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/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: 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/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.

OBJECTIVES/SPECIFIC AIMS: The aim objective of this research work is the study of neurocognitive endophenotypes in the new classifications of mental disorders. METHODS/STUDY POPULATION: Neuropsychological tests,such as the UPSIT test were used. The population was composed by a sample of patients with a diagnosis of schizophrenia RESULTS/ANTICIPATED RESULTS: Olfactory discrimination is a potencial neurocognitive endophenotype in the study of schizophrenia research DISCUSSION/SIGNIFICANCE OF IMPACT: The aim impact of this research work is the study of Dimensional classifications of mental disorders.

OBJECTIVES/SPECIFIC AIMS: Oncostreams represent a novel growth pattern of GBM. In this study we uncovered the cellular and molecular mechanism that regulates the oncostreams function in GBM growth and invasion. METHODS/STUDY POPULATION: We studied oncostreams organization and function using genetically engineered mouse gliomas models (GEMM), mouse primary patient derived GBM model and human glioma biopsies. We evaluated the molecular landscape of oncostreams by laser capture microdissection (LCM) followed by RNA-Sequencing and bioinformatics analysis. RESULTS/ANTICIPATED RESULTS: Oncostreams are multicellular structures of 10-20 cells wide and 2-400 μm long. They are distributed throughout the tumors in mouse and human GBM. Oncostreams are heterogeneous structures positive for GFAP, Nestin, Olig2 and Iba1 cells and negative for Neurofilament. Using GEMM we found a negative correlation between oncostream density and animal survival. Moreover, examination of patient’s glioma biopsies evidenced that oncostreams are present in high grade but no in low grade gliomas. This suggests that oncostreams may play a role in tumor malignancy. Our data also indicated that oncostreams aid local invasion of normal brain. Transcriptome analysis of oncostreams revealed 43 differentially expressed (DE) genes. Functional enrichment analysis of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented GO biological process. Network analysis indicated that Col1a1, ACTA2, MMP9 and MMP10 are primary target genes. These genes were also overexpressed in more malignant tumors (WT-IDH) compared to the less malignant (IDH1- R132H) tumors. Confocal time lapse imagining of 3D tumor slices demonstrated that oncostreams display a collective motion pattern within gliomas that has not been seen before. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, oncostreams are anatomically and molecularly distinctive, regulate glioma growth and invasion, display collective motion and are regulated by the extracellular matrix. We propose oncostreams as novel pathological markers valuable for diagnosis, prognosis and designing therapeutics for GBM patients.

OBJECTIVES/SPECIFIC AIMS: Our aim is to test whether osteocyte-specific CXCL12 expression is critical to exercise-driven bone formation. METHODS/STUDY POPULATION: All procedures were approved by the NEW YORK UNIVERSITY Institutional Animal Care and Use Committee. We generated male and female mice in which CXCL12 was deleted from OCYs (CXCL12ΔOCY) by crossing CXCL12 floxed mice and 10kb DMP1-Cre transgenic mice (gifts from Drs. Geoffrey Gurtner and Lynda Bonewald, respectively). The 10kb DMP1-Cre has been shown to be robustly expressed in odontoblasts and OCYs, with little to no activity in cells from non-mineralized tissues (Lu+ J Dent Res 2007). Growing male and female mice (n=3-8/group) were given fluorochrome labels every two weeks between 4-16 weeks of age, to monitor the role of CXCL12 during development. A second group, of adult 16-week-old mice (n=5/group), were subjected to tibial axial cyclic loading (1200µɛ, 2Hz, 120cycles, 3days/wk for 2 wks) (Liu+ Bone 2018). Basal and load-induced periosteal (Ps) and endosteal (Es) mineralizing surface (MS/BS, %), mineral apposition (MAR, µm/day) and bone formation rates (BFR/BS, µm3/µm2/year) were calculated (Dempster+ JBMR.2013) at mid-length. RESULTS/ANTICIPATED RESULTS: No significant differences were detected in basal bone formation during development. However, relative load-induced Ps MAR (rMAR) was reduced by 50% in female (p=0.02) and 75% in male (p=0.002) CXCL12ΔOCY mice; and similarly, Ps rBFR/BS was reduced by 50% in female (p=0.01) and 70% in male (p=0.001) CXCL12ΔOCY mice (Figure 1). Es bone formation was not affected by CXCL12 deletion. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, osteocyte-specific CXCL12 expression plays a critical role in exercise-driven periosteal new bone formation, suggesting that CXCL12 signaling may positively regulate osteogenic differentiation and/or mature osteoblast function. Further underlying mechanisms are currently being explored. Thus, osteocyte-specific CXCL12 signaling may be a promising target to enhance load-induced bone formation in patients with compromised ability to form new bone.

