12 microarrays for cis-DCE mineralization and new stress genes
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ABSTRACT:
ORGANISM(S): Escherichia coli K-12 Escherichia coli
PROVIDER: GSE13698 | GEO | 2009/11/24
SECONDARY ACCESSION(S): PRJNA109423
REPOSITORIES: GEO
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Project description:Metabolically-engineered Escherichia coli has been used previously to degrade the ubiquitous pollutant cis-1,2-dichloroethylene (cis-DCE), and the impact of the metabolic engineering was assessed by investigating the changes in the proteome. Here, genome-wide transcriptome analysis was performed to confirm that a strong heat shock and/or oxidative stress occurs during enhanced cis-DCE mineralization. Also, seven new stress proteins (YchH, YdeI, YodD, YodC, YgiW, YhcN, and YjaA) that were previously uncharacterized have been identified. Experiment Overall Design: Twelve planktonic RNA samples for the six sets of microarray experiments (set i was repeated) were prepared (Table 1 indicates the rationale of the whole-transcriptome studies): (i) cells of E. coli TG1 with TOM-Green/IsoILR1/GSHI* vs. no cloned genes contacted for 2 h with 1 mM cis-DCE to determine the effect of expressing eight genes (encoding TOM-Green/IsoILR1/GSHI*) on cis-DCE mineralization, (ii) cells of TG1 with TOM-Green/EchA F108L/I219L/C248I vs. TOM-Green contacted for 2 h with 1 mM cis-DCE to determine the effect of the evolved epoxide hydrolase on cis-DCE mineralization, (iii) cells (turbidity of 1.0) of E. coli BW25113 with 2 mM H2O2 vs. no H2O2 contacted for 10 min with shaking to determine gene expression in response to H2O2 stress since cis-DCE mineralization induced many oxidative stress genes (iv) cells (turbidity of 1.0) of the BW25113 ygiW strain with 2 mM H2O2 vs. no H2O2 contacted for 10 min to determine gene expression in response to H2O2 stress in the absence of YgiW, and (v) cells (turbidity of 1.0) of the BW25113 ychH strain with 2 mM H2O2 vs. no H2O2 contacted for 10 min to determine gene expression in response to H2O2 stress in the absence of YchH. Experiment Overall Design: For the cis-DCE degradation system, the exponentially-grown TG1 cells (Table 1) were contacted with 1 mM cis-DCE together with 0.5 mM IPTG and 5 mM succinate (as a carbon/energy source during the cis-DCE degradation) in 60 mL vials for 2 hr as performed previously in the proteomics study J. Proteome Res. 5: 1388 - 1397 (2006). After 2 hr contact with cis-DCE, samples were centrifuged, and the cell pellets were frozen immediately with dry ice and stored at -80°C. For the H2O2 stress system, overnight cultures of BW25113, BW25113 ygiW, and BW25113 ychH were re-grown to mid-log phase in LB (turbidity at 600 nm of 1), then the cultures were split into two cultures and 2 mM H2O2 and water (negative control) were added. After contacting for 10 min, samples were centrifuged and the cell pellets frozen immediately with dry ice and stored at -80°C. RNA was isolated from the biofilm cells as described previously using sonication and a bead beater Appl. Microbiol. Biotechnol. 64: 515-524 (2004).. Experiment Overall Design: For DNA microarray analysis, the E. coli Genechip antisense genome array (Affymetrix, P/N 900381, Santa Clara, CA) and the E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N 900551) were used to study the differential gene expression profile of the K12 cells; the E. coli Genechip antisense genome array contains probe sets for all 4290 open reading frames (ORF), rRNA, tRNA, and 1350 intergenic regions, and the E. coli GeneChip Genome 2.0 Array contains 10,208 probe sets for open reading frames, rRNA, tRNA, and intergenic regions for four E. coli strains: MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933. The same type of microarray was used for each binary comparison. cDNA synthesis, fragmentation, and hybridizations were as described previously J. Bacteriol. 188: 587-598 (2006). Hybridization was performed for 16 h, and the total cell intensity was scaled to an average value of 500. The probe array images were inspected for any image artifact. Background values, noise values, and scaling factors of both arrays were examined and were comparable. The intensities of polyadenosine RNA control were used to monitor the labeling process. