The analysis of genomic data and integration of diverse biological data sources has become increasingly difficult for researches in the life sciences. This problem is exacerbated by the speed with which new data is gathered through automated technology like DNA microarrays. We developed a virtual reality application for visualizing hierarchical relationships within a gene family and for visualizing networks of gene expression data. Integration of other information from multiple databases with these visualizations can aid pharmaceutical researchers in selecting target genes or proteins for new drugs. We found the application of virtual reality to the field of genomics to be successfull.
<p>Supplemental data 1: Cell line information: Summary of the characteristics of cell lines used in this study including origin, tumor type, ALK and MYCN status (wt = wild type, NA = non amplified, A = amplified). Supplemental data 2: GI50 concentration of TAE-Ââ€�684 in neuroblastoma cell lines Supplemental data 3: phospho-Ââ€�ALK and total ALK expression in a panel of 10 neuroblastoma cell lines Supplemental data 4: ALK expression upon ALK inhibition using shALK Supplemental data 6: Expression of 77 ALK regulated genes in a panel of 10 neuroblastoma cell lines treated with ALK inhibitor TAE-Ââ€�684 Supplemental data 7: Expression of 77 ALK regulated genes in SK-Ââ€�N-Ââ€�AS with induced ALK Supplemental Data 8: Progression-Ââ€�free survival versus ALK signature scors Supplemental data 11: ALK signature gene analysis upon inhibition of two ALK downstream pathways Supplemental data 14:Expression of MAPK genes in CLB-Ââ€�GA after ALK inhibition Supplemental data 15: A. RET mRNA expression levels upon FOXO3 activation (+Dox) and Pi3K/AKT inhibition (+PI) in cell line SY5Y (Santo et al., 2013, GEO-Ââ€�id: GSE42762); B. pFOXO3a levels upon ALK inhibition with the LDK-Ââ€�378 compound are downregulated Supplemental data 16: Cell growth upon treatment of neuroblastoma cell lines with different concentrations of Trametinib (MAPK/MEK inhibitor) is only modestly affected Supplemental Data 17: Scheme to clarify our hypothesis</p>
<p>Supplemental data 13: Summary of Gene Set Enrichment analysis results obtained on expression profiling data of CLB-Ââ€�GA on different time points (10' - 30'- 60' - 2h - 4h - 6h) after pharmacological inhibition using TAE-Ââ€�684.</p>
Abstract The development of Alzheimer’s disease (AD) involves central and peripheral immune deregulation. Gene identification and studies of AD genetic variants of peripheral immune components may aid understanding of peripheral-central immune crosstalk and facilitate new opportunities for therapeutic intervention. In this study, we have identified in a Flanders-Belgian family a novel variant p.E317D in the Toll-like receptor 9 gene ( TLR9 ), co-segregating with EOAD in an autosomal dominant manner. In human, TLR9 is an essential innate and adaptive immune component predominantly expressed in peripheral immune cells. The p.E317D variant caused 50% reduction in TLR9 activation in the NF-κB luciferase assay suggesting that p.E317D is a loss-of-function mutation. Cytokine profiling of human PBMCs upon TLR9 activation revealed a predominantly anti-inflammatory response in contrast to the inflammatory responses from TLR7/8 activation. The cytokines released upon TLR9 activation suppressed inflammation and promoted phagocytosis of Aβ 42 oligomers in human iPSC-derived microglia. Transcriptome analysis identified upregulation of AXL, RUBICON and associated signaling pathways, which may underline the effects of TLR9 signaling-induced cytokines in regulating the inflammatory status and phagocytic property of microglia. Our data suggest a protective role of TLR9 signaling in AD pathogenesis, and we propose that TLR9 loss-of-function may disrupt a peripheral-central immune crosstalk that promotes dampening of inflammation and clearance of toxic protein species, leading to the build-up of neuroinflammation and pathogenic protein aggregates in AD development.
Abstract Background The most common TREM2 variant associated with increased risk to develop Alzheimer’s disease (AD) is p.R47H. This variant has been classified as loss‐of‐function, mostly by comparing p.R47H with the common variant in TREM2 over‐expressing cell lines. Whether this holds true when TREM2 is expressed at physiological levels in a disease‐relevant cell type such as human microglia, has not been fully characterized. Method We established and thoroughly characterized human iPSC‐derived microglia endogenously expressing TREM2 to assess the effect of p.R47H on TREM2 cellular distribution, TREM2 signaling and microglial functioning. For this study microglia generated from a set of isogenic gene‐edited iPSC lines homozygous for TREM2 knockout (KO) and TREM2 p.R47H were evaluated and compared to microglia from the parental control iPSC line. In addition, microglia generated from iPSC lines obtained from several p.R47H carriers were compared to microglia from iPSC lines obtained from several healthy control non‐carriers. SNP genotyping confirmed the presence of R47H. Result The p.R47H and common variant TREM2 were present at similar levels at the cell surface of iPSC‐derived microglia, whereas no expression of TREM2 was detected in the TREM2 KO microglia. Microarray profiling indicated that the microglial transcriptome is very similar between the common variant and p.R47H yet distinct from TREM2 KO microglia. Biochemical evaluation of TREM2 signaling indicated no reduction in TREM2 downstream signaling activation in the p.R47H variant expressing microglia. Similarly, soluble TREM2 levels were increased in culture medium of p.R47H microglia. Finally, analysis of microglial functioning including phagocytosis of artificial and disease‐relevant substrates showed no difference between the p.R47H variant and the common variant, in contrast to TREM2 KO microglia. Conclusion We conclude that in cultured human iPSC‐derived microglia, the p.R47H variant shows no evidence of a loss‐of‐function mechanism. Future experiments will determine whether this is also the case in microglia in their natural brain environment. Our long‐term goal is to better understand TREM2 biology to support the development of novel therapeutics for AD and other neurodegenerative diseases characterized by TREM2‐mediated neuroinflammation.