OBJECTIVES/SPECIFIC AIMS: Previously, we showed decreased development of endometriotic lesions in CD44 knockout mice compared to control.(1) CD44 has 10 different variants and a standard form. Menstrual endometrial cells (MECs) from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6) than v3, compared to MECs from women without endometriosis. (2) Here, we assessed the effects of CD44 standard (CD44s), CD44v3 and CD44v6 overexpression (OE) on immortalized human endometrial epithelial (iEECs) and stroma cells (hESCs) in vivo attachment in a nude mouse xenograft model. 1. Knudtson JF, Tekmal RR, Santos MT, et al. Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice. Reproductive sciences (Thousand Oaks, Calif.). 2016;23(1):87-91. 2. Griffith JS, Liu YG, Tekmal RR, Binkley PA, Holden AE, Schenken RS. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants. Fertility and sterility. 2010;93(6):1745-1749. METHODS/STUDY POPULATION: Overexpression of CD44s, CD44v3 and CD44v6 was carried out using lipofectamine and their expression verified with qRT-PCR in iEEC and hESCs. Nude mice, 8-10 week old, were injected with estrogen 1 week prior to injection of iEECs and hESCs (n=7 per group). The cells were counted after transfection and at least 300,000 iEECs and 300,000 hESCs were injected per mouse. The transfected cells were tagged with cell tracker red (iEECs) and green (hESCs). Forty-eight hours after injection into the xenograft, the mice were sacrificed. The cells were counted using fluorescent stereo microscopy (FSM). Percent attachment was calculated based on the number of cells visualized by FSM divided by the number of transfected cells injected. Unpaired student t-test was performed to analyze differences in the percent attachment of the cells. RESULTS/ANTICIPATED RESULTS: The majority of cells were attached to the peritoneum. There was increased attachment of hESCs with OE of CD44v6 compared to control (p=0.03). CD44v6 OE did not change attachment of iEECs. There was no difference in attachment in iEECs or hESCs with OE of CD44s or CD44v3. DISCUSSION/SIGNIFICANCE OF IMPACT: Overexpression of CD44v6 increases attachment of ESCs to PMCs in an in vivo xenograft model. Menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.

OBJECTIVES/SPECIFIC AIMS: High-grade serous ovarian carcinoma (HGSOC) is the most common and malignant histological subtype of epithelial ovarian cancer. While the majority of HGSOC patients initially respond to platinum-based chemotherapy, they often present with recurrent chemoresistant disease, which is extremely fatal. Therefore, there is an urgent need to identify predictive biomarkers of platinum response and to develop rational, targeted therapies to improve the outcome of patients with HGSOC. The objectives of the present study are to profile and assess the clinical significance of MYC network dysregulation in HGSOC. METHODS/STUDY POPULATION: We will conduct a retrospective cohort study of Puerto Rican Hispanics with HGSOC who underwent surgery followed by platinum-based chemotherapy at clinical institutions in Puerto Rico. Medical records, pathology reports, and cancer registries will be reviewed to extract data on clinicopathological features, disease recurrence, and death. For eligible patients, formalin-fixed, paraffin-embedded (FFPE) tissue samples will be processed and analyzed by quantitative Real Time PCR (qRT-PCR) and immunohistochemistry (IHC). RESULTS/ANTICIPATED RESULTS: Expression levels of MYC and MYC-related molecules are expected to correlate with clinicopathological features and prognosis of HGSOC. DISCUSSION/SIGNIFICANCE OF IMPACT: The identification and validation of clinically-relevant alterations in HGSOC, such as dysregulation of the MYC network, will be crucial to guide therapy regimen, maximize clinical benefit, and improve patient outcome.

