OBJECTIVES/SPECIFIC AIMS: This project aimed to offer Citizen Scientists basic knowledge about clinical and translational research, as well as introductory information to the topics a Citizen Scientist may work with at the University of Florida (UF). As part of that goal, several objectives were identified: (1) create a curriculum to operationalize training for new Citizen Scientists, (2) utilize best practices in educational research for curriculum design and implementation, (3) create a resource that can be freely available to other groups, and (4) implement the finished curriculum with Citizen Scientists at UF. METHODS/STUDY POPULATION: Working with an expert in the field of instructional design, an overall design plan was created and implemented. This included first conducting interviews with Citizen Scientists to determine the most appropriate format for the content, creating 10-minute videos with subject matter experts, and crafting learning assessments for each didactic video. Topics for the curriculum were conceptualized by gathering input from CTSI leaders, Citizen Scientists, and staff members, and learning objectives were created to help guide the content creation. The ADDIE (Analysis, Design, Development, Implementation, Evaluation) instructional design model was utilized to help guide the creation process, and this included a formative evaluation of the content, where assessment questions or videos were edited in response to Citizen Scientist feedback. It was important to both CTSI leadership and the Citizen Scientists themselves that the curriculum be widely accessible, so the curriculum was made as an open educational resource, meaning that it is available online for use by anyone with content that can be customized to specific programs or organizations. To implement the curriculum with UF Citizen Scientists, the materials were ported into Canvas, a widely utilized learning management system at UF. Participants were split into two groups, one group with Citizen Scientists already in the program (n=6) and a group with new Citizen Scientists (n=2). IRB approval to conduct this pilot test and share the results was obtained, and implementation ran from July 2017- January 2018. RESULTS/ANTICIPATED RESULTS: Data were taken from participant scores on the curriculum’s 15 didactic learning assessments, and while the number of attempts on assessments was not limited, only first-attempt results were analyzed. Veteran Citizen Scientists scored higher than the new Citizen Scientists, with an overall score across all assessments of 84% vs 74%. The existing Citizen Scientists performed better on most topics, however the newer Citizen Scientists outscored the veterans on two modules: Cultural Competency (90% vs 77%) and Biomedical Informatics and Big Data (80% vs 73%). The newer members also had fewer retakes on these two modules, with the newer members having only one retake for these two modules, compared to seven total retakes across both modules for the established members. Participants were also asked basic questions about learning comprehension, video quality, and assessment item clarity, in addition to offering narrative feedback. Participants across both groups seemed largely pleased with the curriculum, as indicated by results of the course evaluation. Most (75%) Citizen Scientists felt it was easy to understand the information in the video tutorials, while 75% of Citizen Scientists felt that the assessment items were comprehensive, and 62.50% felt that the assessment items matched the learning objectives. The new Citizen Scientists were far more likely to respond favorably to the video and assessment evaluation questions, with 95% of all responses marked as “agree” or “strongly agree”, compared to 57% of the responses from the established Citizen Scientists. DISCUSSION/SIGNIFICANCE OF IMPACT: The performance of new vs veteran Citizen Scientists underscores the need for introductory information on clinical research topics for new community stakeholders, as well as the need for ongoing refresher training. Though the existing Citizen Scientists had been working with these topics already, and some had experienced the modules already through the formative evaluation, some topics remained a challenge for participants, a notion reflected in their assessment scores. Clinical research, particularly translational research, can be difficult to understand, and when a stakeholder understands the fundamentals underlying the research with which they are assisting, communication barriers are eliminated, feedback is well-reasoned and actionable, and there is greater buy-in from stakeholders. Understanding the clinical research process helps community stakeholders better understand their contribution to research and offer critical feedback to aid in implementation of research findings in health-related settings.

