ABSTRACT IMPACT: This study will answer key questions that spine surgeons have regarding techniques used in cement augmentation of vertebral compression fractures and will ultimately advance patient care for such injuries. OBJECTIVES/GOALS: The objective of this study is to determine if a difference exists in load-bearing characteristics and load-to-fracture between injecting cement anteriorly prior to screw placement versus cement augmentation via fenestrated pedicle screws. We also expect differences in load-to-failure characteristics between different cement volumes. METHODS/STUDY POPULATION: This study will be performed in a bioengineering laboratory that has access to a Materials Testing System (MTS). Eight cadaveric specimens will be selected from our stock after pre-screening via CT for inclusion and exclusion criteria. The levels T8-L1 will be dissected from the vertebral column along with any soft tissue structures. The vertebral bodies will be potted in an epoxy mold. From each spine, there are 2 groups of three. One vertebral body from each spine will serve as an internal control, one will be augmented with cement via a cannula and then instrumented with a non-fenestrated screw and the third will be instrumented will a fenestrated screw and then augmented with cement. After appropriate curing time, repeat CT imaging will be completed. The specimens will then be loaded to failure and the results analyzed. RESULTS/ANTICIPATED RESULTS: We hypothesize that we will see a better anterior spread with the cannula/non-fenestrated screw method as compared to the fenestrated screw. The reason being is that we would expect the fenestrated screw to experience more cement extruding from the fenestration rather than being directed anteriorly. We believe a better anterior spread of the cement will lead to a greater load-bearing capacity for the vertebral body. We also believe that a difference will exist in load-to-failure testing with the two volumes being tested, though we cannot predict to what a degree this difference will be impactful as there have been few studies prior looking at this. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is significant because it will aid in determining the optimal technique to implement in the setting of vertebral compression fractures. This will lead to improved patient care as well as a greater understanding of the instrumentation used in such procedures. The results will lay the groundwork for future research on this procedure.

OBJECTIVES/GOALS: Experimental pain testing is used to identify changes in nociceptive processing and outcomes with intervention. This study investigated exercise induced changes in experimental pain and self-reported pain intensity after an acute bout of exercise in participants with fibromyalgia. METHODS/STUDY POPULATION: Ten females with fibromyalgia (55±10yr) were familiarized to study procedures and underwent submaximal (20% maximal voluntary contraction) intermittent eccentric muscle contractions isolated to the right arm for 10-minutes. Self-reported pain intensity (0-10 numerical pain rating scale [NPRS]) of the exercising arm was measured before, during, and after exercise; whole-body pain intensity was measured before and after exercise. Experimental pain testing included measurement of pressure pain thresholds (kPa [PPTs]); temporal summation (TS) of pressure pain with a constant mechanical pressure; and TS of punctate pressure with repeated application of monofilaments before and after exercise. RESULTS/ANTICIPATED RESULTS: Participants reported minimal to moderate arm pain (3.1±2.1) during exercise. Following exercise, arm pain and whole-body pain significantly increased (3.1±2.2 and 1.6±0.5, respectively) [p<0.05]. No change occurred with PPTs at the bicep (138.9±49.5 to 142.8±55.3), PPTs at the quad (212.0±105.4 to 228.1±100.0), TS of mechanical pressure pain (7.6±2.1 to 7.9±1.5), TS of punctate pressure pain at the bicep (2.6±1.7 to 3.0±1.5), and TS of punctate pressure pain at the quad (3.6±1.5 to 3.7±1.4) before to after exercise respectively [p>0.05]. The change in self-reported arm and whole-body pain did not correlate with the change in experimental pain testing. DISCUSSION/SIGNIFICANCE OF IMPACT: In people with fibromyalgia, there is no relation between self-reported clinical pain and experimental pain following a single exercise session. Further research should identify the influence of exercise training on pain perception and if experimental pain testing translates to clinical insight.