OBJECTIVES/SPECIFIC AIMS: Prostate cancer is the second leading cause of cancer-related death among men in the U.S. and over half of all prostate cancer patients receive radiation therapy (RT). RT induces double-strand breaks (DSBs) in DNA which are lethal to cells if not repaired. While potentially curative, 10% of low-risk patients and 50% of high-risk patients treated with RT still experience tumor recurrence. Thus, identification of novel therapeutic targets to enhance RT will likely reduce prostate cancer mortality. The only clinical approach to enhance RT is androgen deprivation therapy, which targets androgen receptor (AR) signaling; however, its use is limited due to systemic side effects. We recently reported that PRMT5 epigenetically activates AR which led us to investigate if targeting PRMT5 sensitizes prostate cancer to RT. The goal of this project is to determine if PRMT5 is a therapeutic target for prostate cancer radiosensitization and analyze its mechanistic role in response to radiation. METHODS/STUDY POPULATION: To evaluate if targeting PRMT5 may sensitize prostate cancer cells to radiation, we performed a clonogenic assay of irradiated cells. To determine if PRMT5 is required for repair of radiation-induced DSBs, we performed foci analysis via immunocytochemistry. We then used RNA-seq, qPCR, western blot, and ChIP to evaluate a potential epigenetic role of PRMT5 in activating the expression of genes critical to DSB repair. To extend our findings, we analyzed clinical data from around 18,000 of cancer patients encompassing 43 cancer types to assess if PRMT5 expression correlates with the expression of its putative target genes. RESULTS/ANTICIPATED RESULTS: Targeting PRMT5 sensitizes prostate cancer cells to radiation independently of AR status. RNA-seq analysis revealed putative PRMT5 target genes including several involved in DSB repair and G2 arrest. Mechanistically, PRMT5 functions as a master epigenetic activator of DNA damage response (DDR) genes: PRMT5 maintains the basal expression of several DDR genes including BRCA1, BRCA2, and RAD51 and is recruited upon radiation to DDR gene promoters to activate their expression via histone methylation. Targeting PRMT5 decreases expression of these genes at the protein level and hinders repair of radiation-induced DSBs in multiple cancer and non-cancer cell types. Clinically, PRMT5 expression positively correlates with the expression of these DDR genes across all 43 cancer types analyzed. DISCUSSION/SIGNIFICANCE OF IMPACT: PRMT5 acts as a master epigenetic activator of genes involved in DDR and is critical for cells to survive radiation treatment. Importantly, PRMT5 epigenetically activates multiple genes that encode for well-characterized core repair proteins involved in HR (RAD51, RAD51AP1, RAD51D, BRCA1 and BRCA2) and NHEJ (NHEJ1, Ku80, XRCC4, and DNAPKcs), which may explain why PRMT5 is essential to repair IR-induced DSBs in several cell lines. As PRMT5 is overexpressed in many human cancers and its overexpression correlates with poor prognosis, our findings suggest that more efficient DSB repair via PRMT5 overexpression in these cancers may confer survival advantages particularly following DNA damaging treatments. Lastly, because targeting DSB repair is a clinically validated therapeutic approach for cancer treatment, our findings also suggest that PRMT5 targeting may be explored as a monotherapy or in combination therapy with radiation therapy or chemotherapy for cancer treatment.

OBJECTIVES/SPECIFIC AIMS: Drugs to attenuate aortic growth are usually not initiated in patients with Marfan syndrome until aortic dilation is already present. Therefore, we measured the impact of drugs (the renin-angiotensin system inhibitors losartan and enalapril) on survival and thoracic aortic growth in a mouse model of Marfan syndrome when extensive aortic dilation was already present. METHODS/STUDY POPULATION: Male and female fibrillin-1 hypomorphic (FBN1 mgR/mgR) mice (n=10-12/group) were stratified into treatment groups by aortic diameter at 6 weeks of age to ensure an equivalent average aortic diameter in each group at the start of the study. Osmotic mini pumps filled with PBS (vehicle), enalapril (2 mg/kg/d), or losartan (20 mg/kg/d) were implanted subcutaneously into mice after stratification. Mini pumps infusing drug or vehicle were replaced every 4 weeks for a total duration of 12 weeks. Wild type littermates (n=10) were infused with PBS as a negative control to the Marfan mouse model. Ascending aortic diameters from male and female FBN1 mgR/mgR mice and their wild type littermates were assessed by ultrasound every 4 weeks from 6 to 18 weeks of age. Aortic diameters were measured luminal edge to luminal edge during diastole. RESULTS/ANTICIPATED RESULTS: 6 week old FBN1 mgR/mgR mice exhibited significantly dilated ascending thoracic aortas at study initiation compared to their wild type sex-matched littermates (in males: FBN1 mgR/mgR = 1.87 +/− 0.07mm, wild type = 1.23 +/− 0.07mm; p <0.001) (in females: FBN1 mgR/mgR = 1.56 +/− 0.07mm, wild type = 1.18 +/− 0.07mm; p <0.001). Baseline mortality of FBN1 mgR/mgR mice infused with PBS was 36% in male and 22% in female mice at the time of study termination. Within sex-matched mgR littermates, there was no significant difference in survival between groups treated with PBS, enalapril, or losartan after 12 weeks (p=0.224 for males, p=0.094 in females). In the same groups, no significant difference in maximum ascending aortic diameter was detected after treatment for 12 weeks (in males: PBS=2.69 +/− 0.19 mm, enalapril=2.04 +/− 0.27 mm, losartan=2.42 +/− 0.28 mm; p=0.24) (in females: PBS = 1.92 +/− 0.13, enalapril=1.89 +/− 0.31, losartan=1.98 +/− 0.17; p=0.86). Furthermore, aortic diameters in the FBN1 mgR/mgR mice were found to demonstrate sexual dimorphism. DISCUSSION/SIGNIFICANCE OF IMPACT: This research shows that losartan is not effective when administered after significant thoracic aortic dilation has already occurred in FBN1 mgR/mgR mice. This has important translational implications because losartan is usually not started in patients with Marfan syndrome until significant aortic dilation is already present. Therefore, more research needs to be done to determine the critical time period within which this medicine will be effective if given to patients. In addition, this research demonstrates that male FBN1mgR/mgR mice have a significantly larger aortic diameter than female FBN1mgR/mgR mice. This sexual dimorphism has recently been observed in patients with Marfan syndrome as well. Additional studies for understanding the mechanism underlying this sexual dimorphism have the potential to elucidate new therapeutic approaches for aortic disease.