The Recruitment Innovation Center (RIC) has created a toolkit of novel strategies to engage potential participants in response to recruitment and retention challenges associated with COVID-19 studies. The toolkit contains pragmatic, generalizable resources to help research teams increase awareness of clinical trials and opportunities to participate; produce culturally sensitive and engaging recruitment materials; improve consent and return of results processes; and enhance recruitment of individuals from populations disproportionately impacted by COVID-19. This resource, the “RIC COVID-19 Recruitment and Retention Toolkit,” is available free online. We describe the toolkit and the community feedback used to author and curate this resource.

OBJECTIVES/GOALS: Aim1: To develop a natural language processing (NLP) algorithm to effectively identify statin associated muscle symptoms (SAMS) in patients’electronic health records (EHRs). Aim2: To develop a machine learning model based on clinical features within EHRs that predict the likelihood of SAMS occurrences. METHODS/STUDY POPULATION: A retrospective cohort of adult patients initiated on statins within the Minnesota Fairview Healthcare System EHRs from 2010 to 2020 will be analyzed. NLP-PIER (Patient Information Extraction for Research) platform will be used to search and identify patients who developed SAMS after statin initiation. Manual annotation of clinical notes will be completed to validate the accuracy of identified SAMS cases. Then, a selection of clinical features within the EHRs will be input as predictors for machine learning algorithms development. Select machine learning classifiers will be deployed to generate models for the prediction of SAMS and the best-performing model will be selected based on model performance. RESULTS/ANTICIPATED RESULTS: The expected outcomes include generation of a fine-tuned NLP algorithm that can rigorously identify SAMS occurrences within EHRs. Further, we anticipate having a practical risk model that accurately predicts patients’risks of developing SAMS when taking statins. DISCUSSION/SIGNIFICANCE: The positive and translational impact of our research will be to equip healthcare providers with such informatics tools to improve statin adherence, ultimately promoting patient optimal health and outcomes by maximizing the tolerance and thus realizing the therapeutic benefits of statins.

OBJECTIVES/GOALS: To maximize health outcomes from their work, basic researchers must understand the process by which lab discoveries are translated into clinical care. We developed an academic course designed to provide students in our Clinical and Translational Sciences PhD program with an in-depth understanding of translational applications of basic research. METHODS/STUDY POPULATION: A preliminary needs assessment was done with students, educators, and clinicians to identify the course content. Based on these data, didactic modules including research question identification, research team development, participant recruitment, and research data collection were piloted in a synchronous, virtual course. Then, for 6 weeks, students shadowed clinical mentors who worked in the students research areas. Finally, with their mentors, students developed and presented clinical research protocols. Student pre- and post-course surveys gauged alignment of course objectives and learning outcomes. A post-course, focus group with students gathered feedback on course content, structure, and students confidence in implementing their experiences from the course into real-world settings. RESULTS/ANTICIPATED RESULTS: Six MD/PhD and PhD students participated in the pilot course. Pre/post-assessments (n=4) showed students were more confident in clinical question/research protocol formulation, development of patient recruitment/enrollment strategies, and integration of research methodologies into their research projects after completing the course. Students asked for additional content on budgeting and grant funding. Post-course focus group participants (n=2) appreciated the experience of writing a clinical protocol and the flipped classroom teaching style, which allowed them to network with clinical faculty leading didactic sessions. Students also noted course content was relevant and motivating, although they suggested adding content about clinical trials measures to enhance their shadowing experiences. DISCUSSION/SIGNIFICANCE: A course that combines didactic and clinical experiential training provides a robust, translational research foundation for basic scientists. This training is critical to help them contribute to the effective/efficient translation of lab discoveries to clinical practice. Future course development will include students from other PhD programs.

