OBJECTIVES/GOALS: To develop an untargeted metabolomics assay that can holistically characterize the small molecule signatures of different inborn errors of metabolism (IEM) for biomarker discovery and identification of novel IEMs, with the goal of implementing the assay into the clinical laboratory to improve testing efficiency. METHODS/STUDY POPULATION: A hydrophilic interaction liquid chromatography (HILIC) column and reverse phase (RP) column were assembled in tandem on a SCIEX X500B quadrupole time-of-flight (QTOF) system to create a dual liquid chromatography (LC), tandem mass spectrometry method. The X500B was operated in data-independent acquisition mode with both positive and negative ionization. A mixture of 165 reference standards from eleven compound classes common to IEMs were used to evaluate the capability of the assay to resolve small molecules. Chromatographic resolution for each standard was determined qualitatively by comparison to a reference spectral database. External validation of the assay will be performed by analyzing a commercial library of reference metabolites. RESULTS/ANTICIPATED RESULTS: A total of 88% (146/165) of the standards were detected by the assay. The RP column alone resolved 71% (117/165) of the standards, the HILIC column resolved 33% (55/165), while 17% (29/165) of the standards were resolved by both columns. The HILIC column resolved standards that were more polar, while the RP column resolved more non-polar compounds. To evaluate matrix effects, the reference standard mixture was spiked into pooled plasma. In the presence of plasma 6/146 (4%) of the standards were suppressed to levels below the limit of detection. We expect external validation with the commercial metabolite library will corroborate these results, and that the high-quality spectral data attained from this reference library can be used to improve identification of unknown metabolites in patient samples. DISCUSSION/SIGNIFICANCE: We have shown our untargeted metabolomics assay can detect known biomarkers for IEMs. Clinical implementation of this method could streamline diagnosis of IEMs while simultaneously improving patient outcomes by leveraging the metabolome for biomarker discovery, and improved understanding of IEM mechanisms to inform novel treatment strategies.

ABSTRACT IMPACT: This study will provide important insight about effective team formation from coming up with an idea to successfully implementing that idea, as well as will highlight the implementation, evolution, and future directions of a team science initiatives. OBJECTIVES/GOALS: The goal of this study is to describe the feasibility of initiating an ideation forum to catalyze team formation, explore the process by which themes and teams are selected to participate in the forum setting, and assess the progress of participating teams post-forum through internal and external funding and other synergistic research activities. METHODS/STUDY POPULATION: Three ideation forums took place between 2018-2019 at Rutgers University, with a defined process and collection of data. The method of intervention to trigger team science, specifically the methodology employed to identify teams and produce new collaborative ideas, will first be described to show the feasibility of such an event to encourage team formation. In post-hoc analysis, we compare various success matrices of participating teams received seed funding versus teams that didn’t receive any funding to assess the progress of teams in the research ideation forum incubation process. RESULTS/ANTICIPATED RESULTS: Triggering team science through ideation forums is feasible and, in fact, quite productive to creating a durable response in formed teams showing continued productivity in publications, fundraising, and other academic metrics. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our case review can illuminate how academic institutions can support team science research through ideation forums. In addition, this study lays an initial foundation for improvements in ideation forum creation and new metrics that can be shared broadly to compare across other institutions.

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.

