Microarray of iPSC-derived macrophages generated in a bioreactor process compared to PBMC-derived macrophages and exposed to P. aeruginosa
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ABSTRACT: Human iPSC-Mac or PBMC-mac were seeded on 24-well plates (500.000cells/well) and cultured over night. On the next day, cells were washed three times with PBS. Subsequently, P. aeruginosa laboratory strain PAO1 in RPMI medium without antibiotics (MOI10) was centrifugated on the cells (600xg) and incubated at 37°C. Cells with medium only served as non-infected controls. After 1h cells were de-attached, washed and resuspended in RNA lysis buffer. RNA isolation was performed with RNAeasy micro Kit (Qiagen), according to manufactures instructions. Human iPSC samples were obtained after sorting of iPSC for TRA-1-60+ to separate iPSCs from feeder cells.
Project description:The bovine mammary epithelial cell line Mac-T has been used to study mammary gland in vitro. The reliability of this system to study mammary gland has not been tested using genomics approaches. In the present experimetn a direct transcriptomics comparison between Mac-T cells and mammary tissue at -30 and at 60 day in milk (DIM) is performed. Data indicated that Mac-T cells and mammary tissue had a substantially different transcriptome with a larger difference between Mac-T cells and lactating mammary tissue (i.e., 60 DIM) compared to non-lactating mammary tissue (i.e., -30 DIM). In addition, data indicated that the Mac-T cells substantially differ with mammary tissue in lactation-specific functions. cDNA from Mac-T cells induced in vitro into lactation for 36h (addition of prolactin) was directly hybridize in the microarray chip with cDNA from mammary tissue at -30 or 60 day in milk from 3 cows.
Project description:Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection within the cystic fibrosis (CF) lungs. The effects of sub-inhibitory colistin on gene expression in P. aeruginosa were investigated by transcriptome microarray and functional analysis. Analysis revealed an alteration in the expression of 60 genes in total from a variety of pathways. Genes associated with bacterial chronic colonisation and virulence such as response to osmotic stress, motility, and biofilm formation, as well as those associated with LPS modification and quorum sensing are the most highly represented. Most striking among these is the upregulation of the PQS biosynthesis operon including pqsH, pqsE, and the anthranilate biosynthetic genes phnAB. Early activation of this central component of the QS-network may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF-lung. Experiment Overall Design: Three independent cultures of the P. aeruginosa strain PAO1 were exposed to 0.15 µg colistin mlâ1. The untreated and treated samples were grown from OD600 0.05 to 0.8 and subsequently total RNA was extracted using the Ambion RiboPureTm- Bacteria kit according to the manufacturerâs instructions.
Project description:Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organised quorum sensing (QS) systems via small acyl-homoserine lactone (AHL) signal molecules. Recently, a third bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the QS systems. To further dissect the various independent regulation levels for many QS induced genes and to evaluate the impact of PQS on the QS circuitry, we performed a transcriptome analysis of PAO1 cultures supplemented with PQS. The global transcriptional profile in response to PQS revealed a marked up-regulation of genes belonging to the tightly interdependent functional groups of iron acquisition and oxidative stress response. Remarkably, not only most of the differentially regulated genes but also the induction of a lacZ transcriptional fusion of rhlR could be traced back to a iron chelating effect of PQS. Nevertheless, although iron deficiency per se induced rhlR, there seems to be PQS specific effects that are independent of the PQS effect on P. aeruginosa iron homeostasis Experiment Overall Design: For RNA extraction bacteria were harvested at early logarithmic, late logarithmic and stationary phase of growth. Three independent cultures of PQS-treated and untreated PAO1 each were pooled and the RNA was immediately stabilized with RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). RNA was isolated using the RNeasy kit (Qiagen), treated with DNaseI (Roche) for 30 min at 37M-BM-0C and re-purified with the RNeasy spin column. The subsequent steps of cDNA generation and Biotin-ddUTP terminal labelling were performed as described in the manufacturerM-bM-^@M-^Ys instructions for the P. aeruginosa GeneChipM-CM-^R. Fragmented and labelled cDNA was hybridised to a GeneChip at 50 M-BM-0C for 16 h. The washing steps, staining and scanning of the microarrays were performed using the Affymetrix GeneChip system. Data analysis was performed using the Affymetrix Microarray Suite Software 5.0 with Affymetrix default parameters. As expression analysis was performed in duplicate, a total of two GeneChips per culture condition was scanned at 570 nm, 3 M-BM-5m resolution in an Affymetrix GeneChip scanner. The signals were multiplied by a scaling factor to make the average signal for all the arrays equivalent. The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. Data were combined with the latest annotation from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at www.pseudomonas.com.