OBJECTIVES/GOALS: To investigate digital behavior patterns before cerebrovascular disease (CeVD), we compared accelerometer-measured physical activity (PA) phenotypes of future CeVD patients versus controls in UK Biobank. METHODS/STUDY POPULATION: Accelerometer data from 76,525 eligible participants monitored for 7-days (Jan. 2013-Dec. 2015) was classified into four categories: sedentary, light PA (LPA), moderate-to-vigorous PA (MVPA), and sleep. Covariables and diagnoses were defined using baseline data and patient records. Daily PA patterns associated with incident CeVD were compared to controls using negative binomial regression models. RESULTS/ANTICIPATED RESULTS: Adult participants with future CeVD (n = 2,163) spent 4.4% less time in MVPA (Incident Rate-Ratio (IRR) 0.956; 95% CI = 0.923-0.992; p = 0.016) compared to controls. During 0:00-5:59h (midnight to 5:59AM), future CeVD patients were less likely to sleep (IRR = 0.985; 95% CI = 0.977-0.992; p <0.001) but more likely to be sedentary (IRR = 1.189; 95% CI = 1.098-1.290; p <0.001) or in LPA (IRR = 1.108; 95% CI = 1.015-1.211; p <0.001). In subgroup analyses, decreased MVPA was observed in current/former smokers (IRR = 0.887; 95% CI = 0.819-0.963), males (IRR = 0.931; 95% CI = 0.870-0.997), and the unemployed/retired (IRR = 0.923; 95% CI = 0.856-0.998), an effect more pronounced in depressed patients (p for interaction = 0.044) and prolonged (> 2 hr/day) screen users (p for interaction = 0.018). DISCUSSION/SIGNIFICANCE: The digital phenotype of PA prior to CeVD is characterized by less sleep during 0:00-5:59h and less daily MVPA, demonstrating the utility of accelerometer data in identifying candidates for preventative intervention.

OBJECTIVES/SPECIFIC AIMS: This study aims to describe adaptability in methods used to apply community input to programming within the field of translational science. The outcomes of community informed programming include opportunities for innovative projects and approaches, and better responsiveness to community needs. It is anticipated that this will result in greater community involvement in research, moving towards greater health equity. METHODS/STUDY POPULATION: The SC CTSI is situated in urban Los Angeles, one of the most diverse communities in the world. Eight SC CTSI Community Engagement Core initiatives that employ community partnership are illustrated. The activities include social marketing campaigns for cervical cancer prevention; use of community-embedded research ambassadors to increase scientific literacy in Latino and Black/African-American communities; use of innovative technologies to educate pediatric patients and families about clinical research; working with the entertainment industry to promote clinical research in popular television shows; a community advisory board that is tailored and embedded in each CSTA core group; a community based research dissemination program; an ad-hoc community advisory group assembled to adapt a research 101 curriculum for Black/African-American communities; and a series of listening sessions conducted throughout Los Angeles. RESULTS/ANTICIPATED RESULTS: Integration of community voices provide direction for future planning, programming and execution of all referenced initiatives. Ultimately, the goal for these discussions with community members is to develop innovative approaches to CTSA programming. DISCUSSION/SIGNIFICANCE OF IMPACT: Racial and ethnic minorities continue to experience underrepresentation in clinical research trials. CTSAs have been tasked with addressing barriers that have historically led to disparities in research participation, and by extension, the effectiveness of medical interventions in diverse populations. Community input is an invaluable source for knowledge and innovative ideas in how to increase involvement in various aspects of the research process, including dissemination, recruitment and enrollment in clinical trials. CTSAs have increasingly augmented Community Engagement programs within their respective cores to address population disparities. The approaches used to engage communities require an element of fluidity and flexibility, and a reliance on the input of community members, in order to maintain relevant and desired community engagement practices.