OBJECTIVES/GOALS: Knocking down the inflammatory response following joint trauma may halt the cytokine cascade and prevent the resulting cyclic degradation of articular cartilage. MK2 inhibiting (MK2i) peptides are an emerging and promising class of pharmaceutical to treat post-traumatic osteoarthritis (PTOA); however, these peptides are susceptible to proteolytic degradation in the extracellular space. Our objective is to encapsulate MK2i in thermoresponsive hollow nanoparticles (hNPs) to knockdown the inflammatory cytokine IL-6 to prevent the cyclic degradation of articular cartilage. METHODS/STUDY POPULATION: NP Synthesis: N-isopropyl acrylamide (NIPAm) cores was initiated by potassium persulfate (KPS) in aqueous solution with sodium dodecylsulfate (SDS) at 70°C under a nitrogen for 2 hours. Then exposed to oxygen for 45 min, followed by a nitrogen purge. NIPAm, 2-acrylamindo-2-methyl-1-propanesulfonic acid (AMPS), N,N’-bis(acryoyl)cystamine (BAC), and Acrylic Acid (AAc), in fluorescent batches rhodamine b isothiocyanate (RBITC), were polymerized around the core to form the shell. NPs were purified using tangential flow filtration. The NPs were dialyzed at 4°C for 14 days to remove the core and form hNPs. Loading & Release: hNPs and MK2i were incubated at 1 mg/ml at 4°C for 24 h. MK2i released into 1x PBS and analyzed on HPLC. IL-6 Expression: Bovine chondrocytes seeded at 10,000 cell/cm2 were stimulated with 20 ng/ml IL-1b daily and treated once with 100 µg/ml MK2i loaded-NP or 100 µg/ml free MK2i treatment on day 2. Analyzed on bovine IL-6 ELISA. In Vivo Intra Articular Injections: 75 µl of 2 mg/ml hNPsRHB or a PBS control was injected into the right knee of 4-month old Fischer 344 (Envigo) rats. Rats were imaged daily for 7 days then euthanized, legs dissected, and imaged. RESULTS/ANTICIPATED RESULTS: Core removal facilitated increased MK2i release from hNPs, Fig 1A, allowing up to 63% after 5 days in PBS. The hNPs generated here offer a continual sustained release of MK2i and hNPs are non-cytotoxic (data not shown) up to 12 mg/ml. MK2i loaded-NPs significantly knocked down IL-6 production after a single treatment after 2 days, Figure 1B, and continued knockdown for up to 4 days. hNPsRBITC was successfully injected into rat joint space and was retained for at least 7-days compared to pre-injection and PBS control, Fig 1 B-C. DISCUSSION/SIGNIFICANCE OF IMPACT: hNPs protect MK2i from ECM degradation and offer continual sustained release into chondrocytes. Core removal allows for MK2i release in vitro with further sustained release compared to previous non-degradable model. The single MK2i treatment lead to a significant IL-6 knockdown bovine chondrocytes for up to 4 days in hNPs. We were able to successfully inject and retain fluorescently labeled hNPs within rat knees for 7 days. Our translational therapeutic shows the promise of delivering a degradable, non-cytotoxic hNP into the joint space to knockdown the inflammatory response to halt the cyclic progression of articular cartilage degradation and progression of PTOA. CONFLICT OF INTEREST DESCRIPTION: The authors declare the following competing financial interest(s): Moerae Matrix, Inc. has a worldwide exclusive license to the CPP (MK2 inhibitor peptide). A. Panitch owns greater than 5% of Moerae Matrix, Inc.

OBJECTIVES/SPECIFIC AIMS: The primary aim is to assess differences in therapeutic effect between MSC and EPC EVs on acute ischemic rat hearts through delivery in a biocompatible and shear-thinning hydrogel. Primary outcomes for therapeutic assessment include an in-vitro angiogenesis assay and in-vivo hemodynamic analysis, mainly identifying differences in ejection fraction and contractility. Secondary hemodynamic outcomes include cardiac output, stroke volume, and end-diastolic pressure volume relationship (EDPVR). Secondary structural outcomes include post-mortem scar analysis and immunohistochemistry (IHC) staining for angiomyogenesis. METHODS/STUDY POPULATION: MSCs and EPCs will be cultured according to previously published protocols. EVs will be isolated from cultured cell lines through precipitation methods with polyethylene glycol. EVs will be qualitatively analyzed with nanoparticle tracking analysis (NTA) and flow cytometry. The shear thinning hydrogel (STG) will be constructed using a hyaluronic backbone conjugated to adamantane or beta-cyclodextrin, ultimately facilitating guest-host interactions with shear thinning properties. Controls and treatment groups mixed with the hydrogel will be injected into the border zone of infarcted Wistar rat hearts immediately following a left anterior descending artery ligation. Hemodynamic assessment will be performed at four weeks through left ventricular catheter based pressure-volume recordings. Ex-vivo analysis will include scar thickness assessment using Masson collagen staining and IHC stain for vessel (anti-vonWillebrand factor; anti-Isolectin) and myocyte formation (anti-cardiac Troponin I). RESULTS/ANTICIPATED RESULTS: We hypothesize that, in-vitro, MSC-EVs will demonstrate non-inferior angiogenic potential as compared to EPC-EVs. We posit that MSC-EVs will demonstrate superior therapeutic effect to EPC-EVs in-vivo as measured by functional hemodynamics and structural assessment. We have successfully isolated MSC and EPC EVs and have validated uniformity across EV populations (Figure 1). Preliminary data from the angiogenesis assay (n=3) demonstrated that MSC-EV and EPC-EV produce non-significantly different angiogenic potential as measured by number of vascular meshing extremes (p=0.144) and length of master vascular segment (p=0.193), with significant differences compared to either positive or negative controls. DISCUSSION/SIGNIFICANCE OF IMPACT: Novel regenerative therapies are needed for patients with a history of AMI given current limitations to therapy and sequelae of ischemic heart disease. Delivery of extracellular vesicles through a shear-thinning gel is a novel “off-the-shelf” translational approach to address the current clinical need.