Objectives/Goals: The NIH Common Fund launched Community Partnerships to Advance Science for Society (ComPASS) to study ways to reduce health disparities by addressing underlying structural factors within communities. ComPASS was designed for community organizations to lead research that addresses community needs. Methods/Study Population: ComPASS awarded five health equity research hubs (Hubs) to provide specialized technical support to ComPASS research projects led by community organizations. Expertise provided by the Hubs to the community-led projects will focus on specific facets of community health, including nutrition access, health care access, and built environment. The Hubs support community-led piloting and testing of structural interventions within community settings by providing subject matter expertise in areas including structural and multilevel intervention study design and methods, implementation science, and community engagement. Results/Anticipated Results: The Hubs will provide expertise and support to the community-led research projects around one or more social determinants of health domains: health care access and quality, education access and quality, economic stability, social and community context, and neighborhood and built environment. The Hubs will help identify strategies for measuring health outcomes and assessing the effects of structural and contextual factors on intervention outcomes. We anticipate the ComPASS program will lead to a better understanding of how structural interventions that leverage multi-sectoral partnerships can advance health equity. Discussion/Significance of Impact: Through community-led research, ComPASS projects are implementing structural interventions to address social determinants and advance health equity. The technical scientific support rooted in health equity provided by the Hubs is essential to the success of these research projects.

OBJECTIVES/GOALS: Diffusion MRI can identify microstructural brain changes and can provide insight into neural development and to potentially prognosticate speech and language outcomes in children with SNHL. The goal of our study was to investigate MRI-based microstructural changes along the brainstem regions of the auditory pathway in pediatric patients with SNHL. METHODS/STUDY POPULATION: We reviewed cohort of pediatric patients with SNHL who obtained MRI at 3T between 2011 and 2019. We identified 16 pediatric patients (age RESULTS/ANTICIPATED RESULTS: We identified significant differences in FA values of the SON between the SNHL cohort and controls (0.377 ± 0.056 vs 0.422 ± 0.052; p=0.009). No other FA or MD values were significantly different between the two groups. Among younger children (ï‚£5 years), MD was significantly decreased in the SNHL cohort compared to controls in the IC (0.918 ± 0.051 vs 1.120 ± 0.142; p5 years), there were no significant differences in MD (1.124 ± 0.198 vs 0.997 ± 0.103; p = 0.119). There were no significant differences in MD or FA in the white matter fibers of the IC-SON tract. DISCUSSION/SIGNIFICANCE: This study is the first to assess microstructural changes in brainstem auditory pathway regions among children with SNHL. Longitudinal studies are warranted to assess the predictive value of these MRI-based findings for long-term outcomes and the efficacy of intervention.

OBJECTIVES/GOALS: The study aims to identify and measure metabolites in irradiation-induced senescent pre-adipocytes induced by the increased secretion of pro-inflammatory cytokines. Through untargeted metabolomics, we seek to understand the alterations to metabolism and mitochondrial activity that occur during irradiation-induced senescence. METHODS/STUDY POPULATION: First, commercially available human primary subcutaneous pre-adipocytes were cultured in vitro, and irradiated to induce senescence. To confirm senescence, cells were stained for beta-galactosidase, which was positive in senescent pre-adipocytes. The cells and their conditioned cultured media were then collected and frozen for untargeted metabolomics to profile metabolites. The sample analysis is currently underway and will be conducted using central carbon isotope tracing and chromatograph mass spectrometry. Principal Component Analysis, fold change analysis, and heat maps will be used to detect and report the changes in metabolite signals. Oxygen consumption rate and extracellular acidification rate measurements are in progress at present using the Agilent Seahorse XF Cell Mito Stress Test. RESULTS/ANTICIPATED RESULTS: We expect that metabolites of central carbon metabolism and oxidative phosphorylation will be upregulated. The concentrations of metabolites of pathways altered by the pro-inflammatory cytokines that have been identified to be secreted by senescent cells are expected to be altered. The metabolites measured in conditioned culture media will provide insight into the changes to the cellular microenvironment caused by senescence. Finally, we anticipate that mitochondrial function, and both aerobic and anaerobic metabolism will be altered in senescent pre-adipocytes compared to controls. Through the present study, we will achieve a better understanding the metabolic alterations that occur as part of cell senescence in pre-adipocytes. DISCUSSION/SIGNIFICANCE: Adipose tissue dysfunction due to the accumulation of senescent cells has been linked to chronic diseases such as diabetes and insulin resistance, and inflammaging. Through this study, we expect to provide new insight into the metabolic alterations of senescence and offer a backbone for prospective translational human studies and clinical trials.

