Project description:Corneal epithelial stem cells reside in the limbus that is the transitional zone between the cornea and conjunctiva, and are essential to maintain the homeostasis of corneal epithelium. However, their characterization is poorly understood. Therefore, we constructed gene expression profiles of limbal epithelial SP and non-SP cell using RNA-sequencing. As a result, limbal epithelial SP cells have immature cell phenotypes with endothelial/mesenchymal cell markers, while limbal epithelial non-SP cells have epithelial progenitor cell markers.
Project description:Puccinia graminis f. sp. tritici is the cause of wheat stem rust. A microarray was designed from genes predicted from the P. graminis f. sp. tritici genome assembly, and gene expression measured for four conditions which include wheat or barley infecting growth stages initiated by urediniospores. mRNA was prepared from fresh urediniospores, uredinospores germinated for 24 hr, wheat seedlings infected with urediniospores for 8 days, and barley seedlings infected with urediniospores for 8 days. The asexual uredinial infection cycle on wheat produces additional urediniospores, which can start new cycles of wheat infection and are readily spread by aerial transport. This expression data is further described in Duplessis et al, Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici
Project description:Targeted therapies against cancer stem cells which are enriched in side populations (SP) involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the murine lung adenocarcinoma cell line A2C12 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment.
Project description:Targeted therapies against cancer stem cells which are enriched in side populations (SP) involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the human lung adenocarcinoma cell line A549 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment.
Project description:Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high.
Project description:In previous work in our group, shotgun genome sequencing of Arthrobacter sp. revealed potential new P450 monooxygenases and many other oxidoreductases with putative hydroxylation activity. A targeted approach to identify enzymes involved in the degradation of certain molecules is proteomic analysis. In the case of growth on certain substances, enzymes like P450s, which are responsible for the observed organism’s capabilities, might be overexpressed or initially induced.
Project description:Macrophages play fundamental roles in regulation of inflammatory responses to pathogens, resolution of inflammation and tissue repair, and maintenance of tissue homeostasis. The long (L) and short (S) isoforms of SP-R210/MYO18A, a macrophage receptor for surfactant protein A (SP-A) and C1q, regulate basal and inflammatory macrophage phenotype at multiple gene expression, translational, and subcellular levels in addition to their SP-A and C1q-mediated functions; disruption of L renders macrophages hyper-inflammatory, although the underlying mechanism had previously been unexplored. We questioned whether disruption of the L isoform would alter the global genomic responses. RNA sequencing analysis of SP-R210L(DN) macrophages revealed basal and influenza induced upregulation of genes associated with inflammatory pathways, including TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockdown of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) compared to WT cells. ChIP-seq analysis of histone H3 methylation showed alterations in both repressive (H3K9me3 and H3K27me3) and transcriptionally active (H3K9me3) histone marks. Influenza A virus (IAV) infection, which stimulates an array of cytosolic and TLR-mediated antiviral mechanisms, resulted in differential redistribution between proximal promoter and distal sites and decoupling of PU.1 binding from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. Our findings suggest that SP-R210L-deficient macrophages are poised with an open PU.1-primed chromatin conformation to rapidly respond to inflammatory and metabolic stimuli.
Project description:Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high. Detection of gene expression variance among Frankia HfpCci3 (Cci3) cells grown in ammonium chloride for three days, five days and HfpCci3 cells grown in nitrogen fixing conditions for three days using mRNA-seq
Project description:Puccinia graminis f. sp. tritici is the cause of wheat stem rust. A microarray was designed from genes predicted from the P. graminis f. sp. tritici genome assembly, and gene expression measured for four conditions which include wheat or barley infecting growth stages initiated by urediniospores. mRNA was prepared from fresh urediniospores, uredinospores germinated for 24 hr, wheat seedlings infected with urediniospores for 8 days, and barley seedlings infected with urediniospores for 8 days. The asexual uredinial infection cycle on wheat produces additional urediniospores, which can start new cycles of wheat infection and are readily spread by aerial transport. This expression data is further described in Duplessis et al, Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici A total of 12 samples were analyzed, including three biological replicates of the four conditions.