OBJECTIVES/SPECIFIC AIMS: To understand the mechanisms of how a non-antimicrobial can reshape a commensal microbe community to cure a ubiquitous human disease. METHODS/STUDY POPULATION: Whole genome sequencing of bacterial isolates, metabolomic investigations of previously collected skin microbe isolates from patients, and structural investigations of a protein from these skin microbes. RESULTS/ANTICIPATED RESULTS: Metabolic pathways associated with adaptation to a changing skin microenvironment, novel antimicrobial characterization, and a structural understanding of a novel nutrient acquisition protein. DISCUSSION/SIGNIFICANCE OF IMPACT: Multiple angles of this investigation are poised to improve current non-antimicrobial dermatologic treatments and they have the potential to impact microbe-related diseases in other human microenvironments.

OBJECTIVES/GOALS: Patient adherence with antidepressant therapy is a critical aspect of clinical management. Drug molecules often interact with other off-target proteins resulting in adverse events. We utilized an interactome model for predicting the likelihood of adverse drug reactions in order to accurately prescribe antidepressant medications to a patient. METHODS/STUDY POPULATION: We utilized an interactome model to study physical interactions between proteins and biological functions disrupted by major depression to understand how changes in genes alter patient-specific drug efficacy and cause adverse drug-reactions. To study how drugs and diseases propagate through the proteins and biological functions, we harnessed diffusion profiles to represent nodes in the graphical model and used a biased random walks model to illustrate how signals propagate in a heterogeneous biological network. The edge weights were defined for the drug, disease, protein, and biological function node types. The interactions studied included drug-protein, disease-protein, protein-protein, protein-biological function, biological function-biological function. RESULTS/ANTICIPATED RESULTS: Based on previous studies, we anticipate that the genetic variants modulating antidepressant response include 5-HTT, STin2, HTR1A, HTR2A, TPH1 and BDNF. Additionally, genetic variants such as SLC6A4 as well as the HPA pathway may play an important role in antidepressant therapeutic response. With regards to medication drug response, the genes, SLC6A4 and HTR2A, have been known for encoding proteins that affect the synaptic cleft of serotonergic neurons. Also, the gene SLC6A4 has been shown to encode for the serotonin reuptake transporter, which is the main pharmacological target of SSRIs. In female patients with major depression, the polymorphism associated with MAOA gene may be involved in the pharmacological response. DISCUSSION/SIGNIFICANCE: The key to successful depression treatment is early adherence but nearly 60% of patients discontinue antidepressants within three months. By developing a strong understanding of the genes that alter treatment efficacy, we can provide patients with more awareness regarding the most effective treatment plan to minimize adverse events.