Background: Rheumatoid arthritis (RA) is a disease characterized by synovial joint inflammation, mainly affecting small joints. Histological findings in synovial biopsies ranges from inflammatory infiltration including ectopic lymphoid structures, to a cell sparse fibroid phenotype. T cells in affected joints are non-naïve and have by flow cytometry approaches been shown to have a wide TCR-beta chain gene usage. New technologies allow for analyses of paired TCR sequences and their antigen-specificities. Objectives: To study the alpha/beta-T cell receptor repertoire in single sorted T cells from synovial biopsies at time of RA-diagnosis. Meth ods: Synovial biopsies were taken, primarily using an ultrasound guided technique, from seventeen patients (12 ACPA+, 5 ACPA-) with rheumatoid arthritis. Fresh biopsies were enzymatically digested, followed by mild mechanical treatment, prior to flow cytometry cell sorting. Single cell index sorting of T cells was made into 384-well plates with PCR-buffer followed by a nested PCR and deep sequencing of the TCR amplicons. TCR-receptor sequences showing clonal expansion from four ACPA+ HLA-DRB1*0401 patients were further cloned into SKW3 cells for studies of their reactivity by in vitro stimulation with peptides of viral and citrullinated origin from the literature. A positive response, as measured by CD69-up regulation or IL-2 production, was used to define specificity. Results: Fourteen of the assessed joints were small (1 MTP, 4 MCP and 8 wrists), whereas the remaining three were large joints (2 knees and 1 ankle), table 1. Individual T cells could be isolated from all of these biopsies, with a variating CD4:CD8 ratio. Based on the flow cytometry phenotyping we could identify CD4 T cells of both Treg and T peripheral helper phenotype already at this early time point. Productive alpha/beta-TCR sequences could be retrieved from 16 out of 17 patients and clonal expansion (>1 copy/TCR) was seen in all but one of these patients, with clone sizes ranging between 2 – 34 copies of each TCR. Table 1. Patient characteristics. Patients Gender (F/M ) HLA-SE alleles Joints Joint swelling prior to biopsy (months ) Stiffness specific joint (median VAS ) Pain specific joint (median VAS ) ACPA+ (n = 12) 9/3 *0401, *0404, *0408, *01 and *10 1 MTP, 4 MCP, 6 wrists, 1 knee 4 (1-12) a 46 (0-84) 45 (22-99) ACPA- (n = 5) 3/2 *0401 1 MCP, 2 wrists, 1 ankle, 1 knee 5 (0.25-7) 59 (15-73) 47 (33-81) SKW3 cell lines (patients n = 4) 4/0 * 0401/0404 n=2 *0401 n=2 1 MTP, 3 wrists 2 (1-6) 50.5 (42-84) 50 (40-99) a Data not available for one patient. One patient with prior RA-diagnosis, but after 9 months of treatment remission lasting for 20 years. Artificial T cell lines were generated from the expanded clones of HLA-DRB1*04:01 RA subjects. Our in vitro stimulation protocol identified virus specific CD4 T cells in all samples. So far, no citrulline reactivity has been found. HCMV, followed by HHV were the most commonly found viral reactivities, whereas others were found only in one donor (e.g. JCV, EBV). The majority of clones are thus “orphans”, to which we are still seeking the driving antigen. Conclusion: Clonally expanded T cells are found in the synovium of early RA patients and include virus-specific CD4+ T cells. Our data show that the local T cell repertoire is broad already at the time of RA diagnosis Disclosure of Interests: Sara Turcinov: None declared, Erik af Klint Paid instructor for: Abbvie (courses and lectures), An De Bondt Employee of: Janssen., Muhammad Sohel Mia: None declared, Anca Catrina: None declared, Frederik Stevenaert Employee of: Janssen, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica
Abstract Motivation: Existing pre-processing methods for DNA microarrays designed to detect copy number variations (CNVs) lead to high false discovery rates (FDRs). High FDRs misguide researchers especially in the medical context where CNVs are wrongly associated with diseases. We propose a probabilistic latent variable model, cn.FARMS, for array-based CNV analysis which controls the FDR without loss of sensitivity. At a DNA region, cn.FARMS constructs a model by a Bayesian maximum a posteriori estimation where the unobserved, latent variable represents the copy number that is measured by observed genetic markers (probes). The latent variable’s prior prefers parameters which represent the null hypothesis, (same copy number for all samples), from which the posterior can only deviate by a high information content in the data. The more probes agree on the region’s copy number, the less is the uncertainty about the latent variable’s value, the higher is the information content. Results: We compared cn.FARMS on a HapMap Mapping250K_Nsp and SNP6.0 benchmark data set to CRMAv2 and dChip. The comparison is based on the sex determination based on the data from the X chromosome, where males possess one copy and females two. The ROC curve serves to compare the FDR for different true positive rates. In both experiments cn.FARMS yielded the best classification results.Availability: This approach is publicly available in R at http://www.bioinf.jku.at/software
