, fluvoxamine, or exact-matching placebo of each medication, participants were randomized 1:1:1:1:1:1 to the 6 treatment allocations. This abstract focuses on whether early treatment with metformin prevented Long Covid. Immediate release metformin was titrated to 1500mg daily over the first 6 days. We assessed the incidence of clinician-diagnosed Long Covid with follow up through 10 months after enrollment. We also assessed where participants were diagnosed with Long Covid, and where they received Long Covid treatment. RESULTS/ANTICIPATED RESULTS: Of 1124 participants, 98 (8.7%) report having a healthcare provider make a diagnosis of long covid. By arm, 6.9% (39/564) of metformin participants report having a diagnosis for long covid as compared with 10.5% (59/560) of matched placebo controls. The absolute reduction attributable to metformin was 3.6% (95%CI, 0.3% to 7.0%; P=0.031) with a relative risk reduction of 34% (95%CI, 3% to 55%). The metformin cost per long covid case averted was $28 (95%CI, $15 to $306). 10-month follow-up data will be available at the time of presentation as well as an analysis of baseline factors associated with the development of Long-Covid, independent of treatment allocation in the trial. DISCUSSION/SIGNIFICANCE: Metformin reduced the incidence of clinician-diagnosed long covid by 34% in a double-blind randomized placebo-controlled trial, and previous research published in-vitro activity by metformin against SARS-CoV-2 and other RNA viruses. Further investigation of metformin as early treatment for SARS-CoV-2 is warranted.

ABSTRACT IMPACT: This study aims to provide insight into naturally acquired immunity against severe malaria, thereby laying the foundation for the design of novel vaccine candidates to prevent severe disease as well as monoclonal antibody therapies to treat severe malaria. OBJECTIVES/GOALS: Severe malaria is caused by parasite surface antigens that contain high sequence diversity. Nevertheless, P. falciparum-exposed individuals develop antibody responses against these antigens. Our goal is to isolate antibodies with broad reactivity to understand how disease protection is acquired. METHODS/STUDY POPULATION: Our study cohort consists of Ugandan adults living in a malaria-endemic region with high transmission intensity, who are protected against severe malaria. Using fluorescently labeled probes of parasite surface antigens, we have isolated antigen-specific B cells from these donors. We then expressed the corresponding monoclonal antibodies in vitro. These antibodies were screened against a library of variant surface antigens to determine antibody breadth and potential to inhibit interaction of the parasite surface antigen with host receptors, a critical step in pathogenesis. Additionally, using a panel of variant surface antigen mutants, we have predicted the epitopes targeted by the broadest monoclonal antibodies. RESULTS/ANTICIPATED RESULTS: We have identified three monoclonal antibodies with exceptionally broad reactivity and inhibitory activity against our panel of severe disease-inducing variant surface antigens. We have identified two major sites targeted by these broadly reactive antibodies. The first site was associated with the largest breadth, but limited inhibitory potential, while the second site showed high-affinity antibody binding and inhibition of receptor binding. Interestingly, two of these three antibodies were very similar in structure, even though they were isolated from different donors. Isolation of antigen-specific B cells from additional donors will enable us to identify how common such broadly reactive antibodies are and allow the identification of additional epitopes DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is the first to isolate broadly reactive antibodies that are likely to protect against severe malaria in naturally immune individuals. Further characterization of antibody-antigen interactions will inform the development of this surface antigen as a vaccine candidate for malaria.

OBJECTIVES/GOALS: The Penn State CTSI has been working diligently to help communities in their fight against COVID-19. As the rapidly evolving COVID-19 situation has unfolded the Penn State CTSI has been able to provide support to our community stakeholders. As our communities have and are faced with unprecedented challenges, our CTSI has been there every step of the way. METHODS/STUDY POPULATION: The Penn State CTSI is unique as it sits in rural Pennsylvania that not only spans a wide catchment area but also many diverse communities. The Penn State CTSI connected with our communities throughout the pandemic to bring timely and culturally appropriate information about the novel COVID-19 pandemic through our own institution and in partnership with community leaders. Stakeholder boards were formed to hear from various communities about hardships and challenges that were and are being faced due to COVID-19. RESULTS/ANTICIPATED RESULTS: The Penn State CTSI provided our communities with information through various different platforms to ensure that needs were being met in dissemination of pertinent information related to COVID-19. No only was information tailored to the specific needs that were discussed during stakeholder boards, the information was provided in different languages and platforms in order to meet cultural and other needs to ensure health equity and literacy were met. DISCUSSION/SIGNIFICANCE: Not only did the Penn State CTSI provide these services to our current community partners new partnerships were formed to create stronger alliances amongst those being serve. Members of the Penn State CTSI were invited to serve on state and local advisory bords and became trusted messengers in our communities.