OBJECTIVES/GOALS: Negative emotions (NE) play a pivotal role in addiction-related processes, including tobacco lapse during a quit attempt. Some NEs (e.g., shame, guilt) are posited to lead to a spiraling effect, whereby lapse predicts increased NEs leading to further lapse. This study goal is to examine associations between NEs and lapse. METHODS/STUDY POPULATION: This study examined associations between tobacco lapse and 13 distinct NEs among people who use tobacco and are trying to quit in two tobacco cessation studies. In Study 1, 220 adult (ages 18-74) cigarette users who identified as Black (50% female) participated in a 14-day study where ecological momentary assessment (with assessments approximately every 4 hours) was used to assess emotions and lapse in real-time and real-world settings. In Study 2, 288 adult (ages 18-71) cigarette users who were low socioeconomic status (51% White, 14% Black, 10% Hispanic, 49% female) participated in a 14-day study with the same study protocol as Study 1. Between and lagged within-person associations testing links between distinct NEs and lapse were examined with multilevel modeling with logistic links for binary outcomes. RESULTS/ANTICIPATED RESULTS: Results from Study 1 suggested that at the between-person level, disgust (OR =1.22, CI: 1.05, 1.42), nervousness (OR=1.23, CI:1.05,1.43), guilt (OR=1.40, CI: 1.16,1.69), and sadness (OR=1.18, CI:1.02,1.36) were predictive of higher odds of lapse, and at the within-person level, shame (OR=1.23, CI:1.04,1.45) was associated with higher odds of lapse. Results from Study 2 were similar and suggested that at the between-person level, disgust (OR=1.35, CI: 1.16, 1.56) and guilt (OR=1.88, CI:1.07,3.30), and at the within-person level, shame (OR =1.31, CI:1.10,1.55), were associated with higher odds of lapse. DISCUSSION/SIGNIFICANCE: The present study uses real-time, real-world data to demonstrate the role of distinct NEs on momentary tobacco lapse and helps elucidate specific NE that hinder the ability to abstain from tobacco use during a quit attempt. Results suggest that disgust, guilt, and shame play consistent roles in predicting lapse among diverse samples of tobacco users.

ABSTRACT IMPACT: Implement and evaluate a fellowship program to foster a new generation of entrepreneurial and collaboratively-minded team scientists, equipped with the knowledge and skills to innovate technology-based solutions for COVID-19 to advance human health OBJECTIVES/GOALS: Mount Sinai Targeted Healthcare Innovation Fellowship (THRIVE) is a 9-month program for participants from diverse professional backgrounds to develop HealthTech innovations related to COVID-19. The program is designed to provide an experiential team science platform for fellows to take an idea from concept to commercially viable innovation. METHODS/STUDY POPULATION: Following a competitive application process, 16 THRIVE fellows comprise four teams working collaboratively in an online forum with input from experts in the field. Success of the program will be evaluated by:

assessing pre- and post- collaborative research orientation among THRIVE fellows using the ROI scale1

using social network analysis (SNA) to investigate the social networks of THRIVE fellows to capture patterns of communication and collaboration related to innovation development

exploring participant experiences of group formation, teamwork and collaboration related to innovation development using one-to-one semi-structured interview

determining team success in innovation development, measured by number of publications, funding awarded, provisional patents and viable products. RESULTS/ANTICIPATED RESULTS: Paired t-tests will determine whether collaborative orientation of THRIVE fellows changes pre- vs. post- program participation, indicating changes in attitude toward multidisciplinary team work. SNA will be used to describe structural patterns of communication that occur at individual and group levels. Network-level indices will provide insight into patterns of communication that exist in innovation development: degree centrality (number of connections per individual), betweenness centrality (number of bridges to others in a network), closeness centrality (closeness to others in a network). We will also test for associations between network characteristics and team success. DISCUSSION/SIGNIFICANCE OF FINDINGS: Understanding patterns of formal and informal relationships, interactions, and perceptions of the collaborative process among individuals in THRIVE teams will elucidate whether such a program can provide an effective forum for team science and innovation development related to COVID-19.

