Project description:This SuperSeries is composed of the following subset Series: GSE40560: Transcriptome analysis in primary fibroblasts from HOIL-1-deficient patients upon TNF-alpha or IL-1beta stimulation GSE40561: Transcriptional analysis of whole blood in patients with auto-inflammatory disorders GSE40838: Transcriptome analysis in peripheral blood mononuclear cells (PBMC) from HOIL-1-deficient patients upon TNF-alpha or IL-1beta stimulation Refer to individual Series
Project description:MCF-7 cells were stimulated with TNF-alpha in order to identify IKKb substrates. conditions: TNF alpha stimulation TNF alpha stimulation + SC-514 IKK (kinase dead mutant) + TNF alpha stimulation IKK(WT) + TNF alpha stimulation basal Already validated IKK substrates were used to train random forest and to predict new substrates. Among other interesting candidates we validated AEG-1 (S298) as an IKKb substrate. We provide evidence that IKKb-mediated AEG-1 phosphorylation is essential for IkBa degradation as well as NF-kB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. (replicate 1 out of at least 2)
Project description:<p>The etiologies of primary immunodeficiencies often yield novel insights about the immune system. Although a genetic etiology has been suspected for patients with abnormally low CD4+ T cells in the absence of HIV infection or any known causes of lymphopenia, no genetic mutation has been described to date for any case of primary CD4 lymphopenia. In this study, we characterized a non-consanguineous family with two non-HIV infected boys exhibiting an inverted CD4 to CD8 T cell ratio and a history of recurrent chronic viral infections since birth. Consistent with a decreased thymic output of CD4+ T cells, the percentage of CD31+ cells in the CD4+ naive population of these patients was decreased. In addition, the activation of T cells was significantly impaired in the patient upon TCR stimulation. Given the mother's T cells show completely skewed X chromosome inactivation, we suspected that the nature of this disease is X-linked. We performed X-chromosome exon-capture targeted single-end Solexa sequencing on two brothers and the mother and found a 10 base pair deletion at an intron-exon junction of Magnesium Transporter 1 (MAGT1), a Mg<sup>2+</sup> specific transporter. We confirmed that this deletion leads to altered splicing, frameshift, early termination of the mRNA, and deficient protein expression in the lymphocytes of the two patients. Moreover, knockdown of this transporter in T cells isolated from healthy donors can recapitulate the observed T cell activation defect while its ectopic expression in the patients' lymphocytes can restore T cell stimulation. Our discovery highlights the significance of this transporter to T cell function.</p>
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to TNF-alpha (100 units/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, TNF monosome and TNF polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (TNF polysome/TNF monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. Keywords: Translation State Array Analysis