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

ABSTRACT IMPACT: This project uses rapid prototyping, 3D printing, and cell seeding to solve the most common post-operative complications that arise from the current methods of urethral elongation during phalloplasty. OBJECTIVES/GOALS: Post-op fistula and stricture formation occur in up to 50% of phalloplasty patients. These complications arise from the mismatch between skin and uroepithelium, or from scarring secondary to ischemia. Here we describe the fabrication of a novel vascularized urothelial flap for phalloplasty that contains discrete urothelial and vascular channels. METHODS/STUDY POPULATION: A custom designed 3D negative mold, with a urethral channel and a vascular inlet and outlet channel was prototyped in Adobe Fusion 360 and printed on a Prusa i3 MK3S printer in PLA. A 2mm diameter pluronic sacrificial macrofiber was used to connect the channels to form a vascular loop, and 1% type-I collagen was extruded over the mold. After solidifying, the scaffold was demolded and seeded with grade I urothelial carcinoma (SW780 cells, at 5-10 x 106 cells/mL) in the urethral channel, and adenovirus-infected E4 endothelial cells (at 3x106 cells/mL) in the vascular channel. The scaffolds were cultured up to 14 days and then fixed for histologic analysis. RESULTS/ANTICIPATED RESULTS: Collagen scaffolds were fabricated reliably using the custom 3D negative molds. After both seven and fourteen days of culture, the urothelial channel contained a robust, stable urothelial monolayer lining throughout the channel. By 14 days urothelial multilayer formation was seen, providing definitive evidence of a more mature urothelial layer. Grooves within the collagen allowed for nests of urothelial cells to develop, leading to increased multilayer formation. In addition, the vascular channels supported a healthy endothelial lining at both seven and fourteen days. There were no significant histological differences between constructs seeded with 5x106 urothelial cells/mL and 107 cells/mL. We anticipate that multilayer formation will increase with time, and that constructs will survive beyond 28 days. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have developed a novel strategy to engineer vascularized urethral tissue. These constructs can be kept for at least 14 days and form stable monolayers and multilayers consistent with native urothelial architecture. Using 3D printing and autologous cell seeding promises to create patient-specific vascularized urethral flaps for phalloplasty.

OBJECTIVES/GOALS: To determine the utility of any standard, one must first evaluate whether or not the standard meets the needs of the use case it is meant to support. We aim to quantify data availability of the HL7 FHIR standard to support data collection for three state-based registries in a rural state and measure the potential effects on data quality and collection time. METHODS/STUDY POPULATION: FHIR mapping will be performed to assess the level of HL7 FHIR standard completeness (or data element coverage) in supporting data collection for the three registries. A systematic approach, previously developed and used by Garza and Zozus, will be used to map registry data elements to corresponding HL7 FHIR standard resources. FHIR coverage will be calculated as a percentage (total data elements “Available in FHIR” vs. total data elements overall) and will be observed across different domain areas (i.e., demographics vs. medications vs. vital signs, etc.) in order to identify the domains with the least and most coverage. RESULTS/ANTICIPATED RESULTS: Although there have been informatics solutions that relied on data exchange standards to improve data collection, none have actually evaluated the coverage of the standard for supporting the needs of clinical data registries. To address this gap, we aim to evaluate the availability of the HL7 FHIR standard to support data collection for three state-based registries. These results will provide insight into the generalizability of a FHIR-based solution to support data acquisition and processing across multiple registries and demonstrate the potential for seamless exchange of that data for secondary use in clinical and translational research. Quantifying the coverage will also be used to further advance its development in order to meet the data collection needs of state and national clinical data registries. DISCUSSION/SIGNIFICANCE: Registries often rely on manual abstraction of EHR data. These manual approaches have had a negative impact on data quality and cost, often due to the complexities associated with collection and mapping of the data to fit the registry model. HL7 FHIR has the potential to address these issues by automating part or all of the data collection process.