OBJECTIVES/GOALS: The objective of the study was to determine whether CLP altered genes associated with mitochondrial function in the diaphragm. METHODS/STUDY POPULATION: A rodent cecal-ligation and puncture (CLP) model used to mimic sepsis-induced critical illness. The CLP model involved ligation of 50% of the cecum below the ileocecal valve in adult C57BL6 mice, followed by needle puncture of the cecum resulting in mid-grade sepsis. Mice survived for 48 hours or more, following injury. Diaphragm and limb muscles were harvested 24 hours following CLP (N = 6) and following a sham CLP procedure (N = 6). RESULTS/ANTICIPATED RESULTS: Gene expression of mitochondrial related genes (mef2c, myh1, pgc1-α), were significantly decreased in the diaphragm of CLP injured animals when compared to controls. In addition, ubiquitin ligases, genes associated with skeletal muscle atrophy murf1 and atrogin were increased in the diaphragm 24 hours after injury (p< 0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that sepsis-induced critical illness significantly impacts the expression of genes implicated in mitochondrial homeostasis and atrophy. Ongoing studies will identify whether CLP injury decreases skeletal muscle mitochondrial function.

OBJECTIVES/GOALS: MGMT methylation status is used to predict the response to TMZ. However, a subpopulation of patients lacking MGMT methylation still respond to TMZ. We applied omics approaches and functional studies to a cohort of GBM patients to identify novel markers that may more accurately predict TMZ response. METHODS/STUDY POPULATION: We applied a combination of omics approaches and functional studies to a cohort of GBM patients to search for novel markers that would predict the response to TMZ treatment more accurately than traditional markers. Using a set of 47 primary and secondary GBM tumor samples, we employed comparative transcriptomics, whole exome sequencing, data independent acquisition (DIA) proteomics, and phosphoproteomics to look for DNA mutations and changes in gene expression and/or protein expression that correlated to response or non-response to TMZ. Subsequently, we performed functional studies and analyzed patient treatment data to validate our results. RESULTS/ANTICIPATED RESULTS: This study is in early stage, but we anticipate that our combination of methods may allow us to identify and validate at least one novel biomarker for TMZ response in patient GBM. For example, comparative transcriptomics or phosphoproteomics may identify a previously unrecognized gene or protein over/under-expression in a subset of patients. In this case we will validate findings using western blotting, IHC staining, and through siRNA of target gene/protein on patient derived GBM cells to examine if removal of this marker leads to TMZ sensitivity. We will further confirm prediction of marker correlation through comparison with matched patient treatment data. DISCUSSION/SIGNIFICANCE: The identification of improved biomarkers to predict response to TMZ treatment is a discovery that could rapidly become standard of care for GBM patients. It would ensure that all responders receive TMZ and avoid exposing nonresponders unnecessarily to TMZ and its potential side effects.

OBJECTIVES/GOALS: We previously reported that genetic polymorphisms in the beta-catenin gene (CTNNB) are associated with the development of Clostridiodes difficile colitis during autologous stem cell transplantation (https://www-ncbi-nlm-nih-gov.proxy.libraries.uc.edu/pubmed/29594489). To biological validate these findings, we sought to evaluate the development of chemotherapy-associated Clostridiodes difficile infections by assessing the effect of C.difficile toxin B (TcdB) and of using melphalan in beta-catenin protein expression in Caco2 cells. METHODS/STUDY POPULATION: To determine the effect of melphalan and/or C.difficile toxin B on expression of Beta-catenin from human gut epithelial cells:

• Adenocarcinoma cells (Caco-2) cells were seeded and allowed to grow into monolayers

• Monolayers were treated with PBS, TcdB, melphalan and/or TcdB + melphalan for 24 hours and then washed with PBS

• Immunofluorescence was measured on the monolayers to visualize three markers -DAPI-Nuclear Stain (blue),Actin-ccytoskeletal stain (red), B-Catenin (green)

• Analysis of images with ImageJ (NIH). Statistical analysis of the effect of TcdB and/or melphalan on β-catenin protein levels was determined by One-way ANOVA