OBJECTIVES/GOALS: The use of P2Y12 receptor inhibitors like Clopidogrel is crucial in the prevention of thrombotic events in patients with coronary artery disease, peripheral arterial disease, and cerebrovascular disease. Variation in the level of platelet inhibition is present in many patients, and it is associated with the occurrence of major adverse cardiovascular events (MACEs). The term High-on treatment platelet reactivity (HTRP) is used to describe impaired antiplatelet inhibition while on Clopidogrel. Multiple factors have been associated with the presence of HTPR in patients with CAD and PAD, including CYP2C19 loss of function polymorphism, drug-drug interactions, and medical comorbidities. Gender differences are another factor that might influence the levels of platelet inhibition while on Clopidogrel and hence, HTPR. Differences by Gender exist in platelet biology, count, and activation. The evidence for the influence of Gender in HTPR is limited, but a possible association has been described. In this study, we described the association of Gender with HTPR and Major Adverse Cardiovascular Events (MACEs) occurrence. The data is from a sample of Hispano-Caribbean patients on Clopidogrel therapy alone or in combination with Aspirin that were retrospectively evaluated from an ongoing trial in Puerto Rico. The result of this study provided evidence of the influence that Gender has on antiplatelet therapy function and MACEs occurrence. METHODS/STUDY POPULATION: The population in the study consisted of Hispano-Caribbean patients using Clopidogrel alone or in combination with Aspirin for coronary artery disease, peripheral arterial disease, or cerebrovascular disease. The sample was obtained from multiple hospital institutions with cardiovascular services in Puerto Rico during the years 2016-2019. Patients were part of the ongoing trial, “Adopting a precision medicine paradigm in Puerto Rico: leveraging ancestral diversity to identify predictors of Clopidogrel response in Caribbean Hispanics.” The sample size consisted of 150 patients. Participants were recruited during routine medical care, pre-admission evaluation for elective cardiac procedures, or during hospitalization in the participating institutions. Platelet reactivity testing was performed with the system Verify Now® to determine PRU values, and High on-treatment platelet reactivity was defined as PRU ≥208. One year after recruitment, the patients were re-evaluated for the occurrence of MACEs. The association of the variables HTPR, occurrence of MACEs, and Gender were assessed using logistic regression in addition to the role of HTPR and Gender for predicting MACE occurrence. The analysis was done using the statistic software Intellectus ©. RESULTS/ANTICIPATED RESULTS: The sample consisted of 67 females and 83 males with and Mean age of 67.87 years and 61.11 years, respectively. The prevalence of HTPR in the sample was 32.67 % (n = 49) with 36% (n = 24) for females, and 30%(n = 25) for males. The mean PRU values were 179.54 for females and 170.81 for males. The percentage of MACEs one year after recruitment was 29.33 % (n = 44) with 43% on females (n = 19), and 57% on males (n = 25). Logistic regression for Gender predicting HTPR was non-significant with a χ2(2) = 0.55, p = .758, and McFadden R2 = 0.00. Also, logistic regression for the effects of Gender and HTPR on the Odds of MACEs occurrence was not significant based on a model with an alpha of 0.05, χ2(2) = 1.99, p = .370, and McFadden R2 = 0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: The sample consisted of 67 females and 83 males with and Mean age of 67.87 years and 61.11 years, respectively. The prevalence of HTPR in the sample was 32.67 % (n = 49) with 36% (n = 24) for females, and 30%(n = 25) for males. The mean PRU values were 179.54 (±70.42) for females and 170.81(±64.89) for males. The percentage of MACEs one year after recruitment was 29.33 % (n = 44) with 43% on females (n = 19), and 57% on males (n = 25). Logistic regression for Gender predicting HTPR was non-significant with a χ2(2) = 0.55, p = .758, and McFadden R2 = 0.00. Also, logistic regression for the effects of Gender and HTPR on the odds of MACEs occurrence was not significant based on a model with an alpha of 0.05, χ2(2) = 1.99, p = .370, and McFadden R2 = 0.01.

OBJECTIVES/GOALS: Extracellular vesicles (EVs) in biofluids can reflect pathology in the brain (like alpha-synuclein protein linked to Dementia with Lewy Bodies) making them attractive disease biomarkers to distinguish disease. Here, we will investigate their potential as non-invasive biomarkers to differentiate Dementia with Lewy Bodies & Alzheimer's Disease. METHODS/STUDY POPULATION: We will leverage the collection of antemortem plasma from autopsy confirmed Dementia with Lewy Bodies (DLB), Alzheimer's Disease (AD) & healthy controls (HC) through the Mayo Clinic Brain Bank. We will characterize antemortem brain-derived plasma EVs (bPEVs) & post-mortem brain-derived EVs (bEVs) of the same patient using nanoparticle tracking, electron microscopy, & western blotting for EV-associated proteins. We will then measure levels of total & phosphorylated alpha-synuclein (asyn) using AlphaLisa. We will assess EVs' ability to initiate asyn aggregation using a real-time quaking-induced conversion (RT-QuIC) assay & a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Once characterized, this will allow differentiation of bpEVs in DLB, AD, or healthy controls. RESULTS/ANTICIPATED RESULTS: We hypothesize that asyn levels and seeding capacity will discriminate DLB from AD. With AlphaLisa assay, we expect higher total and phosphorylated asyn levels in DLB bpEVs and their corresponding post-mortem bEVs. Using RT-QuIC and FRET assay, we expect EVs isolated from DLB patient samples to support seeded aggregation, whereas EVs from AD and HC will not. DISCUSSION/SIGNIFICANCE: To date, no diagnostic or less invasive biomarkers can distinguish DLB from AD. The successful completion of the aims outlined in this proposal will identify characteristics of bpEVs that differentiate DLB from AD or HC and support the development of bpEVs as a non-invasive, early biomarker to diagnose patients presenting with dementia from DLB.