OBJECTIVES/GOALS: This study uses diffusion kurtosis imaging (DKI) to investigate the structural profiles of basal ganglia (BG) motor circuitry in Vascular Parkinsonism (VP), Parkinson’s disease (PD), and healthy aging controls (HC). VP is a clinical diagnosis of lower body predominant parkinsonism without significant benefit from levodopa. VP is distinct from PD, yet the concept of VP remains debated due to the inability of prior studies to identify specific causative changes. One reason for this may be limitations in measuring intricate BG connectivity in vivo. Given the predominant lower body parkinsonism symptoms in VP, we hypothesized that VP would be associated with decreased connectivity specifically within the BG motor loop. METHODS/STUDY POPULATION: We obtained DKI brain imaging in subjects with VP (N = 7), PD (N = 21), and HCs (N = 58), the latter of which had cardiovascular risk factors but no neurological symptoms. The VP and PD groups were evaluated by a parkinsonism-focused motor exam and brief cognitive testing. We compared BG motor loop connectivity between groups and investigated for correlation between connectivity and clinical scores. To account for differences in fiber counts due to the different imaging scanners and protocols between cohorts, we used a BG motor loop proportion, which was the ratio of the BG motor loop fiber count over a control loop, the visual processing pathway. We used Kruskal-Wallis rank sum test with post-hoc Dunn tests to assess imaging findings between subject groups, and Pearson’s correlation to look for correlation between clinical scores and fiber counts. RESULTS/ANTICIPATED RESULTS: The whole brain connectome showed the fewest number of fibers in VP, followed by PD, and then HC (p<0.0001). The BG motor loop proportion fiber count of the BG motor loop was lower in the VP group, compared to the PD and HC cohorts (p = 0.031). In the VP group, the whole brain connectome fiber count correlated with a gait and balance subscore of the Movement Disorders Society - Unified Parkinson Disease Rating Scale (R = −0.87, p = 0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: This study indicates that VP is associated with decreased structural connectivity, with a disproportionate degree of loss in the BG motor circuitry. While the etiology for this susceptibility to injury and preferential damage to BG remains to be defined, these findings can provide an important starting point for a biological understanding of VP, and a potential future marker for diagnosis and tracking disease progression.

OBJECTIVES/GOALS: Molar hypomineralization (MH) is a highly prevalent dental disease that leads to rapid enamel decay even with preventative measures. While the harm is apparent, the etiology of the condition is not. Further, MH is difficult to study due to limited intact extracted teeth. Therefore, we need a method to study MH non-destructively and intraorally. METHODS/STUDY POPULATION: Recent work has shown excess proteins not commonly found in enamel are present in teeth with MH. This is theorized to be due to disruption and infiltration in the cells that form enamel leading to leftover protein and under mineralized enamel. We hypothesizethese proteins have specific spectroscopic signatures that can be detected using light. We further hypothesize that modern fiber optic probes can provide a method for non-destructive, pain-free, and rapid intraoral examination of MH. Initially, Micro Raman spectroscopy was used to detect specific vibrational bands associated with organics in teeth with MH followed by the collection of spectra of teeth with MH and heathy enamel using a spectrophotometer. These spectra were examined for any obvious differences. RESULTS/ANTICIPATED RESULTS: Currently 12 teeth were collected, and micro-computed tomography reconstructions confirmed location in 3D of MH lesions. Micro Raman of a MH-affected tooth revealed clear organic associated Amide I and III vibrational bands when compared to a synthetic hydroxyapatite (mineral in enamel) powder standard. We determined the wavelength of light that can be used to detect spectral differences between healthy enamel and teeth with MH. The next steps include optimization of the protocol of the intraoral spectrometer with the determined wavelength for implementation in clinic to allow for collection of spectra without the need for tooth extraction. DISCUSSION/SIGNIFICANCE: We hope that this work will lead to advancements in our understanding of the mechanism of MH as well as act as a proof of concept for a MH diagnostic tool. In the long term, the goal is to ideally lead to improvements in dental health care, decrease dental costs, and improve overall quality of life for the children with this condition.

OBJECTIVES/SPECIFIC AIMS: Parents often make errors in comprehending and executing their child’s inpatient discharge instructions, putting their child at risk for adverse post-discharge outcomes. Suboptimal provider-caregiver communication has been linked to errors in comprehension and execution of provider instructions, especially for parents with limited health literacy. Few studies have systematically examined features of pediatric inpatient written discharge instructions that may contribute to errors. Our objective was to assess the readability, understandability, and actionability of pediatric inpatient written discharge instructions. METHODS/STUDY POPULATION: This was a cross-sectional analysis of the written discharge instructions (standardized template, content not standardized) provided to parents at an urban public hospital, enrolled as part of a prospective cohort study (n=171) focused on parent ability to comprehend their child’s discharge instructions. Inclusion criteria were: English/Spanish-speaking parents of children ≤12 years old discharged on ≥1 daily medicine. Discharge instructions were assessed for: 1) Readability (Average of 5 formulas [Flesh Reading Ease, Flesch-Kincaid, Gunning Fog, Simple Measure of Gobbledygook, Forcast]), 2) Understandability and actionability (AHRQ Patient Education Materials Assessment Tool [2 independent reviewers; κ>0.8 for both]). RESULTS/ANTICIPATED RESULTS: Mean (SD) reading grade level was 11.4 (0.7); none of the instructions were written at a recommended reading level of 6th to 8th grade or below. Mean (SD) understandability was 37.7 (6.9)%; mean actionability was 41.7 (8.4)%. All 171 sets of instructions used medical terminology without adequate plain language explanations and included information that was not relevant to the child’s diagnosis and associated care (e.g., obesity counseling, smoking cessation given to a child with appendicitis). None of the sets of instructions presented information in a logical sequence (e.g., diet instructions in more than one location) or included any pictographic information or other visual aids to support the text (e.g., diagram of medication dose within a dosing tool). DISCUSSION/SIGNIFICANCE OF IMPACT: Written discharge instructions provided in the pediatric inpatient setting were suboptimal. Use of a systematic approach to improve discharge instructions, using a health literacy perspective, has the potential to improve post-discharge outcomes in children.