OBJECTIVES/GOALS: Despite calls for the integration of Education for Sustainable Healthcare (ESH) into health professional training programs, most curricula have yet to adapt accordingly. This qualitative study sought to understand faculty perspectives on ESH knowledge, interest, barriers, and facilitators. METHODS/STUDY POPULATION: From 2018 to 2020, 71 health professional education faculty from 6 University of California (UC) campuses participated in ESH integration workshops. Using purposeful sampling based on gender and campus, a subset of workshop faculty participants were selected to participate in individual interviews. Interviews were conducted via Zoom using a structured interview guide, eliciting participants experience integrating ESH, perceived barriers and facilitators, and perspectives on student, faculty, and health science leadership knowledge and interest. Transcripts were double coded with an inductive-deductive approach using Dedoose, reconciled, and analyzed to identify themes. RESULTS/ANTICIPATED RESULTS: Participants included 17 faculty at 6 UC campuses representing diverse health disciplines. Although participants noted high general awareness of and interest in climate change among students and faculty, they observed a lack of specific, health-relevant knowledge, resulting in discomfort communicating with others on climate and health. Perceived barriers to expansion of ESH included limited curricular space, competing topics, and lack of faculty expertise. Participants posited that framing climate change in health terms, establishing learning objectives and protected faculty time, identifying connections to ESH within existing research and curriculum, and obtaining commitments from campus leadership would facilitate successful ESH integration. DISCUSSION/SIGNIFICANCE: Our findings reinforce student and faculty interest in ESH curricular integration and identify important barriers and facilitators, lending context for educators planning ESH infusion. Training of faculty on climate health is urgently needed for ESH integration in health professional education.

ABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value < 10-5. For each SNP, we identified the region that was in linkage disequilibrium (r2 > 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases.

OBJECTIVES/GOALS: Our goal was to distinguish SARS-CoV-2 CD8+ T cell epitopes of spike, membrane, and nucleocapsid products in 27 of the most frequent HLA-Aand -B alleles.We hypothesize that differences mediated by variation in SARS-CoV-2 and host HLA genetics affect the differential clinical severity and presentation of acute infection and PASC. METHODS/STUDY POPULATION: Genomic sequences of SARS-CoV-2 variants were blasted against the original Wuhan strain using Ensembl’s SARS-CoV-2 browser. We examined 16 COVID variants: 2 Alpha (B.1 and B.1.1.7), 5 Delta (AY.100, AY.25, AY.3, AY.3.1, and AY.44), and 9 Omicron (BA.1, BA.1.1, BA.2, BA.4, BA.5, BQ.1, BQ.1.1, XBB.1, and XBB.1.5), sequenced from the Louisiana patient population. cDNA sequences were translated using the Expasy tool. To predict MHC-I epitope binding, we used the Immune Epitope Database and Analysis Resource, via TepiTool utilizing the IEDB recommended default prediction and the 27 most frequent HLA-A and -B alleles. In silico peptide docking was conducted on FoldX, utilizing HLA-B*15:01 structures (n= 7) from the Protein Data Bank. RESULTS/ANTICIPATED RESULTS: CD8+ epitope conservation was estimated at 87.6-96.5% in S, 92.5-99.6% in M, and 94.6-99% for N. As the virus mutated, an increasing proportion of S epitopes experienced reduced predicted binding affinity: 70% of Omicron BQ.1- XBB.1.5 S epitopes experienced decreased predicted binding, as compared to ~ 3% and ~15% in Delta AY.100-AY.44 and Omicron BA.1-BA.5 respectively. Additionally, we identified several novel candidate haplotypes that may be susceptible to severe disease, notably HLA-A*32:01, -A*26:01, -B*58:01, and -B*53:01, and relatively protected from disease, such as -A*01:01, -A*02:01,-A*31:01, -B*15:01, -B*40:01, -B*44:03, and -B*57:01. In silico analysis of COVID peptides and HLA-B*15:01, a common allotype in the United States, largely matched predicted binding patterns. DISCUSSION/SIGNIFICANCE: To elicit long term COVID-19 immunity and prevent PASC, it is important to understand the relationship between T-cells, viral variants, and HLA genetics. This project is one of the first to explore the interaction between CD8+ epitope diversity and viral genetics for the majority of the United States population.