OBJECTIVES/SPECIFIC AIMS: The purpose of this case series is to show how helping parents instill a non-restrictive, structure-based (i.e., authoritative) approach to feeding is useful in addressing family food conflicts in a clinical child obesity treatment program. METHODS/STUDY POPULATION: Case reports are presented for 3 young children (two 8-year-old males and one 7-year-old female) with obesity (BMI ≥ 95th percentile for age and sex). Patients underwent family-based treatment at Brenner FIT® (Families In Training), an interdisciplinary tertiary weight management clinic. RESULTS/ANTICIPATED RESULTS: All patients experienced a period of rapid weight gain and/or severe onset obesity. Parents reported a combination of problematic eating behaviors (e.g., sneaking food, frequent complaints of hunger, vomiting from rapid consumption). Families implemented structure-based feeding with a meal-snack schedule and allowed children to eat until they were full from the food provided at meal-snack times. BMI z-score decreased from 2.19 to 2.07 in patient 1 and from 2.43 to 2.09 in patient 2 (follow-up weight was not available for patient 3). DISCUSSION/SIGNIFICANCE OF IMPACT: The improvements observed by our clinical program after families lifted restriction and instituted authoritative feeding is anecdotal evidence for the ecological validity of existing empirical work. Randomized controlled trials are needed to examine causality.

OBJECTIVES/SPECIFIC AIMS: Chlamydia trachomatis (CT) infection can lead to reproductive morbidity in women. Animal models suggest that protection against CT is mediated through the cytokine interferon-gamma (IFN-γ), produced by CD4+ T-cells, which clears CT through intracellular tryptophan depletion. In humans, correlates of protection remain to be elucidated, which hinders chlamydia vaccine development. Natural clearance of CT infection (e.g., clearance before antibiotics) may be an immunological correlate of protection, evidenced by (1) CT clearance without antibiotics; and (2) a 4-fold reduced risk of CT reinfection within 6 months. We have identified women with and without natural clearance of CT infection. By comparing these two groups of women, the role of IFN-γ-mediated natural clearance of CT infection will be investigated. METHODS/STUDY POPULATION: Through collaboration with a cohort study of CT-infected women, we have access to stored specimens from women who naturally cleared CT or had persisting CT infection. Using peripheral blood mononuclear cell (PBMC), we will assess whether natural clearance of CT infection is associated with IFN-γ-producing CD4+ T-cells by stimulating PBMC ex vivo with CT antigens using intracellular cytokine staining. We will also use cervicovaginal lavage (CVL) and untargeted High-Performance Liquid Chromatography-Mass Spectrometry to assess for tryptophan-dependent and -independent metabolic pathways associated with natural clearance of CT infection. RESULTS/ANTICIPATED RESULTS:: To date, IFN-γ has been measured in 10 women who did not clear CT infection, demonstrating that <20% of these women produced significant levels of IFN-γ. Women who naturally cleared CT have yet to be studied. Untargeted HPLC-MS has been performed on 6 women (3 who cleared matched to 3 with persisting CT infection). To date, 11 pathways that are significantly associated with natural clearance have been identified. DISCUSSION/SIGNIFICANCE OF IMPACT: The outcome of natural clearance of CT infection is distinct from women with persisting chlamydia. These studies may inform whether IFN-γ, produced by CD4+ T-cells, or tryptophan-dependent or -independent metabolic pathways are associated with natural clearance, which may advance chlamydia vaccine development.