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 11-15 probe pairs (p-value less than 0.05), these genes were discarded. Experiment Overall Design: Extract protocol: To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 50 seconds at the maximum speed in a mini bead beater (cat. no. 3110BX, Biospec). The total RNA was isolated by following the protocol of the RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase I (Qiagen). Experiment Overall Design: Label protocol: The total RNA samples were first converted into cDNA through a reverse transcription reaction with poly-A RNA controls spiked into the same reaction mixture to monitor the entire target labeling process. The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50-200 bp fragments, which was checked by running the fragmented cDNA on a 2% agarose gel. The fragmented cDNA was labeled at the 3’ termini by the Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP (Affymetrix, P/N 900181). Experiment Overall Design: Hybridization protocol: The biotin-labeled target was hybridized to the Affymetrix GeneChip E. coli array at 45C for 16 hour at 60 rpm using the Hybridization Oven 640 (Affymetrix), then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure. Experiment Overall Design: Scanning protocol: The microarray was scanned at 570 nm to get an image file by the GeneChip Scanner 3000 (Affymetrix). Using GeneChip® Operating Software, total cell intensity was scaled automatically in the software to an average value of 500.
2009-11-27 | E-GEOD-13698 | biostudies-arrayexpress
Project description:12 microarrays for cis-DCE mineralization and new stress genes
| PRJNA109423 | ENA
Project description:Cartilage mineralization is a tightly controlled process, imperative for skeletal growth and fracture repair. However, in osteoarthritis (OA), cartilage mineralization may impact the joint range of motion, inflict pain and may increase chances for joint effusion. Here we attempt to understand the link between inflammation and cartilage mineralization by targeting SIRT1 and LEF1, both reported to have contrasting effects on cartilage.
2022-05-16 | GSE200522 | GEO
Project description:Rosiglitazone-targeted transcriptional networks as gene expression signature of physiological and pathological mineralization – a top-down approach to identify novel targets for bone and vascular health Clinical studies have recently revealed detrimental skeletal and vascular effects of the insulin sensitizer rosiglitazone. At the cellular level, we have shown earlier that rosiglitazone accelerates osteoblast differentiation from human mesenchymal stem cells (hMSC) at the expense of increased oxidative stress and apoptosis. In calcifying human vascular cells, rosiglitazone stimulates pathological mineralization, an effect that was diminished by the antioxidant resveratrol. In order to elucidate the transcriptional networks underlying the rosiglitazone-enhanced mineralization phenotype, we performed genome-wide transcriptional profiling of osteogenic hMSCs treated with rosiglitazone for short-term periods of 1 up to 48h during the first two days of culture time, a phase that we show is sufficient to mediate the stimulatory effect of rosiglitazone on mineralization. Microarray-based mRNA expression analysis revealed 190 probes that were differently expressed in at least one condition compared to vehicle-treated control. This rosiglitazone gene signature contained confirmed primary PPAR targets and was also endogenously regulated during osteogenic differentiation from hMSCs and osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) into calcifying vascular cells (CVCs). Further comparative analysis revealed rosiglitazone targets that were commonly enriched in osteoblasts and CVCs or specifically enriched in either osteoblasts or CVCs. Finally, we compared the expression patterns of CVC-specific genes with patient expression data from carotid plaque versus intact adjacent tissue, and identified five rosiglitazone targets that were differentially regulated in CVCs and carotid plaque but not osteoblasts when compared to their respective non-mineralizing counterparts. These targets, i.e PDK4, SDC4, SPRY4, TCF4, and DACT1 may specifically control extracellular matrix mineralization in vascular cells and hence provide interesting candidates for further investigations to improve vascular health.
2023-10-05 | GSE67518 | GEO
Project description:Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases, including osteoporosis and bone tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (MSCs). CASC2 knockdown using small interfering RNA (siCASC2) increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining levels, or the expression of early osteogenic transcripts including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at either transcriptional and protein level. Inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP levels. The results indicate that lncRNA CASC2 regulates late osteogenesis and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests lncRNA CASC2 as a potential new therapeutic target in bone mineralization.