Objectives/Goals: Mentoring is essential for the development of investigators, and while many mentorship programs exist, few focus exclusively on the challenges associated with community-engaged research (CER). This program focused on forging lasting mentoring relationships with CER experts and strengthening connections with community partners. Methods/Study Population: The 9-month program focused on: Exclusive focus on CER mentorship. The curriculum drew on best practices from CTSC partner institutions (Emory University, University of Wisconsin-Madison) and local expertise from the NIH-funded U54 Investigator Development Core. Prior work identified the lack of CER-specific mentorship as a barrier to success, prompting the creation of this dedicated program. Small cohorts with community mentors: Each bi-monthly cohort comprised four mentees and one community mentor, supported by CTSC staff. This structure created a safe space for sharing project challenges and offered grounded perspectives on community-partner needs and expectations. Mentor coaching mentors received targeted coaching, enhancing their capacity to guide mentees and boosting overall mentor satisfaction. Results/Anticipated Results: Evaluation surveys captured satisfaction, confidence, and likelihood of continued participation. Key findings include * High Satisfaction: Both mentors and mentees reported strong satisfaction with program elements. * Skill Development: Mentees cited support across project design, grant development, networking, and policy work. * Future Participation: 100 % of mentors and mentees indicated they would join again or recommend the program to colleagues. * Community Impact: Participants highlighted the program’s sense of community, passion, and peer support. Discussion/Significance of Impact: This CER mentoring program addresses a critical gap: traditional career mentors often focus on clinical or academic specialties, overlooking the unique challenges of building equitable community partnerships. By centering mentorship on CER and incorporating coach-driven mentor development, the program fosters bidirectional growth.

OBJECTIVES/GOALS: The goal of this study is to reveal strong candidate genes for inherited retinal diseases (IRDs) in humans to better understand the mechanisms behind IRD development and reveal potential therapeutic targets. We hope these findings will help improve our understanding of IRDs and, subsequently, diagnostic accuracy and prognosis for IRD patients. METHODS/STUDY POPULATION: The goal of the International Mouse Phenotyping Consortium is to identify the function of all protein-coding genes in the mouse genome via generation and phenotypic characterization of single gene knockout (KO) mice. Using this database, we identified all KO strains associated with abnormal retinal phenotypes, removed all RNA coding genes and pseudogenes, converted to human orthologues, and conducted a literature search for existing research regarding candidate IRD genes and retinal function/abnormalities. A similar process was used for RetNet genes. Subsequently, we performed bioinformatics analysis, including functional annotation (e.g. panther-db), pathway analysis (e.g. KEGG), and string-db to visualize known and predicted protein-protein interactions between the two data sets. RESULTS/ANTICIPATED RESULTS: Analysis of the IMPC database revealed, out of 8481 phenotyped genes, 572 unique protein coding genes were associated with 14 categories of retinal abnormalities such as abnormal retinal vasculature and abnormal retinal thickness, 377 of which have never been associated with retinal pathology in humans or mice. Pathway analysis of the IMPC database highlighted a general metabolism pathway as well as PI3K-Akt and MAPK pathways, not found in RetNet pathway results. Unique clusters from functional annotation clustering of the IMPC include DNA methylation and protein ubiquination. Visualization of protein-protein interactions in string-db between the IMPC (mouse) and RetNet (human) revealed 4 clusters of interest with gold standard RetNet IRD proteins interacting with candidate IMPC IRD proteins. DISCUSSION/SIGNIFICANCE: IMPC analysis revealed 572 candidate IRD genes, 377 of which are novel with no existing independent research related to the retina outside of the IMPC. Bioinformatic analysis reveals 4 strong clusters of interest through string-db where a gold standard RetNet gene interacts with candidate IRD genes as well as many functional pathways of interest.