OBJECTIVES/SPECIFIC AIMS: The first aim of the study is to evaluate the accuracy of serum biomarkers of acute GVHD measured after four weeks of corticosteroid therapy to predict 6 month NRM. The second aim of this study is to compare the accuracy of the biomarker algorithm to that of clinical response to corticosteroids after four weeks. The third aim of the study is to develop a novel regression model that uses weekly biomarker measurements over the first month of corticosteroid therapy to predict 6 month NRM. METHODS/STUDY POPULATION:. Patients who received HCT at one of 22 IRB-approved centers and provided blood samples to the Mount Sinai Acute GVHD International Consortium (MAGIC) biorepository and developed GVHD between January 2008 to May 2018 are included in this study. Patients were divided by time into a training set (Jan 2008-Dec 2015, n=233) for model development and a validation set (Jan 2015-May 2018, n=357) to evaluate the predictive performance of the model. The later time of the validation set was chosen deliberately to model contemporaneous GVHD treatment practices. The size of each group was designed so that there would be roughly equal numbers of deaths in both groups. RESULTS/ANTICIPATED RESULTS:. Serum concentrations of GVHD biomarkers after one month of corticosteroid therapy were measured in the validation set, and the predicted probability of NRM ($\hat{\rm p}$) was computed according to the previously published algorithm: $\log[-\log(1 - \hat{\rm p})]=-11.263 + 1.844({\rm logST}2)+ 0.577({\rm logREG}3\alpha)$. The performance of the biomarker algorithm was evaluated by creating receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC) in the validation set. The AUC of the biomarker algorithm was a significantly better predictor of 6 month NRM than clinical response to treatment after four weeks of corticosteroids (0.84 vs. 0.64, p<0.001), which is a clinically relevant improvement in accuracy. To evaluate serial biomarker monitoring, serum biomarker concentrations will be measured weekly at five time points from treatment initiation to one month after corticosteroid therapy. We will use these values in the training set to develop a regression model for 6 month NRM that accounts for repeated biomarker measurements. The performance of this model will be tested in the validation set and the accuracy of the serial biomarker measurements will be compared to the accuracy of measuring biomarkers at the single time point after four weeks of corticosteroid therapy. An AUC improvement of 0.05 would be considered clinically significant. DISCUSSION/SIGNIFICANCE OF IMPACT: Clinical response to treatment after four weeks has been the standard endpoint in GVHD interventional trials for decades. If biomarkers measured at the same time more accurately predict long term mortality, this study would provide the basis for a novel endpoint in GVHD trials and enable more accurate determination of effect size of experimental interventions. An accurate biomarker algorithm will prove useful in guiding immunosuppressive treatment decisions for patients with GVHD. Patients identified by the algorithm as low-risk may benefit from reduced-dose corticosteroid therapy, potentially reducing lethal opportunistic infections. Patients identified as high-risk will be candidates for more intensive immunosuppression or investigational therapies. This precision medicine approach tailors therapy to the individual patient’s biology.

OBJECTIVES/SPECIFIC AIMS: The aims of this study are threefold. Firstly, we are examining the effects of increased in vitro afterload (a proxy for hypertension) on human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) response to sunitinib in a durable and dynamic cardiac microtissue culture system. Secondly, we are exploring effects of repeat exposure and recovery of both sunitinib and afterload throughout the lifetime of the hiPSC-CM microtissue. Finally, we are assessing methods to prevent and treat sunitinib induced cardiotoxicity. Primary outcomes for this study are commonly utilized metrics of cardiotoxicity: degree of caspase activation, electrophysiology benchmarks for minimum voltage threshold and maximum capture rate, and microtissue force generation. METHODS/STUDY POPULATION: HiPSC-CMs are cultured and matured as 3D cardiac microtissues (CMTs) on a microtissue array. After maturation, cells are exposed to sunitinib doses of 0µM, 0.5µM, 1µM or 5µM for 12 hours. Concurrently with sunitinib dosing, increases in microtissue array stiffness are created with application of an external magnetic field. Afterload spring constants are fixed at pre-determined physiologic values ranging from 0.5µN/µm, to 5µN/µm. For Aim 1: Half of the CMTs are harvested at 8 hours after sunitinib dosing to conduct the caspase 3/7 assay, and the remainder are examined for 3 days following drug exposure to track temporal changes in electrophysiology and force generation. For Aim 2: After CMT maturation, 12-hour exposures to sunitinib are repeated three times at a fixed dose, with doses separated by one week. Concurrently with sunitinib dosing, increases or decreases in microtissue stiffness are created by changing the strength of an applied external magnetic field to create “ramp up” or “ramp down” stiffness conditions. Caspase assay and contractility metrics are measured at each timepoint. For Aim 3: Experimental conditions are conducted as described in Aim 1. Prior to the introduction of sunitinib, either carvedilol or an AMP-kinase activator is added to the CMT culture media at physiologic concentrations. Primary outcomes are examined as in Aim 1. RESULTS/ANTICIPATED RESULTS: Aim 1: We hypothesize that increases in microtissue afterload, synchronized with sunitinib exposure will augment sunitinib toxicity in cardiomyocytes resulting in elevations of caspase 3/7 activity and minimum voltage capture as well as decreases in maximum capture rate and maximum force generation. Aim 2: We hypothesize that repeat exposures to both sunitinib and to increases in afterload will augment sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Additionally, we hypothesize that decreases in afterload will decrease effective sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Aim 3: We hypothesize that exposure to an AMP-kinase activator but not carvedilol will decrease the effects of sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of small molecule, targeted chemotherapeutic agents is increasingly common. Many of these agents cause cardiotoxic side effects, the mechanisms of which are incompletely understood. Our lab has developed a novel 3D tissue engineering platform capable of supporting durable in vitro cardiac microtissues that experience dynamic alterations in their biomechanical load. By using this platform to examine the cardiotoxic effects of sunitinib, insight into treatment and prevention of this common problem will be developed.