2023-06-14 | PXD040463 | Pride
Project description:We report the application of next generation sequencing technology for high-throughput profiling of mRNA, circRNA and lncRNA in hFOB 1.19 cells during the mineralization. Osteoblast RNA profiles in different process of mineralization, which is day 0, day 3, day 6 and day 9, respectively. The transcriptome sequencing on an Illumina HiSeq X Ten platform were carried out at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. In the process of osteoblast mineralization, a total of 8464 dysregulated circRNAs were identified, of which 6332 circRNAs were upregulated and 2132 circRNAs were downregulated. In addition, 1633 differentially expressed mRNAs were also identified, with 1027 mRNAs upregulated and 606 mRNAs downregulated. This study provides a framework for the application of NGS profiling towards characterization of osteoblast mineraliztion.
2020-11-08 | GSE148203 | GEO
Project description:In the present study, the Agilent-016251 Sparus aurata oligo microarray platform (GPL6467) was used to compare expression profiles of mineralization-induced VSa13 cell cultures against untreated ones. ECM mineralization was induced for 4 weeks by supplementing medium with 50 µg/ml of L-ascorbic acid, 10 mM β glycerophosphate and 4 mM CaCl2. For each group, total RNA was extracted from three (3) independent biological replicates, each consisting of pools of cells. Data analysis demonstrated that expression profiles were strongly affected by ECM mineralization with hundreds of genes differentially expressed with relevant fold-change.
2011-06-01 | GSE18915 | GEO
Project description:We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone. We isolated total RNAs from long bones of both WT and Hyp mice at 12 days of age. Since the RNA yields from the long bones are very low, we combined 2 bone samples with same genotype (WT or Hyp) for one RNA extraction. We will compare the difference of the gene expressions between Hyp and WT. We will use 4 samples in each animal condition.
2009-12-21 | E-GEOD-14242 | biostudies-arrayexpress
Project description:Purpose: The goal of this study is to compare (RNA-seq) transcriptomes of in vitro cultured human bone marrow-derived mesenchymal stem cells (hMSCs) and fallow deer antler-derived skeletal progenitors (FD RM Cells) under multiple conditions to identify candidate proliferation and mineralization genes responsible for fast antler regeneration Methods: hMSCs and FD RM Cells were cultured in vitro under 1) serum-free (0% serum) or serum (10% serum) conditions for 2.5 days or 2) Control (0 ng/mL BMP-2 and 0 nM dexamethasone) and osteogenic (100 ng/mL BMP-2 and 100 nM dexamethasone) media for 24 days. mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. The sequence reads were analyzed at the transcript isoform level STARS followed by Cufflinks. Validation for genes of interest was performed using immunofluorescence staining. Results: Comparison of human and fallow deer skeletal progenitor datasets yielded proliferation and mineralization gene candidates Conclusions: Our study represents the first detailed analysis of human and fallow deer transcriptomes of skeletal progenitor cells under proliferation and mineralization conditions, with biologic replicates, generated by RNA-seq technology. Our in vitro comparative approach circumvent some of the logistical and technical challenges in identifying candidate proliferation and mineralization genes responsible for rapid deer antler regeneration. We conclude that in vitro comparison of RNA-seq based transcriptomes identified candidate proliferaiton and mineralization genes to advance bone biology and holds promise to rapidly regenerate large bone volumes for regenerative medicine. The comparative approach utilized here can be adapted for almost any tissue to study a specific phenomenon of interest.
2018-11-16 | GSE89796 | GEO
Project description:In the present study, the Agilent-016251 Sparus aurata oligo microarray platform (GEO accession: GPL6467) was used to compare expression profiles of mineralization-induced VSa16 cell cultures against untreated ones. ECM mineralization was induced for 4 weeks by supplementing medium with 50 µg/ml of L-ascorbic acid, 10 mM β glycerophosphate and 4 mM CaCl2. For each group, total RNA was extracted from three (3) independent biological replicates, each consisting of pools of cells. Data analysis demonstrated that expression profiles were strongly affected by ECM mineralization with hundreds of genes differentially expressed with relevant fold-change.
2011-06-01 | GSE18941 | GEO