Transcription profiling of human oligodendrocytes identifies o Tmem10/Opalin as a novel marker
Ontology highlight
ABSTRACT: Background; During the development of the central nervous system, oligodendrocytes generate large amounts of myelin, a multilayered insulating membrane that ensheathes axons, thereby allowing the fast conduction of the action potential and maintaining axonal integrity. Differentiation of oligodendrocytes to myelin-forming cells requires the downregulation of RhoA GTPase activity. Results; To gain insights into the molecular mechanisms of oligodendrocyte differentiation, we performed microarray expression profiling of the oligodendroglial cell line, Oli-neu, treated with the Rho kinase (ROCK) inhibitor, Y-27632 or with conditioned neuronal medium. This resulted in the identification of the transmembrane protein 10 (Tmem10/Opalin), a novel type I transmembrane protein enriched in differentiating oligodendrocytes. In primary cultures, Tmem10 was abundantly expressed in O4-positive oligodendrocytes, but not in oligodendroglial precursor cells, astrocytes, microglia or neurons. In mature oligodendrocytes Tmem10 was enriched in the rims and processes of the cells and was only found to a lesser extent in the membrane sheets. Conclusions; Together, our results demonstrate that Tmem10 is a novel marker for in vitro generated oligodendrocytes. Experiment Overall Design: 2 Subexperiments: Experiment Overall Design: 1. olineu in absence of neurons (control) vs. olineu in presence of neurons Experiment Overall Design: including dye swap design with 6 technical replicates Experiment Overall Design: 2. olineu in presence of neurons (control) vs. Y27631 treated olineu in presence of neurons
Project description:Objective: Vascular malformations affect 3% of neonates. Venous malformations (VMs) are the largest group representing more than 50 % of cases. In hereditary forms of VMs gene mutations have been identified, but for the large group of spontaneous forms the primary cause and downstream dysregulated genes are unknown. Methods and Results: We have performed a global comparison of gene; expression in slow-flow VMs and normal saphenous veins using human whole genome micro-arrays. Genes of interest were validated with qRT-PCR. Gene expression in the tunica media was studied after laser micro-dissection of small pieces of tissue. Protein expression in endothelial cells (ECs) was studied with antibodies. We detected 511 genes more than 4-fold down- and 112 genes more than 4-fold up-regulated. Notably, chemokines, growth factors, transcription factors and regulators of extra-cellular matrix (ECM) turnover were regulated. We observed activation and arterialization of ECs of the VM proper, whereas ECs of vasa vasorum exhibited up-regulation of inflammation markers. In the tunica media, an altered ECM turnover and composition was found. Conclusions: Our studies demonstrate dysregulated gene expression in tunica interna, media and externa of VMs, and show that each of the three layers represents a reactive compartment. The dysregulated genes may serve as therapeutic targets. Experiment Overall Design: - 4 samples Experiment Overall Design: - samples are replicates with dye swap
Project description:The cystic leukoencephalopathy without megalencephaly has been defined as distinct autosomal-recessive syndrome of quasi-static encephalopathy with normo- or microcephaly, impaired psychomotor development and characteristic pattern on brain MRI. We identified mutations in the gene encoding the RNASET2 glycoprotein in seven affected individuals as the cause of disease. The results suggest that RNASET2 plays an important role in central nervous system myelination and development. 2-Color dye swap design independent for two different families including technical and biologial replicates for affected family members (disease) and not affected family members (controls).
Project description:One of the most important vectors of the Brazilian Spotted Fever, the tick Amblyomma aureolatum in Brazil was used in this study. We laboratorial controlled the infection of adult females of A. aureolatum with the virulent brazilian strain Taiacu of Rickettsia rickettsii. The group of ticks was divided into 2 testing groups, group 2 (G2) composed of adult females incubated at 35°C for 3 days that were not fed after molting to adults and group 3 (G3) composed of adult females fed on its favorite natural host, the dog (Canis familiaris) also for 3 days. Right after incubation or feeding, ticks were individually dissected and all internal organs were transferred to RNAlater® Solution (Life Technologies) until gDNA and total RNA simultaneously isolation. A total of 14 ticks of each group were analyzed in two biological replicates (7 ticks each). Dye-swap was also applied to construct the technical replicate of each biological sample total RNA from both experimental samples (G2 and G3) was used for hybridization to dual channel arrays. Two biological replicates were used for each experimental group.