OBJECTIVES/SPECIFIC AIMS: Previous studies suggest that genetic variants in the oxytocin receptor (OXTR) may alter oxytocin dose requirement for labor induction and may increase risk for preterm labor and neurodevelopmental disorders. However, the mechanisms of actions of these variants remain unknown. The goal of this study was to functionally characterize common missense and noncoding variants in OXTR. First, we aimed to determine the effects of missense variants on two major aspects of receptor function: calcium signaling and β-arrestin recruitment. Second, we used allelic expression imbalance assays in an effort to identify regulatory single nucleotide polymorphisms (SNPs) in noncoding regions of OXTR that alter OXTR mRNA expression. METHODS/STUDY POPULATION: We used the Exome Aggregation Consortium database to identify the 12 most prevalent missense single nucleotide variants in OXTR. To determine the functional effects of these variants, we transfected human embryonic kidney cells (a common model system used to study receptor function) with wild type OXTR, variant OXTR, or empty vector control. We used the calcium-sensitive dye Fluo4 to quantify intracellular calcium flux in response to oxytocin treatment, and used bioluminescence resonance energy transfer assays to measure recruitment of the signaling partner β-arrestin to the receptor. To investigate potential effects of noncoding SNPs on OXTR mRNA expression, we quantified allele-specific expression of OXTR in human uterine tissue obtained from participants at the time of Cesarean section. We used next-generation sequencing (Illumina MiSeq) to count alleles of a reporter SNP in OXTR exon 3. RESULTS/ANTICIPATED RESULTS: Of the 12 most prevalent missense single nucleotide variants, four were predicted to be deleterious by PolyPhen variant annotation software. We anticipate that these variants will alter receptor signaling through calcium or β-arrestin pathways. We further observed that a reporter SNP in OXTR exon 3 exhibits significant allelic expression imbalance in a subset of our myometrial tissue samples, indicating that OXTR expression may be regulated by a functional SNP. Our current work focuses on discovering the functional SNPs in OXTR responsible for the pattern of allelic expression imbalance seen in mRNA. In the future, we will seek to explore the effects of these variants on uterine function by using genome editing of uterine smooth muscle cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that both missense and noncoding variants may affect OXTR expression and function. Future studies may suggest that OXTR sequencing, genotyping, or expression analysis would be useful to identify individuals likely to respond or fail to respond to safe doses of oxytocin for labor induction. Personalizing approaches for labor induction in this way would increase the safety of oxytocin and potentially reduce maternal morbidity and mortality.