Objectives/Goals: Oral health is an important and understudied part of overall health. Poor oral health is linked to many systemic conditions, but little has been done to explore these issues in large electronic health records data sources that include dental health records. Here we report on our exploration of data readiness and completeness of three of these data sources in the Clinical and Translational Science Awards (CTSA) network. Methods/Study Population: Three CTSAs from the Consortium of Rural States (CORES) with diverse geographies, demographics, and data ecosystems can integrate medical and dental records, but it is unknown if the target population having both dental and medical records have sufficient completeness and similarity to enable dental/medical health studies. Here we use descriptive analytics to characterize the demographics, and the “complete data” approach presented by Weber et al. to evaluate differences between the completeness of the general populations and the one having both dental/medical records. We accomplish this by identifying patients with dental records in commonly used research networks and performing empirical patient statistics in comparison to the entire population available at the three institutions. Results/Anticipated Results: This poster will present the results of using the Weber et al. approach to compare the completeness of records of the general patient population in the Iowa, Kentucky, and Utah medical/dental health care systems to those for which they have also dental records. The completeness of the records of these two subpopulations is also associated with different demographic characteristics, as it has been established that the populations served by the dental clinics is biased by dental insurance considerations. The work will show what retrospective studies can (or not) be done using these populations when taking into account that it is well established that studies of populations with different level of completeness can be inconsistent. Discussion/Significance of Impact: This study provides an informatics framework to assess similarity and completeness of patient records with and without dental records. Establishing the level of similarity and completeness in these patient populations is critical to justify the validity of studies that utilize a combined record.

OBJECTIVES/SPECIFIC AIMS: In this study, a semi-targeted metabolomics approach is used to identify metabolic markers of methotrexate (MTX) response in juvenile idiopathic arthritis (JIA) and in vitro. METHODS/STUDY POPULATION: A comparative metabolomic analysis was used to identify metabolomic markers and metabolic pathways associated with MTX activity in vitro and in vivo. Cell-based studies assessed metabolomic profiles in K562 erythroblastoid cells with or without MTX treatment. In vivo analysis utilized plasma samples from JIA patients treated with MTX (n=30) and included samples collected prior to the initiation of MTX and after 3-months of MTX treatment. Plasma samples were from an IRB-approved single center prospective cohort study of biomarkers of MTX response in patients with JIA and were stratified based on American College of Rheumatology pediatric (ACR Pedi) response criteria. Semi-targeted global metabolomic profiles including over 800 metabolites across three analytical platforms at the NIH West Coast Metabolomics Center at UC-Davis and were analyzed by univariate and multivariate analysis using MetaboAnalyst 3.0. RESULTS/ANTICIPATED RESULTS: In K562 cells, MTX treatment was associated with statistically significant changes in 550 of the 850 intracellular metabolites detected (false discovery rate less than 0.05). Major metabolic pathways inhibited by MTX included branched-chain amino acid metabolism, purine and pyrimidine biosynthesis, and lipid metabolism including the inhibition of arachidonic acid metabolism. In patients with JIA, far fewer plasma metabolites were significantly altered following the initiation of MTX and included only 15 of the 833 plasma metabolites detected. Interestingly, MTX treatment was associated with the inhibition of arachidonic acid synthesis, inhibition of purine metabolism, and a dramatic reduction in plasma levels of various exogenous metabolites. In particular, MTX treatment was associated reductions in known metabolic markers of intestinal microbiota metabolism, including: biotin and dehydrocholic acid. Further, stratification of patients based on ACR Pedi response demonstrated that clinical response was associated with a greater reduction in plasma dehydrocholic acid levels following the initiation of MTX. DISCUSSION/SIGNIFICANCE OF IMPACT: This work demonstrates that MTX therapy is associated with a number of biochemical changes in vitro and in vivo, including: inhibition of purine metabolism, inhibition of arachidonic acid metabolism, and an apparent inhibition of gut microbiota metabolism. Most notably, inhibition of gut microbiota metabolism appears to demonstrate a relationship with the observed clinical efficacy of MTX in JIA.