OBJECTIVES/GOALS: Variants in voltage-gated sodium channels (VGSC) are a common cause of severe early onset epilepsy. Changes in CSF neurotransmitters (NT) were identified in 2 cases of VGSC-related epilepsy. Here we investigate NT changes in patients and a novel mouse model of VGSC-related epilepsy. METHODS/STUDY POPULATION: We conducted a single site IRB approved retrospective chart review of patients with VGSC-related epilepsy who underwent CSF NT testing for diagnostic purposes. In parallel, we examined NT levels from the brains of wild-type (WT) and a novel VGSC-related epilepsy mouse model after obtaining IACUC approval. We rapidly isolated forebrain, cortex, striatum, and brainstem from 5-6 animals per sex and genotype. A combination of HPLC with electrochemical detection and mass spectrometry were used to quantify NT levels from brain samples. RESULTS/ANTICIPATED RESULTS: We identified 10 patients with VGSC-related epilepsy who received CSF NT testing. Two of these patients had abnormal NT results including changes to dopamine (DA) or serotonin (5-HT) metabolites. We analyzed NT levels from four brain regions from male and female WT and VGSC-related epilepsy mice. We anticipate that most of the NT levels will be similar to WT, however subtle changes in the DA or 5-HT metabolites may be seen in VGSC-related epilepsy. DISCUSSION/SIGNIFICANCE OF IMPACT: Patients with VGSC-related epilepsy often have autism spectrum disorder, sleep, and movement disorders. Understanding the role of aberrant NT levels in VGSC-related epilepsy may provide additional therapeutic targets that address common neuropsychological comorbidities as well as seizures.

OBJECTIVES/GOALS: Development of a user friendly home kit that enables kidney transplant recipients to process urine at home and post the lysate containing RNA to a Core Laboratory would simplify urinary cell mRNA profiling and facilitate longitudinal monitoring. We report our home processing protocol and investigation of its diagnostic performance characteristics. METHODS/STUDY POPULATION: We developed a home processing protocol (HPP) consisting of urine filtration and lysis of urinary cells, both performed at home by the kidney transplant recipients (KTR) themselves, followed by isolation of total RNA from the lysate and mRNA enrichment using a silica-membrane-based cartridge, both performed at the Core Laboratory. Using the HPP, total RNA was isolated from kidney allograft biopsy-matched urines and absolute copy numbers of CD3εmRNA, CXCL10 mRNA, and 18S rRNA, components of the Clinical Trials in Organ Transplantation 04 (CTOT-04) three-gene TCMR diagnostic signature, and urinary cell BKV VP1 mRNA copy number, were measured using customized RT-qPCR assays. RESULTS/ANTICIPATED RESULTS: CTOT-04 three-gene TCMR diagnostic signature scores in urine processed using HPP discriminated KTR with TCMR (12 TCMR biopsies from 11 KTR) from KTR with no TCMR/BKVN (29 No TCMR/No BKVN biopsies from 29 KTR) (P=0.0005, Mann-Whitney test), and AUROC was 0.84 (95% CI, 0.69 to 0.98). TCMR was diagnosed with sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of -1.213 (P=0.0016,Fisher exact test). BKV VP1 mRNA copy number in urine processed with HPP discriminated KTR with BKVN (n=7) from KTR with no TCMR/BKVN (n=29) and AUROC was 1.0 (95% CI, 1.00 to 1.00). BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5x10^8 BKV VP1 mRNA copies/μg of RNA (P DISCUSSION/SIGNIFICANCE: Urine processed using the HPP predicted TCMR and BKVN in KTR. The HPP represents not only a significant advance towards portability of urinary cell mRNA profiling but also should improve patient management by minimizing visits for urine collection.