OBJECTIVES/SPECIFIC AIMS: In the first aim, we will evaluate the proportion of highly HIV-susceptible memory CD4+ T cells present in the rectal mucosa, based on the proliferation status and expression of the HIV susceptibility markers, CCR5 and α4β7, between HIV-negative adolescent MSM and adult MSM engaging in RAI. The second aim will assess differences between the two study groups in the ratio of Th17 cells (CD4+ IL17+) to Treg cells (CD4+ FoxP3+ CD25+) in the rectal mucosa as a determinant of mucosal inflammation. Finally, in the third aim, we will utilize ex vivo rectal biopsy explant challenge experiments to examine whether HIV target cell availability and the Th17/Treg ratio influence rectal mucosal HIV susceptibility. METHODS/STUDY POPULATION: Rectal biopsy specimens are being collected from healthy, HIV-negative men that comprise the two study groups: 40 adolescent MSM 18-21 years of age who have engaged in RAI at least once previously in their lifetime and 40 adult MSM ≥35 years of age who have engaged in RAI for the previous 5 consecutive years with a minimum of 12 episodes annually. To identify CD4+ subsets of interest for aims 1 and 2, rectal mucosal mononuclear cells are isolated and phenotyped with CD45, CD3, CD4, CD45RA, CCR7, CD69, CCR5, α4β7, Ki67, FOXP3, and CD25 antibodies. To identify the Th17 cell subtype, the cells are stimulated with PMA/Ionamycin and stained with an antibody specific to IL-17A. Using cross-sectional analyses, we will compare the frequencies of mucosal CD4+ T cells that express certain phenotypic characteristics and evaluate differences in the Th17/Treg ratio between adolescent and adult MSM. For aim 3, rectal biopsy specimens are inoculated with HIV virus and the culture supernatant is assayed for p24 concentration on days 3, 7, 10 14, and 18. Longitudinal analyses will be performed to detect differences in p24 concentration at each time point and assess associations with mucosal target cell availability and with the Th17/Treg ratio. RESULTS/ANTICIPATED RESULTS: We hypothesize that younger age will be associated with enhanced memory CD4+ T cell proliferation and increased expression of HIV susceptibility markers (CCR5 and/or α4β7). In addition, we expect that the rectal mucosa of adolescent MSM will demonstrate a higher Th17/Treg ratio as compared to adult MSM, which could facilitate HIV transmission. It is also anticipated that rectal mucosal immune phenotypes characterized by increased HIV target cell availability and high Th17/Treg ratios will be associated with enhanced mucosal HIV susceptibility in the explant challenge model. DISCUSSION/SIGNIFICANCE OF IMPACT: There is a paucity of information regarding the mechanisms of rectal HIV transmission, and no studies to date investigate the immunologic effects of aging on transmission in the rectal mucosa. The results from this study will provide important information regarding age-related differences in the immune cell composition of the rectal mucosa as a critical step in better understanding immunologic factors that influence rectal HIV transmission.

OBJECTIVES/SPECIFIC AIMS: We previously demonstrated that products released by cultured B cells from patients with Multiple Sclerosis (MS) are cytotoxic to neurons and oligodendrocytes, while minimal toxicity was observed in response to B cell secretory products from age- and sex-matched normal controls. The goal of this proposal is to identify the range of brain cells susceptible to MS B cell-mediated cytotoxicity, to define the cytotoxic factor(s) released by MS B cells, and to determine whether particular subset(s) of MS B cells harbor the greatest pathogenic potential. METHODS/STUDY POPULATION: The toxicity of B cell products will be demonstrated by incubating primary rat cultures of neurons, oligodendrocytes, and oligodendrocyte progenitor cells (OPCs) with B cell supernatants. B cells will be isolated from the peripheral circulation of untreated relapse-remitting MS (RRMS) patients and age- and sex-matched normal controls. The identification of specific toxic factor(s) in MS B cell supernatants will be achieved through a combination of exosome-depletion/enrichment of conditioned media, proteomics, next generation sequencing, and lipidomics. Determining pathogenic B cell subsets will be achieved by cell sorting into memory and naïve B cell subsets prior to collection of supernatants. RESULTS/ANTICIPATED RESULTS: We hypothesize that the toxicity of MS B cell products is mediated, at least in part, by extracellular vesicles, such as exosomes. We expect depletion of these exosomes from the B cell conditioned media or inhibition of their biogenesis will mitigate the observed toxicity. Furthermore, differences in B cell-derived exosomal content, such as proteins, (mi)RNAs, or lipids, likely explain the differences in observed toxicity. Lastly, we hypothesize that memory B cells, which are enriched in the CNS of MS patients and demonstrate a more pro-inflammatory profile than naïve B cells, are responsible for the toxicity observed in supernatants of total B cells. DISCUSSION/SIGNIFICANCE OF IMPACT: MS is the most prevalent chronic inflammatory disease of the CNS, affecting more than 2 million people worldwide. Although over a dozen disease-modifying therapies are approved for the treatment of RRMS, none are meaningfully effective at limiting disease progression. This proposal will provide new insight into immune-CNS interactions in progressive MS and provide much-needed novel targets for therapeutic intervention, either via blocking identified toxic molecule(s) or by selectively depleting pathogenic B cell subsets.