Project description:Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into GABA and succinate before entering the TCA cycle in support of cell growth as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were identified to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown indispensable for polyamine utilization. The newly identified dadRAX locus, encoding the regulator, alanine transaminase and racemase respectively, coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintain alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, the alternative gamma-glutamylation pathway for the conversion of putrescine into GABA was also discussed. Subsequently, GabD, GabT and PA5313 were identified for GABA utilization. Growth defect of PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA was also demonstrated in vitro. Polyamine utilization in general was proven independent of the PhoPQ two-component system even the expression of which was induced by polyamines. Multiple potent catabolic pathways as depicted in this study could serve pivotal roles in control of intracellular polyamine levels. Experiment Overall Design: P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of L-glutamate alone or with the addition of putrescine, agmatine or GABA at 20 mM. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Only genes showing consistent expression profiles in duplicates were selected for further analysis.
Project description:In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity. In this study, primary monocytes were differentiated into macrophages using M-CSF (M-Mac) or with a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in the macrophages, four of which were expressed higher in I-Mac (miRNAs 2.1, 8.1, 9.1 and 14.2) whilst three were detected in both M-Mac and I-Mac (miRNAs 9.3, 13.6 and 15.8). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Gene expression microarrays from 3 samples of microphage treated with M-CSF (M-MAC) were compared with 3 samples of micropahge treated with M-CSF + IL-27 (I-MAC)
Project description:In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity. In this study, primary monocytes were differentiated into macrophages using M-CSF (M-Mac) or with a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in the macrophages, four of which were expressed higher in I-Mac (miRNAs 2.1, 8.1, 9.1 and 14.2) whilst three were detected in both M-Mac and I-Mac (miRNAs 9.3, 13.6 and 15.8). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. screening novel and known miRNAs which may have antiviral properties in 2 different treatments in 2 donors.
Project description:Differential expression of regulatory genes of reference strain P. aeruginosa PA01, involved in the quorum sensing was analyzed using Microarray. Total RNA was isolated from reference strain P. aeruginosa PA01, grown with or without bacterial extracts (0.1 mg/ml) using TRI reagent. Total RNA was quantified, and 10 µg RNA was converted to cDNA, fragmented and labelled by following GeneChip® P. aeruginosa PA01 genome array user manual . Labelled cDNAs were hybridized with P. aeruginosa genome array gene chip , washed and stained. Hybridized chips were scanned, processed and analyzed using expression console and transcriptome analysis console.
Project description:Addressing the functionality of predicted genes remains an enormous challenge in the post-genomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism and cell division, during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins.
Project description:Many patients with Cystic Fibrosis suffer from Gastroeosphgeal Refux Disease (GERD). This disease may lead to the aspiration of bile into the lungs. We invesigated the effect of bile on the CF lung pathogen P. aeruginosa. We analysed gene expression profiles, comparing bile treated and untreated cells to identified distinct classes of up/down-regulated genes. P. aeruginosa PAO1 was grown to log stage for RNA extraction and hybridization on Affymetrix microarrays. We conducted three biological replicates as to increase the resolution of expression profiles.