OBJECTIVES/GOALS: Recent studies indicate B cells are involved in dermal fibroblast activation and collagen deposition in the skin. However, B cell distribution in epidermal and dermal layers is unknown. Here, We aim to characterize the distribution of B cells residing in normal skin and keloidal scars. METHODS/STUDY POPULATION: One abdominal normal skin sample and two keloid samples (ear and shoulder) were obtained from the University of Colorado Biorepository Core Facility and from the Plastic Surgery Clinics. Five micron sections from formalin-fixed paraffin-embedded samples were prepared for multiplex fluorescence immunohistochemistry by the Human Immunology & Immunotherapy Initiative. We stained for CD20+, CD19+, and DAPI. Slides were imaged using Vectra®3 scanning system from PerkinElmer. Images were analyzed in InForm®Tissue Finder, phenotpr, phenoptrReports by Akoya biosciences. RESULTS/ANTICIPATED RESULTS: We found a significant increase in the percentage of CD20+ and CD19+ B cells in keloid skin compared to normal skin tissue (14.50% and 14.20% vs 6.47% and 7.56% of the total cells), respectively. Interestingly, we found that in the epidermis of keloid skin CD20+ cell were more abundant (14.46%) whereas in the epidermis normal skin CD20+ cells were less predominant (5.14%). In the dermis of keloid skin, CD20+ and CD19+ were in equal proportions (13%) whereas in normal skin CD19+ cells were more predominant (10.44%) compared to CD20+ cells (7.04%). Dual positive B cells, CD19+/CD20+ cells, were more abundant in keloid dermis (11.06%) compared to normal skin dermis (1.24%). DISCUSSION/SIGNIFICANCE OF IMPACT: B cells are involved in fibroblast activation in diseases such as scleroderma and rheumatoid arthritis. With the increase of CD19+/CD20+ B cells in keloids, the role of B cells in keloid pathogenesis warrants further study. CD27 staining may determine if these are activated or follicular B cells.

Objectives/Goals: This scoping review aims to synthesize current literature on post-deployment monitoring of AI-enabled digital health solutions within clinical practice. Findings identify existing approaches and gaps that inform guidance for post-deployment monitoring in clinical practice. Methods/Study Population: We conducted a scoping review in accordance with PRISMA-ScR guidelines to characterize the current landscape of post-deployment monitoring in healthcare systems. A PubMed search targeted peer-reviewed articles in English published between 2015 and mid-2025, including text or MeSH terms on 1) health system/hospital; 2) artificial intelligence; 3) post-deployment; and 4) evaluation/monitoring. We performed a thematic analysis to identify common challenges, gaps, and opportunities in AI oversight. Additionally, we reviewed guidelines addressing post-deployment AI monitoring. All analyses were conducted using Rayyan.ai and Microsoft Word. Results/Anticipated Results: Among the six studies included after the full-text review, five provide recommendations to ensure transparency, safety, and model performance. These recommendations encompassed monitoring model performance and real-time case report, post-market surveillance, adverse event reporting, end-user training, data standardization and documentation, and interdisciplinary collaboration. One study reports a framework for post-deployment impact grading. Currently, no guidelines addressing post-deployment of AI monitoring in health systems exist. Our findings highlight the urgent need for structured post-deployment processes to ensure AI in healthcare systems is safe, effective, and trustworthy. Discussion/Significance of Impact: The absence of post-deployment guidelines raises concerns. This review underscores the need for interdisciplinary collaboration to establish a post-deployment monitoring process with scientific rigor, scalability, and sustainability that aligns with operational realities.

