{"paper_id":"db9885a6-7a1c-45ab-bc8f-a8e1e9e4edf9","body_text":"regulated synthesis of target proteins was analyzed using autoradiography and\nWestern blot techniques. Platelets from patients with HIV+ were examined in\nparallel. RESULTS/ANTICIPATED RESULTS: We identified that highly puri fied,\nisolated platelets from healthy subjects possess eRT activity. eRT activity was\nblocked with the non-nucleoside RT inhibitor nevirapine at concentrations within\nthe therapeutic drug range. L1 elements are bicistronic, containing 2 open reading\nframes (ORFs), ORF1 and ORF2. Thus, we next identi fied that human platelets\nexpress full-length L1 mRNA containing ORF1 and ORF2. In human platelets, eRT\nactivity was localized to L1 protein containing ribonucleo particles. Platelet eRT\nreverse transcribed exogenous RNAs, a process inhibited by nevirapine, acting in\ntrans using the 3 ′-UTR of exogenous mRNAs as a template. To dissect the\nfunction of eRT in platelets, we next examined cytoskeletal and protein synthetic\nevents in the presence or absence of nevirapine. Inhibition of eRT in isolated\nplatelets led to characteristically beaded platelets in appearance, strongly\nresembling bone marrow proplatelets. Parallel increases in platelet reactivity\nwere also observed. As these changes occurred over hours, not minutes, we\nhypothesized that inhibition of eRT would affect platelet protein synthetic events.\nConsistent with this, RT inhibition resultedin upregulation of global platelet protein\nsynthesis. We validated upregulation of the synthesis of specific proteins (mitofilin,\np-selectin, and L26—a component of the 60S ribosomal subunit essential for mRNA\ntranslation). RNA-DNA hybrids, noncanonical nucleic acid structures that regulate\ngene expression, are enriched in regions where L1 is abundant. RNA-DNA hybrids\nwere present in platelets and expression confirmed via differential digestion of RNAs\n( e g ,w i t hR N a s eAa n dR N A s eI ) .N e x t - g e n e r a t i o ns e q u e n c i n go fp u l l e dd o w n( e g ,\nimmunoprecipitated) platelet RNA-DNA hybrids identified numerous differentially\nexpressed transcripts and we focused on MAP1LC3B (LC3B), a primary regulator of\nautophagy. Hybrid sequencing results for LC3B were validated using qPCR and we\nconfirmed that LC3B RNA binds to L1-encoded RNA binding protein. Platelets\ntreated with nevirapine had increased total LC3B protein expression. As RT\ninhibition is an important mechanism to control HIV infection, we examined platelet\nmorphology, activation, and LC3B expr ession in platelets from HIV + subjects\ntreated with nevirapine. HIV + patients treated with RT inhibitors had higher\nnumbers of platelets that were beaded in appearance at baseline, increased platelet\nreactivity, and differential LC3B expr ession compared with healthy controls.\nDISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, these results\ndemonstrate that platelets possess eRT activity that regulates platelet morphology,\nplatelet hyperreactivity, and protein synthetic events. We postulate that eRT activity\nin platelets may be a new post-transcriptional regulatory checkpoint. Moreover, our\nfindings have implications in HIV + patients treated with RT inhibitors, where off-\ntarget effects may contribute to platelet activation and an increased risk of\nthrombosis.\n2114\nThe role of estrogen receptor- β in the development of\nthe early endometriotic lesion\nJennifer Knudtson, Ya-Guang Liu, Marlen Tellez Santos,\nRajeshwar Tekmal, Ratna Vadlamudi and Robert Schenken\nOBJECTIVES/SPECIFIC AIMS: To further elucidate the role of estrogen\nreceptor β (ER-β) in the early endometriotic lesion attachment. METHODS/\nSTUDY POPULATION: EECs were immortalized using a telomerase vector.\nImmortalized cells and parental cells were characterized by genotyping, and\nexpression of ER- β as well as other epithelial cell markers. ER- β was knocked-\ndown in immortalized EECs using lentivirus-mediated shRNA transduction. ER-\nβ knockdown was confirmed by RT-qPCR and Western analysis. EEC cells with\nor without ER- β knockdown were used to assess their attachment to PMCs in\nan established in vitro assay (Lucidi, 2005). Results were analyzed with Student\nt-test. RESULTS/ANTICIPATED RESULTS: Genotyping using karyotype assay\nconfirmed a normal chromosomal profile. Also positive staining for cytokeratin\nand lack of any staining with vimentin confirms the epithelial origin of these cells.\nER-β knockdown has a signi ficant decrease in attachment compared to control\n(p = 0.02). DISCUSSION/SIGNIFICANCE OF IMPACT: Primary and immorta-\nlized cells were 46XX, cytokeratin positive, and vimentin negative con firming\ntheir epithelial origin. ER- β knockdown has a signi ficant decrease in attachment\ncompared with control.