ABSTRACT IMPACT: Characterizing specialized pro-resolving lipid mediators (SPMs) of inflammation in a cohort of adult patients with traumatic brain injury (TBI) will potentially identify novel biomarkers of disease and would generate hypotheses regarding the role of SPMs in the pathophysiology and recovery after brain injury OBJECTIVES/GOALS: Primary aim: evaluate the association between plasma and cerebrospinal fluid (CSF) SPM levels with inflammatory markers known to mediate blood brain barrier (BBB) disruption. Secondary aim: evaluate the association between SPM levels and disease severity, 14-day Glasgow Coma Scale score (GCS) and survival. METHODS/STUDY POPULATION: This is a single-center, prospective, observational cohort study (target N=50). Adult participants with moderate-to-severe non-penetrating TBI will have blood and CSF sampled serially for laboratory measures of SPMs (resolvins, protectins, lipoxins, maresins) and inflammatory peptides (TNF-α, IL-1β, IL-6, MMP-9, TIMP-1) by liquid chromatography’‘ mass spectrometry and RT-PCR & ELISA, respectively. Baseline patient characteristics, admission GCS, and 14-day GCS and mortality will be prospectively collected. To evaluate the association between SPMs and other continuous variables, Pearson’s and Spearman’s correlation will be used. Cox regression will be used to evaluate the association between SPM lipidomes and 14-day survival. RESULTS/ANTICIPATED RESULTS: As the primary outcome measure, the association between SPM levels with assayed inflammatory markers will be determined at 24 and 72 hours post-TBI. We hypothesize that SPMs will be negatively correlated with TNF-α, IL-1β, IL-6, MMP-9 and positively correlated with TIMP-1. As secondary outcome measures, the association between SPM levels with disease severity by admission GCS will be determined at 24 hours post-TBI, and the association between SPM levels and GCS and mortality will be determined at 14-days. We hypothesize that SPM levels will be positively associated with admission and 14-day GCS and will be independently associated with 14-day survival. DISCUSSION/SIGNIFICANCE OF FINDINGS: Using the SPM lipidome as a biomarker of disease is a novel tool for future translational research. It will inform a foundational mechanistic framework for TBI pathophysiology and attenuation of neuroinflammation post-TBI, providing rationale for pre-clinical and clinical research focused on novel therapeutics

OBJECTIVES/GOALS: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatologic disease childhood and a cause of pain and potential disability. JIA has a strong genetic component and no known cure. The goal of this study is to evaluate allele-dependent effects of a novel JIA risk variant at 1q24.3. METHODS/STUDY POPULATION: JIA patients meeting criteria for the two most common disease subtypes (oligoarticular and RF neg polyarthritis) were genotyped using the Immunochip, an Illumina array with dense coverage of the HLA region and 186 other loci previously reported in autoimmune diseases. Phase I association findings (Hinks, 2013) and Phase II analysis (unpublished) of an expanded cohort (4,271 JIA and 14,390 controls) identified new risk loci, including rs78037977 at 1q24.3. We prioritized rs78037977 and predicted possible impacted mechanisms based on Bayesian predictions of attributable risk, the surrounding chromatin landscape, and transcription factor binding data. A luciferase reporter assay was used to assess allele-dependent enhancer activity. RESULTS/ANTICIPATED RESULTS: rs78037977 is located between FASLG and TNFSF18 at chromosome 1q24.3 is associated with JIA (p = 6.3x10−09), and explains 94% of the posterior probability at this locus; no other SNPs in linkage disequilibrium (r2>0.6). The chromatin landscape around rs78037977 contains H3K4Me1 and H3K27Ac marks, which are indicative of enhancer activity. Further, >160 transcription factors have chromatin immunoprecipitation followed by sequencing (ChIP-seq) peaks overlapping rs78037977 in various cellular contexts. In luciferase reporter assays, the region around rs78037977 containing the reference A allele had ~2-fold increased enhancer activity compared to the non-reference allele. DISCUSSION/SIGNIFICANCE OF IMPACT: This work provides in vitro evidence to support allele-dependent enhancer activity of a novel JIA-risk variant at 1q24.3. Our ongoing work investigates the effect of the DNA-containing region of rs78037977 on gene expression and differential transcription factor binding at rs78037977.