OBJECTIVES/GOALS: CASK-related microcephaly with pontine and cerebellar hypoplasia (MICPCH) is a rare X-linked neurodevelopmental disorder caused by mutations in calcium/calmodulin-dependent serine protein kinase (CASK). We aim to rescue CASK expression via an CRISPR/dCas9 epigenetic therapeutic and create iPSC-based CASK relevant in vitro model systems. METHODS/STUDY POPULATION: As females have two X-chromosomes, disease causing mutations present with a 50/50 expression of mutant and wildtype, due to the mosaicism caused by random X-chromosome inactivation (XCI). This project will adapt an established CRISPR/dCas9 epigenetic approach to rescue expression from the silenced, wild-type CASK allele. We aim to accomplish this through testing different dCas9 orthologues and a guide RNA screen targeting the CASK promoter. Constructs will be tested for optimal targeting efficacy in vitro and assessed via RT-qPCR. Additionally, epigenetic modifications from our approach will be analyzed through bisulfite sequencing. We also aim to apply this epigenetic rescue technology in disease relevant cell lines and eventually in engineered patient mutation iPSC-derived neurons. RESULTS/ANTICIPATED RESULTS: Our results show the ability to target CASK and assess gene expression changes with CRISPR/dCas9 paired with an epigenetic modifier and transcriptional activator. Additionally, our fibroblast model with nonpathogenic single nucleotide polymorphisms within CASKallow for allele specific analysis of our targeted reactivation. We anticipate that following an increase of CASK expression, there would be a decrease in region specific promoter methylation. Further, with the identification of clinically described disease-causing point mutations that result in a loss of function of CASK protein, induction of the mutant sequence onto a healthy cell background will result in a similar reduction of CASK protein in our cell model. DISCUSSION/SIGNIFICANCE: This project will demonstrate the first therapeutic avenue for CASK-related MICPCH, and the potential to utilize targeted X-reactivation as a platform approach for X-linked disorders. Further, investigation of smaller dCas9 orthologues prepares our approach for future translational applications such as packaging into AAV for delivery.

Community engagement (CE) is critical for advancing health equity and a key approach for promoting inclusive clinical and translational science. However, it requires a workforce trained to effectively design, implement, and evaluate health promotion and improvement strategies through meaningful collaboration with community members. This paper presents an approach for designing CE curricula for research, education, clinical care, and public health learners. A general pedagogical framework is presented to support curriculum development with the inclusion of community members as facilitators or faculty. The overall goal of the curriculum is envisioned as enabling learners to effectively demonstrate the principles of CE in working with community members on issues of concern to communities to promote health and well-being. We highlight transformations needed for the commonly used critical service-learning model and the importance of faculty well-versed in CE. Courses may include didactics and practicums with well-defined objectives and evaluation components. Because of the importance of building and maintaining relationships in CE, a preparatory phase is recommended prior to experiential learning, which should be guided and designed to include debriefing and reflective learning. Depending on the scope of the course, evaluation should include community perspectives on the experience.

Conducting clinical trials is often complex and involves many individuals from a variety of services, each with a specific role in ensuring its successful implementation. Although an experienced clinical trialist may anticipate many of the challenges, others may be unexpected and detrimental to the successful completion of a study. We describe the use of simulation during preparation for initiation of a randomized clinical trial of a new preparation of antiseizure medication in neonates with seizures. The process of identification of stakeholders and roles, scenario development, and identification of challenges are described. Lessons learned included the potential benefits of simulation exercises, simulation challenges, and challenges associated with the study itself. We posit that going through the steps of a study, rather than merely reading them from a manual of procedures, will help identify potential barriers, complexities, and contingencies that are not readily apparent and may result in fewer protocol deviations and violations.