\n2136\nGut microbiome alterations in children undergoing\nhematopoietic stem cell transplantation\nMuna Qayed, Dalia Arafat, Samridhi Banskota, John Horan,\nEdmund Waller and Gregory Gibson\nOBJECTIVES/SPECIFIC AIMS: Aim 1: To compare microbiome diversity\namong patients who develop BSI post hematopoietic stem cell transplantation\n(HSCT) and patients without BSI. Aim 2: To compare microbiome\ndiversity among patients who develop moderate to severe acute GVHD post\nHSCT and patients without GVHD. Aim 3: To describe alterations in speci fic\nbacterial orders (Enterobacteriaceae, Clostridia, and Lactobacillales) in\npediatric patients undergoing HSCT. METHODS/STUDY POPULATION:\nNext-generation sequencing of the hypervariable V3 region of the 16s rRNA\ngene isolated from stool specimens collected at baseline (start of preparative\nregimen to transplant day), early (day 7 –14 post HSCT), and late (day 21 –28\npost HSCT) from 46 children was performed. Microbiome diversity was\nassessed by the Shannon index as well as UniFrac analysis, and compared among\npatients with and without GVHD/BSI. Baseline bacterial diversity was compared\namong patients by primary diagnosis, race, timing of antibiotic introduction and\nmethod of supplemental nutrition. RESULTS/ANTICIPATED RESULTS: Median\nage was 9 years (range 0.5 –19.2 years). There were 36 patients with\nhematologic malignancies. Patients with nonmalignant disease had a character-\nistic pattern of microbiome diversity (and microbiota order distribution) at\nbaseline that persisted throughout the first month of transplant ( p = 0.004).\nOver all patients, there was an early and persistent drop in microbiome\ndiversity throughout the transplant course. Early introduction of broad\nspectrum antibiotics (prior to transplant day) negatively impacted microbiome\ndiversity ( p = 0.02). There was no difference in microbiome diversity among\npatients who developed BSI, when compared with patients without BSI.\nSimilarly, we did not find an association between microbiome diversity and the\ndevelopment of moderate to severe acute GVHD. Ongoing analysis is\nexamining the individual variations in microbiome at the species level, and\ntheir relation to transplant characteristics and clinical outcomes. DISCUSSION/\nSIGNIFICANCE OF IMPACT: In our analysis, microbiome diversity decreased\nduring HSCT, but in contrast to published data, mainly in adult HSCT\npopulations, we found no association between gut microbiome diversity and\nGVHD or BSI. There are ongoing clinical trials in children and adults using\nprobiotics in HSCT with the aim of decreasing GVHD. Further analysis of our\ndata at the species level may further inform the relationship between gut\nmicrobiome alterations and HSCT complications in children and guide clinical\ninterventions.\n2139\nPBX1 mRNA expression is a prognostic biomarker of\nand clinical indicator by RT-qPCR and RNAScope ®\nin situ hybridization in neuroblastoma\nNilay Shah and Julia Selich-Anderson\nThe Ohio State University, Columbus, OH, USA\nOBJECTIVES/SPECIFIC AIMS: (1) Correlate PBX1 mRNA expression as\nmeasured by RNAScope in situ hybridization, at an RNA number/cell\nmeasurement, Versus by RT-qPCR by the ddCt method. (2) Validate PBX1\nmRNA expression in a second independent cohort of neuroblastoma tumor\nsamples, and correlate with patient outcomes. We expect that PBX1\nexpression will correlate whether detected by RNAScope or by RT-qPCR.\nThis work has the promise of validating a novel biomarker of disease severity,\nand for clinical translation as the RNAscope technology has been CLIA-certified\nfor clinical use for other genes. METHODS/STUDY POPULATION: Primary\nneuroblastoma tumor samples were acquired through the Children’s Oncology\nGroup Tumor Bank, The Cooperative Human Tissue Network Tumor bank,\nand the Westmeade Tumor Bank (Westmeade, Australia), with patient\noutcomes annotated but sequestered until experiments are completed. RT-\nqPCR is performed using 1 μg total RNA isolated from each sample by\nNucleospin RNA kit (Clontech), reverse transcribed by SuperScript VILO\n(ThermoFisher Scientific) and ampli fied using KiCqSTART SYBR Green qPCR\nmix (Sigma Aldrich). RNAScope was performed on sections of fresh frozen\ntumor, in triplicate, per manufacturer protocol (ACDBio) using company-\ndesigned probes. Statistical analyses performed using GraphPad Prism5.\nRESULTS/ANTICIPATED RESULTS: PBX1 mRNA expression as measured\nRNAScope correlated well with matched RT-qPCR values, with most PBX1\ntranscripts identi fied within the malignant cells and not in tumor stroma.\nCorrelation with patient outcomes is ongoing (expected to be available at the\ntime of presentation), but as the RNAScope values correlate with R > 0.9 with\nRT-qPCR values, we expect good correlation with outcomes in our primary\ndata set and matching validation set. DISCUSSION/SIGNIFICANCE OF\nIMPACT: PBX1 mRNA expression is an accurate prognostic biomarker of\noutcome in low and intermediate-risk neuroblastoma, and testing on an\nadditional validation set is planned based on thresholds established by\nRNAScope. RNAScope is a method readily translatable to clinical use and its\ninclusion in future clinical trials will be further studied. It provides an additional\nbenefit that concomitant immunohistochemistry can also be performed.\nAnalysis of high-risk neuroblastomas for responsiveness to retinoic acid based\non PBX1 expression is planned.\ncambridge.org/jcts 57\nhttps://doi.org/10.1017/cts.2017.203 Published online by Cambridge University Press","source_license":"CC0","license_restricted":false}