OBJECTIVES/GOALS: Under enrollment of trials is a continued challenge in clinical research. In response, the Oregon Clinical and Translational Research Institute (OCTRI), the CTSA at Oregon Health & Science University (OHSU), launched a central resource, OCTRI Recruitment, to equip researchers with the knowledge and tools needed for recruitment success. METHODS/STUDY POPULATION: OCTRI Recruitment focused programmatic development in response to the voice of OHSU researchers. In 2018, a qualitative assessment project, “Clinical Research Recruitment Methods at OHSU”, was launched, which included a survey (N=100) and optional interview (N=24), to determine recruitment method utilization and experience, along with opinions on the needs and culture of recruitment at OHSU. In 2022, as part of the same protocol project, a second survey was deployed (N=31), to determine changes in recruitment method use and to identify further recruitment challenges. OCTRI Recruitment also obtains continual informal input on perceived recruitment challenges and opportunities through engagement within the OHSU research team community. RESULTS/ANTICIPATED RESULTS: 2018 survey and interviews showed: many researchers relied on their clinic’s patient population for recruitment (74%); were unaware of available tools to recruit OHSU patients, especially informatics tools (5-22%); and were not aware of and minimally use methods to recruit outside OHSU (<40%). In response, OCTRI Recruitment developed and began recruitment consultations, guidance materials, and educational seminars. In 2022, survey results showed an increase in the use of informatics-based recruitment tools (2-14%+) and increased use of methods focused on individuals outside of OHSU (1-7%+). Additionally, a review of studies post OCTRI Recruitment consultation over three years (N=51) showed that of those studies, 40% increased enrollment numbers and 61% increased team’s confidence level post consult. DISCUSSION/SIGNIFICANCE: This approach to program creation allowed for a uniquely targeted development of services in response to the voice of OHSU researchers and recruitment challenges. Based on additional data, efforts have begun to address the recruitment challenges of a study opportunity website, participant compensation methods, and community-based recruitment.

OBJECTIVES/SPECIFIC AIMS: Cardiovascular diseases (CVD) is the leading cause of death worldwide in both men and women due to lack of cardiac regeneration after disease or damaged is caused. There are many challenges to studying CVD since native cardiomyocytes cannot be cultured in vitro. With the advancements in biomaterial and pluripotent stem cells research, scientists are now able to produce engineered cardiac tissue models in vitro that mimic the native myocardium. This study shows our methods for producing engineered cardiac tissue with potential applications in cardiac regeneration, disease modeling, and scalable production. METHODS/STUDY POPULATION: In this study, human induced pluripotent stem cells (hiPSCs) were combined with two different photocrosslinkable hybrid biomaterials, poly (ethylene)- glycol fibrinogen (PF) and gelatin methacrylate (GelMa), in various tissue geometries to form 3D human engineered cardiac tissues (3D-hECTs). To study tissue growth and contraction, image and video analysis was performed at specific timepoints. To analyze differentiation efficiency and cell population, flow cytometry was performed using cardiac markers. To evaluate gene expression, qPCR was performed using pluripotency and cardiac specific primers. RESULTS/ANTICIPATED RESULTS: Direct cardiac differentiation of encapsulated hiPSCs resulted in synchronously contracting 3D-hECTs in both biomaterials and all tissue geometries. Spontaneous contractions started on Day 7 and increased in velocity, frequency, and synchronicity over time. 3D-hECTs had high cell viability with > 70% of cells positive for cardiac markers. Engineered tissues showed appropriate temporal changes in gene expression over time with pluripotency gene expression decreasing and cardiac gene expression increasing. DISCUSSION/SIGNIFICANCE OF IMPACT: This study shows the potential for direct differentiation of encapsulated hiPSCs to produce physiologically relevant engineered cardiac tissues. Resulting 3D-hECTS showed features of mature myocardium with appropriate cardiomyocyte populations, mechanical motion, and gene expression. Using this platform, we are able to produce engineered cardiac tissue in a variety of biomaterials and tissue geometries to study new therapeutics, mechanism of disease, and scalable tissue culture.