OBJECTIVES/GOALS: The Think Tanks’ aim to assist faculty innovators along the path from discovery to commercialization; serving as a one-stop-shop where investigators can access advice, pilot funds, and direction toward other available resources within the Indiana CTSI. METHODS/STUDY POPULATION: Faculty receive guidance from a pool of advisors in the drug and device industry, as well as their respective university commercialization offices; who serve as an essential resource for a wide range of scientific, technical, clinical, business, and regulatory questions. Investigators submit a simple intake form to connect with a project facilitator for detailed guidance prior to formal meetings. Projects are guided from start to finish with robust tracking and milestone-based funding to help generate data for Investigational New Drug (IND) or Investigational Device Exemption (IDE) applications. Project tracking is conducted using REDCap, which is used for all investigator submissions, with internal processes also tracked in REDCap. RESULTS/ANTICIPATED RESULTS: From Feb-Aug 2021; the Think Tanks provided feedback to 15 drug projects and 6 medical device projects. This included 12 from Indiana University; 4 from Purdue University; and 5 from the University of Notre Dame. Interestingly; drug innovations were submitted primarily by tenure track faculty (100%) with a history of NIH/NSF funding (50%); while device innovations were submitted primarily by clinical faculty without a history of NIH/NSF funding (66%). Based on first-round feedback, a total of approximately $3;400 in pilot funds were provided. Efforts are underway to obtain survey-based feedback from all applicants to date; which will be used to inform future program modifications. DISCUSSION/SIGNIFICANCE: The budding “Think Tank” program provides faculty with a broad perspective of the entire drug and medical device development process, helping investigators understand the critical interplay development stages. Future work seeks to enhance faculty engagement in, and understanding of the commercialization process across Indiana CTSI institutions.

OBJECTIVES/SPECIFIC AIMS: To examine the rate of medical and social service referral utilization among community members who are enrolled in HealthStreet - a community engagement initiative at University of Florida. METHODS/STUDY POPULATION: HealthStreet utilizes the CHW model to conduct health needs assessment, provide referrals to medical and social services and link them to health research at UF. Across two follow-up schedules, these participants are contacted to assess their rate of referral utilization. RESULTS/ANTICIPATED RESULTS: From October 2011-October 2018, HealthStreet completed 10,829 health needs assessments and provided a total of 15,723 medical and/or social service referrals with an average of 1.48 referrals per person. About a third of people completed first and second follow-up respectively (n=3,461; 32.0% and n=3,477; 32.1%), and another third (n=3,891; 35.9%) completed neither. The total number of follow up attempts was 40,863, with an average of 3.85 attempts per person. The overall service utilization rate was 17.02%. The top barriers to utilization included, could not schedule an appointment (26.3%), busy on the date of appointment (21.9%), transportation (9.4%), and already received the service from elsewhere (4.7%). Others (28.3%) did not identify a specific barrier for non-utilization. DISCUSSION/SIGNIFICANCE OF IMPACT: Findings show that those who need services are still hampered by barriers to care that CHWs and other service providers could help them overcome. Facilitating the appointment and providing transportation would assist over a third of those needing services.

Objectives/Goals: In 2021, Mayo Clinic Center for Clinical and Translational Science (CCaTS) launched CE Studios to enhance community-informed research across a multisite institution. This study applied the Proctor’s implementation outcomes framework to evaluate the implementation efforts across diverse settings. Methods/Study Population: From June 2022 to October 2025, we held 59 CE Studios across 19 sites in Arizona, Florida, and Midwest. CE Studios were done in-person and virtually via Zoom. Researchers and community members completed a survey assessing their experience and perceived influence of communities’ feedback on the research project. Data were collected and analyzed from June 2022 to October 2025. To assess the implementation, we applied Proctor’s Implementation Outcomes Framework, which examines eight constructs: Acceptability, Adoption, Appropriateness, Feasibility, Fidelity, Implementation Cost, Penetration, and Sustainability. Results/Anticipated Results: Thirty (51%) researchers and 286 (67%) community members completed the survey. Both groups indicated high satisfaction with CE Studio planning, format, and facilitation, emphasizing meaningful contribution of community feedback on research. All researchers noted at least one stage of their research was influenced by community input: pre-research (28%), study design (28%), implementation (22%), infrastructure (15%), analysis (1%), and dissemination (6%). Over time, CE Studios requests increased, reflecting recognition of their value in enhancing research. The framework revealed missing structured coordinators’ feedback, a key for understanding implementation and highlighted the need to revise evaluation tools and identify additional data sources aligned with the broader framework. Discussion/Significance of Impact: Applying the Proctor’s Implementation Outcomes Framework revealed areas to strengthen CE Studio implementation such as coordinator feedback, refinement of evaluation tools, data source, and strategies to assess CE Studio sustainability and institutional impact. These insights offer a model to guide similar efforts at other institutions.