OBJECTIVES/SPECIFIC AIMS: National concerns about IRB-related research delays have led to re-assessment of IRB review processes at institutional levels. We sought to address whether a dedicated IRB Liaison Service at the Irving Institute’s central location could provide additional useful staff support to the investigator community for interactions with the IRB at various levels of protocol submission. METHODS/STUDY POPULATION: We evaluated the results of a user satisfaction survey and performed a focused in-depth analysis of Liaison Service impact. An online tracking and satisfaction survey was implemented for researchers to complete following each consultation. The goal was to gauge the uses, user types and usefulness of the Service, and to follow-up with researchers who might have additional questions. Data was gathered about users of the Service and their affiliations, and the topics and questions that were discussed. A terse summary was drafted to categorize each consultation that was conducted during office hour sessions. Additionally, surveys were emailed to researchers to gauge their experience with the Service and their overall satisfaction. Users completed the survey either in person at the end of the consultation, or by email request sent immediately following each in-person consultation. The impact of the IRB Liaison Service on IRB protocol approval times was analyzed for a random sub-sample of protocols for which consultations were provided. Consultations for studies with an associated IRB protocol number (i.e., at least initially submitted) from May 2015-June 2017 had been assigned a number in an Excel file. Using a randomization formula, a subset of 90 protocols was identified for further analysis. Protocols that did not result in an IRB submission and duplicate entries were removed. The final dataset consisted of 67 protocols. Those protocols were assessed by type of review process (expedited versus full board review), by status (new submission, first return, second return, etc.), and by which of the seven IRB committees completed the review. Consultations for each protocol included in this subset were reviewed using the notes about that consultation. IRB records in Columbia’s online research oversight system, Rascal, were also reviewed to assess the timing of and issues raised in subsequent IRB review. Factors examined included whether the protocol was approved at next submission and if not, whether questions raised in subsequent IRB returns were related to the topics discussed in the consultation. RESULTS/ANTICIPATED RESULTS: Since its inception in January 2015 through June 2017 (2.5 years), a total of 501 in-person consultations have been performed, usually 25-30 per month. Users were primarily study coordinators and investigators. Most requests concerned new protocol development, policy questions or assistance in addressing IRB comments from submitted protocols. Survey response rate was 43%. Results of 215 competed satisfaction surveys were 100% positive. Of 67 unique protocols analyzed for outcomes of the consultation, 73% were subsequently approved within 14 days. DISCUSSION/SIGNIFICANCE OF IMPACT: Overall, we have found the Liaison Service to be a popular addition to research support, and plan to continue the service. We will continue to evaluate its user satisfaction and usefulness. Additional focus will be placed on whether the Service can improve approval times for human subjects research for protocols using the Liaison Service.

OBJECTIVES/SPECIFIC AIMS: Because clinical trial results are instrumental in the approval of a new drug or changes to the practice of medicine, ensuring the accuracy and validity of collected data is critical in the clinical trial process. This function, routinely carried out by clinical trial monitors in industry-sponsored trials, is often lacking in investigator-initiated trials (IITs) conducted in academia. To address this challenge, we have developed a self-study module that can be used to cross-train academic researchers in essential concepts and practical approaches to monitoring. Furthermore, we are applying a framework drawn from implementation science in the development and launch of this initiative. This framework, as used in other educational programs, is employed here to close the gap between initiative and practice, thereby effectively disseminating this training would improve the quality of clinical trials in academia. METHODS/STUDY POPULATION: This research project applied exploration, installation and implementation stages of the implementation science process by 1) exploring the need for a new initiative, 2) disseminating results, 3) engaging stakeholders, 4) creating standard operating procedures (SOPs) for installation and implementation, 5) studying user satisfaction and effectiveness, 6) addressing feedback and 7) conducting implementation. RESULTS/ANTICIPATED RESULTS: From literature review and internet searches we determined that although numerous GCP training resources exist, most are too broad and lack the practical approaches to meet the complex requirements of monitoring. Moreover, most of the offerings identified are costly or inaccessible. With only about 65% of IITs reported as being monitored (Figures 1 and 2), it appears that there is a clear need for training tools that are easily available to a broader audience. And because monitoring skills are substantially different from those associated with research coordination, it is not surprising that research professionals believed that they would need additional training to become proficient. To address this need, we began developing a monitoring module. We engaged key stakeholders from academia and industry to gain insights into their needs. The results indicated that although our training module was effective, supplementary information on the fundamentals of clinical trials should be included for those new to the field. After incorporating suggested changes and completing the module, we conducted user testing to determine if our module is ready to be broadly disseminated (Figures 3 and 4). Following positive feedback from the group, we are currently in the process of disseminating our module and studying its impact. DISCUSSION/SIGNIFICANCE OF IMPACT: IITs are instrumental in translating academic research into product development. Deficiencies in the quality control of these trials can lead to inadequacies in data accuracy and validity that could lead to significant delays in bringing innovative therapies to patients. Recent NIH policies require data and safety monitoring for all of the trials it supports. The latest addendum to ICH GCP, E6(R2), discusses a need for quality management across the clinical trial lifecycle. As we continue to disseminate and share information during the development of our self-study monitoring module, we are engaging key stakeholders from academia, government, and private institutions to understand and address quality challenges in conducting clinical trials. Finally, this research informs dissemination and implementation research, specifically for creating training for academic research professionals.