OBJECTIVES/SPECIFIC AIMS: Key factors causing irreproducibility of research include those related to inappropriate study design methodologies and statistical analysis. In modern statistical practice irreproducibility could arise due to statistical (false discoveries, p-hacking, overuse/misuse of p-values, low power, poor experimental design) and computational (data, code and software management) issues. These require understanding the processes and workflows practiced by an organization, and the development and use of metrics to quantify reproducibility. METHODS/STUDY POPULATION: Within the Foundation of Discovery – Population Health Research, Center for Clinical and Translational Science, University of Utah, we are undertaking a project to streamline the study design and statistical analysis workflows and processes. As a first step we met with key stakeholders to understand the current practices by eliciting example statistical projects, and then developed process information models for different types of statistical needs using Lucidchart. We then reviewed these with the Foundation’s leadership and the Standards Committee to come up with ideal workflows and model, and defined key measurement points (such as those around study design, analysis plan, final report, requirements for quality checks, and double coding) for assessing reproducibility. As next steps we are using our finding to embed analytical and infrastructural approaches within the statisticians’ workflows. This will include data and code dissemination platforms such as Box, Bitbucket, and GitHub, documentation platforms such as Confluence, and workflow tracking platforms such as Jira. These tools will simplify and automate the capture of communications as a statistician work through a project. Data-intensive process will use process-workflow management platforms such as Activiti, Pegasus, and Taverna. RESULTS/ANTICIPATED RESULTS: These strategies for sharing and publishing study protocols, data, code, and results across the spectrum, active collaboration with the research team, automation of key steps, along with decision support. DISCUSSION/SIGNIFICANCE OF IMPACT: This analysis of statistical methods and process and computational methods to automate them ensure quality of statistical methods and reproducibility of research.

Community involvement in research is key to translating science into practice, and new approaches to engaging community members in research design and implementation are needed. The Community Scientist Program, established at the MD Anderson Cancer Center in Houston in 2018 and expanded to two other Texas institutions in 2021, provides researchers with rapid feedback from community members on study feasibility and design, cultural appropriateness, participant recruitment, and research implementation. This paper aims to describe the Community Scientist Program and assess Community Scientists' and researchers' satisfaction with the program. We present the analysis of the data collected from 116 Community Scientists and 64 researchers who attended 100 feedback sessions, across three regions of Texas including Northeast Texas, Houston, and Rio Grande Valley between June 2018 and December 2022. Community Scientists stated that the feedback sessions increased their knowledge and changed their perception of research. All researchers (100%) were satisfied with the feedback and reported that it influenced their current and future research methods. Our evaluation demonstrates that the key features of the Community Scientist Program such as follow-up evaluations, effective bi-directional communication, and fair compensation transform how research is conducted and contribute to reducing health disparities.

Structural racism in the USA has roots that extend deep into healthcare and medical research, and it remains a key driver of illness and early death for Black, Indigenous, People of Color (BIPOC). Furthermore, the persistence of racism within academic medicine compels an interrogation of education and research within this context. In the spirit of this interrogation, this article highlights a unique model of community-engaged education that integrates cultural humility. As an individual and institutional stance, cultural humility denotes lifelong learning and self-critique, the mitigation of power imbalances, and accountability. The integration of cultural humility emphasizes that when space is created for BIPOC communities to lead the way, education regarding healthcare and research can be effectively reimagined. Demonstrating this effectiveness, six community partners led the development and implementation of a five-module Structural Racism in Healthcare and Research course. Using a cohort model approach, the pilot course enrolled 12 community members and 12 researchers. The curriculum covered topics such as history of racism in healthcare and research, and introduced participants to a cultural resilience framework. Evaluation results demonstrated a significant increase in participants’ knowledge and ability to identify and take action to address inequities related to racism in healthcare and research.

OBJECTIVES/SPECIFIC AIMS: Historically, logistic regression algorithms (LRAs) have failed to differentiate strangulated small bowel obstructions (SBOs) from nonstrangulated SBOs. Our hypothesis is that a machine learning algorithm (MLA) can differentiate strangulated from simple SBOs better than an LRA can. METHODS/STUDY POPULATION: We used records of patients presenting with acute SBO and managed with exploratory laparotomy to test and train algorithms. We compared MLA to LRA via area under the receiver operating characteristic curve (AUROC) and cut-off points maximizing sensitivity and specificity. RESULTS/ANTICIPATED RESULTS: With 192 patient records, the AUROC of the MLA was 0.85. At the sensitivity cutoff, the MLA had 100% sensitivity and 55% specificity. At the specificity cutoff, the MLA had 45% sensitivity and 100% specificity. We anticipate improvements as more records are incorporated, and that LRA will underperform MLA across all measures. DISCUSSION/SIGNIFICANCE OF IMPACT: Our MLA represents a significant improvement over past LRAs, and may provide decision assistance to surgeons managing SBO. If this MLA maintains its high sensitivity, it may be used in the future to prevent unnecessary surgeries.