ABSTRACT IMPACT: This research will promote understanding the role of the Type-I Interferon signaling pathway during embryo implantation, potentially leading to a new diagnostic or treatment target in early pregnancy failure. OBJECTIVES/GOALS: Studies suggest interferon signaling regulation is tightly balanced between physiologic and pathophysiologic growth in early pregnancy. We propose to determine the impact of interferon-mediated inflammation on embryo implantation and early pregnancy failure in normal conditions and chronic inflammatory diseases in a novel mixed-mouse model. METHODS/STUDY POPULATION: To probe the role of type-I interferons (IFNs) in implantation, we will utilize a mouse model and non-surgically transfer both Ifnar1-/- and Ifnar1-/+ embryos into an immune-competent pseudopregnant wild-type female recipient. This will allow analysis of a litter with distinct genotypes within the same, immune-competent, uterine environment. Type-I IFN stimulation will be systemically induced with Poly-(I:C) at various time points around implantation. A similar approach will be used in mouse models of chronic inflammatory disease states associated with early pregnancy loss (e.g. systemic lupous erythematous). With this model, we will be able to control for deficiencies in maternal immune response to specifically determine the embryonic response to inflammation during implantation and development. RESULTS/ANTICIPATED RESULTS: We anticipate the Ifnar1-/+ embryos - those able to respond to Type-I IFN - and their surrounding implantation sites will exhibit more maternal-fetal barrier dysfunction in the form of impaired trophoblast fusion, improper formation of the microvascular architecture, and increased permeability of the maternal-fetal barrier, compared to embryos unable to respond to IFN. We will also conduct similar analyses in mouse models of chronic inflammatory diseases. We hypothesize these mice to have baseline endometrial inflammation that stimulated the IFN-pathway in IFN-capable embryos, producing breakdown of the maternal-fetal barrier. In these mice, we predict Ifnar1-/- embryos will show improved molecular outcomes when compared to Ifnar1-/+ embryos, and thus improved associated pregnancy outcomes. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work can insight into the immunological mechanisms that govern embryo implantation and early placentation. This could provide more pointed means for management and intervention of early pregnancy failure and/or disease states that are commonly associated with poor reproductive outcomes.

OBJECTIVES/GOALS: Writers with high self-efficacy perform better than writers with low self-efficacy regardless of writing ability. We investigated whether Shut Up & Write® (SUAW), a less-intensive writing intervention, produced gains in writing self-efficacy similar to those reported by intensive, longer-term interventions. METHODS/STUDY POPULATION: Meetings were held 2x/wk for 5 wks via Zoom, for 1 hour. Participants were encouraged to attend at least 1x/wk. The 1st few mins were devoted to a discussion of what each person planned to work on. Then, a timer was set and each writer muted themselves, shuttered their webcam, and wrote. When the alarm sounded, everyone returned to the group, and discussed what was accomplished. We measured writing self-efficacy before & after participating in SUAW using a pre-post survey design and used two-tailed paired t-tests to test for significant differences between pre- and post-test means. SUAW participants (n=23) were in 1 of 2 categories: 10 were self-selected LEADS scholars from MSIs, and 13 were medical students in a palliative care program. 86% were URB, 78% were female. RESULTS/ANTICIPATED RESULTS: Seven (30%) SUAW participants completed both the pre- and post-survey. Individuals showed significantly higher agreement from pre-to-post on the self-efficacy item “I have a generally positive attitude toward writing” (p=0.047) using a 5-point Likert scale from “completely agree” to “completely disagree.” Most other items did not indicate significant change between pre- and post-survey. The mean of the question “How satisfied were you with this Shut Up & Write activity?” which appeared only on the post-survey (n=10) was 1.10 (1=extremely satisfied, 5=extremely dissatisfied). Anticipated result: We suspect that the benefits of SUAW are best actualized by ongoing attendance, and that benefits are cumulative. DISCUSSION/SIGNIFICANCE: We found that participation in SUAW promotes writing self-efficacy in early-career URB researchers. This is an exciting finding because publishing ones research is essential for academic advancement, and research supports a relationship between writing self-efficacy and writing production. This may curtail URB scientists’ rate of attrition.