Project description:The homeobox transcription factor Prox1 is expressed in embryonic hepatoblasts and remains expressed in adult hepatocytes. Prox1-null mice show severe deficiencies of liver development, but the underlying mechanisms are unknown. We studied the effects of Prox1 on the transcriptional profile of embryonic day-14 (ED14) met-murine-hepatocytes (ED14-MMH). These immortalized murine hepatoblasts express numerous hepatoblast markers, but not Prox1. We performed stable transfection with Prox1 cDNA, analyzed the transcriptome with Agilent mouse whole genome microarrays and validated genes by qRT-PCR. We observed more than 12-fold up-regulation of 22 genes and down-regulation of 232 genes. Numerous of these genes are involved in metabolic hepatocyte functions and may be regulated by Prox1 directly or indirectly, e.g. by down-regulation of HNF4a. Prox1 induces down-regulation of transcription factors, which are highly expressed in neighboring endodermal organs, suggesting a function during hepatoblast commitment. Prox1 does not influence proliferative activity of MMH but regulates genes involved in liver morphogenesis. We observed up-regulation of both type-IVa3 procollagen and functionally active matrix metalloproteinase-2 (MMP-2), which places Prox1 in the centre of liver matrix turnover. This is consistent with MMP-2 expression in hepatoblasts during liver development, and persistence of a basal lamina around the liver bud in Prox1-deficient mice. Our studies suggest that Prox1 is a multifunctional regulator of liver morphogenesis, hepatocyte function and commitment. Experiment Overall Design: - two samples Experiment Overall Design: - Dye Swap design with 6 arrays
Project description:One of the most important vectors of the Brazilian Spotted Fever, the tick Amblyomma aureolatum in Brazil was used in this study. We laboratorial controlled the infection of adult females of A. aureolatum with the virulent brazilian strain Taiacu of Rickettsia rickettsii. The group of ticks was divided into 2 testing groups, group 1 (G1) composed of adult females incubated at 25°C for 3 days and group 2 (G2) composed of adult females incubated at 35°C for 3 days. Right after incubation of both groups, ticks were individually dissected and all internal organs were transferred to RNAlater® Solution (Life Technologies) until gDNA and total RNA simultaneously isolation. A total of 14 ticks of each group were analyzed in two biological replicates (7 ticks each). Dye-swap was also applied to construct the technical replicate of each biological sample total RNA from both experimental samples (G1 and G2) was used for hybridization to dual channel arrays. Two biological replicates were used for each experimental group.
Project description:Sugarcane plants were grown in soil in a 12h light/ 12h dark photoperiod and 26oC for 3 months. Then, the plants were transferred to constant light conditions and 24 h later, leaves were harvested every 4 h for 48 h. Samples were collected from 24 h in constant light to 68 h in constant light and were labelled accordingly. Two biological replicates of each time point were made, with a dye swap with the reference. The reference used was a equimolar solution of all samples.
Project description:Sugarcane one-eyed seed sets obtained from a variety sensitive to drought conditions (cv. SP90-1638, CTC, Brazil) were cultivated on moist sand for 15 days. The plants were transferred to pots containing moist sand and were irrigated with Hoagland's solution (Hoagland and Arnon, 1950). Regular watering was maintained for 90 days and suppressed after this period for the experimental group. Leaves were collected 24 h, 72 h and 120 h after exposure to drought conditions for the control and experimental groups. Samples were collected and immediately frozen in liquid nitrogen. Leaves from six plantlets were used for each time point. Extraction of total RNA was performed separately on each sample pool. Keywords: time course of stress response RNA from experimental sample and from its respective control sample (untreated plants) was used for hybridization to dual channel arrays. Two biological replicates were used for each experimental point. The quantification of each hybridization was submitted in two files, one for each slide side (technical replicates).
Project description:Cord blood-derived stem cells were in vitro cultured in the presence of 40 ng/ml SCF, 20 ng/ml IL6 and 2 microM lysophosphatidic acid for 5 week. Then mast cells were magnetically isolated and further cultured for 4 days in the presence or absence of 2 ng/ml TGF-betaI. Total RNA was isolated and processed according to the Agilent Low-input RNA Linear Amplification Kit and microarray hybridization protocol. Experiment Overall Design: 5 biological replicates from 5 donors and one technical replicate (dye-swap)
Project description:The alternative sigma factor sigmaB has an important role in the acquisition of stress-resistance in many gram-positive bacteria. In the foodborne pathogen Bacillus cereus sigmaB is activated strongly upon a heat shock and other stress conditions. Here we describe the identification of the set of sigmaB-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. A total of 24 genes could be identified as being sigmaB-dependent as witnessed by (i) significantly lower expression-levels of these genes in mutants deleted for sigB and rsbY (encoding the alternative sigma factor sigmaB and a PP2C phosphatase that acts as a crucial positive regulator of sigmaB-activity, respectively) than in the parental strain B. cereus ATCC 14579, and (ii) increased expression of these genes upon a heat shock. Newly identified members of the sigmaB-regulon of B. cereus include an ECF-sigma factor (sigmaZ), a histidine kinase and two genes that have predicted functions in spore germination. Our data indicate that the sigmaB-regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses of sigmaB in which sigmaB in the B. cereus group was suggested to be close to the ancestral form of sigmaB in gram-positive bacteria. Three comparisons were performed: WT-DsigB, WT-DrsbY and WT30degrees-WT42degrees