Transcription profiling of mouse mRNA to test stoichiometric interaction between cDNA sample sequence and array feature by using the Genisphere branched DNA dendrimer labeling system for the Cy5 channel and a reference sequence to measure the amount of array feature in the Cy3 channe
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ABSTRACT: This experiment was designed in order to measure the stoichiometric interaction between cDNA sample sequence and array feature by using the Genisphere branched DNA dendrimer labeling system for the Cy5 channel and a reference sequence to measure the amount of array feature in the Cy3 channel.
Project description:Analysis of gingival crevicular fluid (GCF) samples may give information of the identity of unattached (planktonic) subgingival bacteria, the 35 forefront candidates for systemic dispersal via ulcerated periodontal pocket epithelium. Our study represents the first one targeting the identity of bacteria in gingival crevicular fluid. Methodology/Principal findings: We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla 45 were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, 46 per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance: Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms. The microbial profiles of GCF and subgingival plaque were analyzed from 17 subjects with periodontal disease.
Project description:Identification of new cancer-associated genes/proteins, characterization of their expression vari-ation, the interactomics-based assessment of differentially expressed genes/proteins (DEGs/DEPs), and understanding the tumorigenic pathways and biological processes involved in BC genesis and progression are necessary and possible by rapid and recent advances in bioin-formatics and molecular profiling strategies. Taking into account the opinion of other authors, as well as based on our own team’s in vitro studies, we sustain that JTB protein might be consid-ered as a tumor biomarker for BC and should be studied as a target for BC therapy. In this study we have identified the differentially expressed proteins (DEPs), carcinogenic pathways and bio-logical processes associated with JTB silencing, using 2D-PAGE coupled with nano-liquid chro-matography tandem mass spectrometry (nLC-MS/MS) proteomics applied to MCF7 breast cancer cell line, for complementing and completing our previous results based on SDS-PAGE, as well as in-solution proteomics of MCF7 cells transfected for JTB downregulation. The functions of significant DEPs have been analysed using GSEA and KEGG analysis. Almost all DEPs exert protumorigenic effects in JTBlow condition, sustaining the tumor suppressive function of JTB. Thus, the identified DEPs are involved in several signaling and metabolic pathways that exert protumorigenic roles: EMT, ERK/MAPK, PI3K/AKT, Wnt/β-catenin, mTOR, C-MYC, NF-κB, IFN-γ and IFN-α response, UPR, and glycolysis/gluconeogenesis. These pathways sustain cancer cell growth, adhesion, survival, proliferation, invasion, metastasis, resistance to apoptosis, cytoskeleton reorganization, maintenance of stemness, metabolic reprogramming, survival into a hostile environment, and a poor clinical outocome. In conclusion, JTB silencing might increase the neoplastic phenotype and behavior of MCF7 BC cell line.
Project description:The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2 or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possible also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for hydrolysis of ATP (CLPC1-TRAP). Based on homology to non-plant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released, i.e. they are trapped. When expressed in wild-type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared to affinity purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates and/or new adaptors. Several of these trapped proteins over-accumulated in clp mutants and/or were found as interactions for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR and CLPT subunits, indicating stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.
Project description:The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2 or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possible also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for hydrolysis of ATP (CLPC1-TRAP). Based on homology to non-plant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released, i.e. they are trapped. When expressed in wild-type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared to affinity purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates and/or new adaptors. Several of these trapped proteins over-accumulated in clp mutants and/or were found as interactions for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR and CLPT subunits, indicating stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.
Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. To understand this process better, we set out to quantify the distribution of cellular proteins between protrusions and cell-body by proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with SILAC proteomics. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from heavy and light SILAC labelled cells then allows for reciprocal mixing and quantification of proteins between protrusions and cell-body. In this study, we determined the relative distribution of 3240 proteins between protrusions and cell-body from two SILAC mixes. Associated ArrayExpress data: E-MTAB-2546.
Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. Apart from proteins, several mRNAs are known to be localised to cell protrusions but to what extent RNA localisation and local translation contributes toward front-back assymetry is unknown. We set out to quantify the relative translation rates of cellular proteins between protrusions and cell-body by Pulsed-SILAC proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with pulsed-SILAC. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from SILAC heavy vs. medium label pulsed cells then allows for reciprocal mixing and quantification of nascent proteins between protrusions and cell-body. We determined the relative local translation rates of 1150 proteins between protrusions and cell-body from two pulsed-SILAC mixes.
Project description:The 3' ends of most Drosophila melanogaster genes are poorly annotated or are determined by only a single EST or cDNA clone. To enhance the annotation of poly(A) site use in Drosophila, we performed deep sequencing on RNA isolated from 29 dissected tissues using an approach designed to enrich for poly(A) spanning reads. From these experiments, we identified 1.4 million poly(A) spanning reads leading to the identification of many new poly(A) sites and the identification of many tissue-specific poly(A) sites. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf RNA from 29 dissected Drosophila melanogaster tissues (in duplicate) were used to prepare polyA enriched RNA-Seq libraries. Briefly, total RNA was poly(A) selected, fragmented, and ligated to 5' and 3' RNA linkers. These libraries were amplified using Illumina paired-end primers, and subsequently reamplified using a 3' primer complementary to the 3' adapter but containing 6 Ts at the 3' end. The libraries were also multiplexed and up to 12 samples mixed per lane and sequenced on an Illumina GAIIx using paired-end 76 bp reads, or an illumina HiSeq 2000 using paired-end 100 bp reads. All reads were mapped to the Drosophila melanogaster genome to identify unmapped reads. Unmapped reads containing at least 10 A residues at the 3' end were identified, the terminal A residues trimmed, realigned to the genome to identify uniquely mapped reads. Such reads were identified as polyA spanning reads
Project description:Molecular mechanisms behind the green insect camouflage have puzzled researchers for ages. Here, we isolated water-soluble green protein from the integument of bush-cricket Tettigonia cantans. De novo sequencing and partial cloning revealed a fragmented form of vitellogenins, ubiquitous glycolipoproteins essential for embryonic development.
Project description:We have demonstrated that a newly evolved TRS within the Nucleocapsid gene of SARS-CoV-2 (termed N.iORF3) leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production. Using reverse genetics-derived viruses we show N.iORF3 contributes to viral fitness during infection and observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Competition assays were performed to determine the relative fitness of different virus mutants and amplicons were analysed to quantify the proportions of different viruses in tissue culture.