OBJECTIVES/GOALS: Craniosynostosis is the premature fusion of one or more cranial sutures that produces brain growth constraints and typically requires surgical treatment. We present an age- and sex-specific method to evaluate surgical outcomes using non-invasive 3D photogrammetry that brings objectivity to the current approach for clinical assessment. METHODS/STUDY POPULATION: First, we created standardized head anatomy representations for 2,020 patients (1,081 males, 939 females, age 3.14 ± 3.05 years) without cranial pathology from their computed tomography (CT) images based on our previous methods. We used principal component regression stratified by sex to establish age-specific normative ranges of anatomical variability and we designed a new metric called cranial shape abnormality (CSA) index that calculates the number of standard deviations from normality of a given patient’s head anatomy. We calculated our CSA index in a group of 56 patients (44 male, 12 female) with sagittal craniosynostosis who underwent sagittal craniectomy from their pre- (22 ± 30 days before surgery) and post-surgical (267 ± 63 days after surgery) 3D photograms to evaluate surgical outcomes. RESULTS/ANTICIPATED RESULTS: We observed a reduction in the CSA index from 1.28 ± 0.26 before surgery to 0.87 ± 0.22 after surgery (p < 0.001 with a paired Wilcoxon test). The CSA index decreased in 53 of 56 patients (94.6%), who consistently showed head shape improvements after corrective surgery during clinical evaluation. Linear temporal regression indicates a CSA index decrease of 0.43 ± 0.05 during the first year after surgery. We found no significant correlation between a patient’s age at surgery and the patient’s CSA index after surgery (Pearson’s correlation coefficient 0.17, p = 0.20) or the patient’s change in CSA index before and after surgery (Pearson’s correlation coefficient 0.22, p = 0.11), suggesting that sagittal craniectomy is equally effective for all patients who are between 85 and 331 days old at the time of surgery. DISCUSSION/SIGNIFICANCE: Our new CSA index is a sex- and age-specific metric of head shape anomalies built upon the observed statistical distributions in the normative pediatric population. Our metric can objectively evaluate pre- and post-surgical head shapes and will allow the investigation of the reported variability in surgical outcomes among patients and procedures.

OBJECTIVES/GOALS: There are178 million bone fractures globally each year, and 46% of fractures that have accompanying vascular damage (ischemia) will not heal without surgical intervention. Using single cell RNAseq we identified Cdk8 as a gene upregulated under ischemic fracture. Our work seeks to inhibit Cdk8 to assess its potential as a therapeutic target. METHODS/STUDY POPULATION: Most bone injuries heal through a cartilage intermediate that requires mesenchymal progenitor cells (hMSCs) to become cartilage forming chondrocytes. hMSCs underwent pelleted 3D chondrogenic differentiation in the presence of Cdk8 inhibitor, Senexin B. Chondrogenic gene expression was assessed via gene analysis of Aggrecan, Collagen II, and Collagen X. Content of sulfated glycosaminoglycans (sGAGs) was quantified through DMMB analysis. With IACUC approval, C57Bl/6 WT mice underwent femoral artery isolation and resection to create an ischemic environment prior to a transverse tibia fracture. Mice underwent intraperitoneal injections of Senexin B from day -1 to 8 and were harvested at 10 days post fracture. Control mice received vehicle injection. Calluses were analyzed through µCT and histomorphometry. RESULTS/ANTICIPATED RESULTS: At 14 days, Senexin B increased chondrogenic gene expression and improved sGAG content in hMSCs. This persisted to day 21, suggesting that Cdk8 inhibition via Senexin B promotes chondrogenesis and matrix deposition. Histomorphometric analysis reveals thatin vivotreatment with Senexin B increases cartilage content and reduces mineralization of the fracture callus compared to the Control. µCT analysis corroborates this, with distinctly less peri-cortical mineral present in Senexin B-treated calluses, and a decrease in total bone volume. These results suggest an altered progression of cartilage formation and endochondral ossification with Cdk8 inhibition. DISCUSSION/SIGNIFICANCE: Our findings reveal that increased Cdk8 is associated with poor healing in ischemic fractures. Inhibition of Cdk8 appears to increase chondrogenesis of hMSCsin vitroand in the murine fracture callusin vivo. Targeting Cdk8 offers potential to improve callus formation in impaired healing scenarios.