Project description:Transcriptome sequencing of mice treated with Chebulae Fructus hydrosoluble extract ethyl acetate fraction and water were performed, and the gene expression profiles were compared.
Project description:Inflammatory Bowel Disease (IBD) is a term describing a collection of conditions characterised by chronic inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. Four kiwifruit extracts, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa), have previously demonstrated anti-inflammatory activity using in vitro models of IBD. This study examined whether these kiwifruit extracts had immune modulating effects in vivo against inflammatory processes known to be increased in patients with IBD. KFEs were used as a dietary intervention in Il10-/- mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 x 2 factorial design. While all Il10-/- mice developed significant colonic inflammation compared to the C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. Whole genome gene and protein expression level profiling indicated that KFEs influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, adaptive immune pathways were reduced by three out of four kiwifruit extracts, with greater reduction seen in the C57BL/6J mice. This suggests that while immune-modulating activity was present in vivo, KFEs did not reduce inflammatory processes relevant to IBD. Experimental design. Two experiments were conducted, one using extracts from gold kiwifruit and one using extracts from green kiwifruit. Within each experiment, both Il10-/- and C57 mice were randomly divided into three diet treatment groups, to ive the following 12 treatments: 1) Gold Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 2) Gold Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 3) Gold Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 4) Gold Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 5) Gold Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 6) Gold Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 7) Green Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 8) Green Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 9) Green Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 10) Green Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 11) Green Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 12) Green Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) Six biological replicates were analysed from each treatment group, except for groups 3 and 7 where five replicates were analysed due to array quality issues with the sixth replicate. Each replicate contained mRNA from one mouse. A reference design was used, where each slide was hybridised with mRNA from one sample (green channel) and mRNA from a common reference pool (red channel).
Project description:The PKS14-knockout strain was inoculated into PDB for 7 days. The fungal cells were extracted with methanol, and the culture broth was extracted with ethyl acetate.
Project description:Metabolomic study of B. cenocepacia J2315 (produces pyomelanin) and K56-2 (lacks pyomelanin) WT strains and mutant strains in which the pyomelanin production phenotype are reversed were cultured in synthetic CF sputum media (SCFM2). Metabolites were extracted from cultures via ethyl acetate and C18 SPE extractions, and extracts were pooled before acquiring UPLC/HRMS/MS data to evaluate pyomelanin-specifc metabolism.
Project description:The apratoxin gene cluster was recovered from fosmid DNA library. The gene set responsible for the biosynthesis of the polyketide backbone was introduced in E. coli BAP strain and expressed at 30C for 24 hours. Two sets of control and experimental samples with culture broth pH adjusted at 8 and 12 respectively was extracted with ethyl acetate and dried.
Project description:The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, the single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for a 10 ng, 250ng and 10µg peptide sample, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the robust approach even for simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
Project description:Investigation of whole genome gene expression level changes in Aspergillus nidulans AN1599 (PbcR) overexpression mutant, compared to the FGSC A4 wild-type strain. Overexpression of the Zn(II)2Cys6 M-bM-^@M-^Stype transcription factor, AN1599.4 (PbcR, pimaradiene biosynthetic cluster regulator), activates a secondary metabolite gene cluster in Aspergillus nidulans. Activation of the pathway in Aspergillus nidulans lead to a production of ent-pimara-8(14),15-diene. 12x135K array of two separate cultures of FGSC A4 and two separate cultures of oe:AN1599(PbcR) with three separate RNA extractions from each culture. Each 135K array measures expression level of 10,546 genes with 6 probes/transcript. In addition, the array format contains tiling probes for 36 longer transcripts. All probes are in duplicates, giving the total number of 137,562 probes per array.
Project description:1. Summary: SfrAB, the soluble ferric reductase of Geobacter sulfurreducens, is a two subunit complex that can catalyze the NADPH-dependent reduction of chelated Fe(III) and is encoded in a two gene cluster in which the sfrB gene (GSU_0510) is 126 bp upstream of the sfrA gene (GSU_0509)[Kaufmann and Lovley (2001) J. Bacteriol. 183:4468-4476]. In order to gain insight into the physiological function of SfrAB, a knockout mutant was constructed by replacing a 3.6 kb stretch of sequence encompassing the C-terminal 72% of the SfrB and the N-terminal 80% of the SfrA coding sequences with a kanamycin resistance cassette via homologous recombination. The SfrAB knockout mutant (Delta sfrAB::kan) was found to be initially unable to grow in media in which acetate served as the sole electron donor. However, following prolonged incubation (2-3 weeks) in acetate:fumarate medium, an acetate-adapted SfrAB-null strain with the ability to grow on fumarate with acetate serving as the sole electron donor was isolated. This acetate-adapted, SfrAB-null strain and wild type G. sulfurreducens were cultured in parallel in chemostats at an intermediate growth rate (0.05 hr-1) in acetate-limited freshwater fumarate medium (5 mM acetate:27.5 mM fumarate). After the chemostats reached steady state, cells were harvested by centrifugation and RNA was extracted. In order to identify genes that were involved in growth on acetate in the absence of SfrAB, this RNA was used to perform microarray analysis comparing gene expression in the acetate-adapted, SfrAB-null and wild type strains. 2. Growth condtions: Cells were cultured in chemostats at a dilution rate of 0.05 hr-1 in acetate-limited freshwater fumarate medium (5 mM acetate:27.5 mM fumarate) and harvested when the chemostats were at steady state. 3. Mutant designation: acetate-adapted SfrAB-null strain, genotype= Delta sfrAB::kan. (Note: there is an unadapted Delta sfrAB::kan strain which cannot grow on acetate and an adapted one which can. The acetate-adapted strain was used for the microarray analysis).