OBJECTIVES/GOALS: The objective of this study is to examine the associated changes in the EHR use patterns after the widespread implementation of telehealth in the ambulatory care setting after the COVID pandemic. METHODS/STUDY POPULATION: The study sample will be all attending ambulatory care physicians at UCSF Health. Signal measures captured by Epic Systems are markers of EHR use pattern that characterize EHR use at the individual provider level in terms of time spent performing certain activities, time spent at particular times of the day, and the number of EHR tools being used. We will use the Single Interrupted Time Series framework to analyze the changes in Signal measures that occur after the widespread implementation of telehealth with pre-telehealth time period defined as Jan 2018 – Feb 2020 and post-telehealth time period defined as March 2020 – present. RESULTS/ANTICIPATED RESULTS: The outcomes of this study will reveal how the increased use of telehealth following the COVID pandemic has changed the way providers utilize various functions within EHR (e.g. time in EHR at particular time of day, documentation, medication and non-medication orders, chart review, etc). These results can, in turn, inform us potential impacts of increased telehealth use on physician burnout given that a number of markers of EHR use pattern (i.e. Signal measures) in previous studies have been associated with burnout. In addition, a stratified version of Interrupted Time Series by specialty and clinical work volume may inform us how different subgroups of providers exhibit varying EHR use patterns in response to the increased use of telehealth. DISCUSSION/SIGNIFICANCE: The use of telehealth will likely remain a strong presence in health care delivery in the post-COVID era. This study can serve as a baseline study on the influence of telehealth on EHR use. Future studies may focus on potential targeted interventions to best support the usage of telehealth.

OBJECTIVES/SPECIFIC AIMS: The study aimed at assessing whether M. bovis BCG infection and inflammation exacerbates the development of atherosclerosis in Ldlr-/- mice. METHODS/STUDY POPULATION: Twelve-week old male Ldlr-/- mice (n=10) were infected with M. bovis BCG (0.3–3.0x10^6 colony-forming units (CFUs)) via the intranasal route, to simulate a natural respiratory route of infection. Mice were subsequently fed a western-type diet (WD) containing 21% fat and 0.2% cholesterol for 16 weeks. Age-matched uninfected Ldlr-/- mice (n=10) fed with an identical WD served as controls. Mice were euthanized after 16 weeks of WD to examine atherosclerotic lesions in aortic root sections and en face aorta using Oil Red O staining. Plasma cholesterol and triglyceride levels were measured by enzymatic assays and lipoprotein distribution was assessed using fast protein liquid chromatography. Because of the important role of T cells and monocytes in atherosclerosis development, we assessed these cell subsets in blood using flow cytometry at 8 and 16 weeks. Experiments were conducted in duplicate. We used unpaired Student’s t-test for group comparisons of numeric variables and flow cytometry data. RESULTS/ANTICIPATED RESULTS: M. bovis BCG infection significantly increased atherosclerotic lesions in en face aorta (plaque size per aorta area ratio; 0.15±0.13 vs. 0.06±0.02; P<0.01), but not in the aortic root. There were no significant differences in plasma cholesterol (1,160 mg/dL vs. 1,278 mg/dL; P = 0.36), triglycerides (340 mg/dL vs. 413 mg/dL; P = 0.28), or lipoprotein profiles between infected vs. uninfected mice at 16 weeks. M. bovis BCG increased circulating T lymphocytes (1,490 cells/uL vs. 1,227 cells/uL; P = 0.03) and monocytes (901 cells/uL vs. 414 cells/uL; P<0.01) within 8 weeks post-infection. When we assessed T lymphocyte subsets, M. bovis BCG infection increased total CD4+ T cell counts (556 cells/uL vs. 416 cells/uL; P<0.01) but not CD8+ T cells. No differences in the proportion of CD44+CD25+ activated T lymphocytes were noted between groups. When we assessed monocyte subsets, M. bovis BCG infection increased the numbers of Ly6Chigh (709 cells/uL vs. 362 cells/uL; P<0.01) and Ly6Clow (145 cells/uL vs. 35 cells/uL; P<0.01) monocytes. Infection was associated with an increased proportion of Ly6Clow monocytes at week 8 (17% vs. 8%; P<0.01) and week 16 (19% vs. 5%; P<0.01), compared to uninfected mice. DISCUSSION/SIGNIFICANCE OF IMPACT: M. bovis BCG infection increased the extent of atherosclerosis formation in the aortas of WD-fed hyperlipidemic Ldlr-/- mice after 16 weeks. Lipid profiles were similar between infected and uninfected mice, and therefore do not explain the observed differences in atherosclerosis. Compared to uninfected controls, M. bovis BCG-infected mice exhibited increased CD4+ T cell and monocyte driven inflammation. Interestingly, M. bovis BCG-infected mice had a higher proportion of non-classical Ly6Clow monocytes, suggesting a pro-atherogenic contribution of these cells in our model. Overall, our results support a pathogenic role of mycobacterial infection in atherosclerosis development and ASCVD.