Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling of control of P. aeruginosa PAO1 (RNA-seq) to transcriptome profiling of DAS-treated and DAE-treated P. aeruginosa PAO1 and to evaluate protocols for optimal high-throughput data analysis. Results: The transcriptome sequencing data of control, DAS-treated and DAE-treated groups were compared and analyzed in the form of the following: DAE vs DAS, control vs DAS, and control vs DAE. The details of the differentially expressed genes among the three groups showed that the amount of differential expressed genes between DAE-treated group and DAS-treated group was high (total 2195, up-regulated 1608, down-regulated 587). There were many differentially expressed genes in the PAO1 strain after DAS treatment (total 2771, up-regulated 1905, down-regulated 866), while the amount of differentially expressed genes after DAE treatment was relatively small (total 770, up-regulated 349, down-regulated 421).
Project description:Graphene has been selected as a candidate for synthetic feeder-free culture substrate guiding human/mouse multipotent stem cell lineage specification, and culturing pluripotent stem cells in a number of studies. However, conventional graphene is not an ideal biomaterial to maintain the pluripotency of human pluripotent stem cells (hPSC) including hESCs/hiPSCs due to its intrinsic hydrophobicity and relatively flat surface topography. Here, we applied morphology-controlled nanocrystalline graphene (NG) coating onto the culture substrates via diffusion-associated synthesis (DAS) process and cultivated hPSCs. It is found that enhanced hydrophilicity and controlled surface roughness of DAS-NG enabled tight focal adhesion of hPSCs onto the DAS-NG coated culture substrate and retained pluripotency for over 2 weeks. It is also found hPSCs grown on DAS-NG shared comparable global gene expression profile with hPSCs grown on mouse embryonic fibroblast (MEF). Importantly, the similarities in cell adhesion gene expression between hPSCs grown on DAS-NG and hPSCs on MEF suggest DAS-NG may provide comparable physical cues with MEF for sustaining pluripotency. Taken together, our findings show a new reliable method for culturing hPSCs in feeder-free condition using DAS-graphene. Human iPSCs and human ESC H9 were seeded onto DAS-NG coated glass, ITO or QU, CVD-grown graphene coated glass, uncoated glass and mitomycin-C treated CF1. Human pluripotent stem cells seeded on each culture substrate were cultured in human embryonic stem cell medium composed of knockout DMEM (GIBCO-10829) supplemented with 20% knockout serum replacement (GIBCO-10828), 1mM L-glutamine, 1% penicillin/streptomycin (PAA-P11-010), 1% MEM-non essential amino acid (PAA-M11-003), 0.1mM beta-mercaptoethanol, and 5ng/ml human basic fibroblast growth factor.
Project description:Aristolochic acid nephropathy (AAN) is characterised by rapidly progressive tubulointerstitial nephritis culminating in end stage renal failure and urothelial malignancy. microRNAs (miRs) are small endogenous post-transcriptional regulators of gene expression implicated in numerous physiological and pathological processes. We aimed to characterise the mechanism of AA induced cell cycle arrest and its regulation by miRs. The microarray experiment was performed to identify differentially regulated microRNAs in human proximal tubulal epithelial cells treated with aristolochic acid (AA).
Project description:Leaf senescence is an essential developmental process that involves altered regulation of thousands of genes and changes in many metabolic and signaling pathways resulting in massive physiological and structural changes in the leaf. The regulation of senescence is complex and although several senescence regulatory genes have been identified and characterized there is little information on how these individual regulators function globally in the control of the process. In this paper we use microarray analysis to obtain a high-resolution time course profile of gene expression during development of a single leaf over a three week period from just before full expansion to senescence. The multiple time points enable the use of highly informative clustering tools to reveal distinct time points at which signaling and metabolic pathways change during senescence. Analysis of motif enrichment in co-regulated gene clusters identifies clear groups of transcription factors active at different stages of leaf development and senescence. A novel experimental design strategy (A Mead et al, in preparation), based on the principle of the “loop design”, was developed to enable efficient extraction of information about key sample comparisons using a two-colour hybridisation experimental system. With 88 distinct samples (four biological replicates at each of 22 time points) to be compared, the experimental design included 176 two-colour microarray slides, allowing four technical replicates of each sample to be observed. Half of the slides were devoted to assessment of changes in gene expression between time points, using a simple loop design to link 11 samples from either the 7h time points or the 14h time points across the 11 sampling days, directly comparing samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, 27 DAS with 29 DAS, etc.), and directly comparing the samples collected at 39 DAS with those collected at 19 DAS. Four separate loops were constructed for the 7h time points and for the 14h time points, using the arbitrary biological replicate labelling to identify the samples to be included in each loop. The remaining slides provided assessment of differences between the 7h and 14h samples and between the arbitrarily labelled biological replicates, with some further assessment of changes between sampling days. All direct comparisons (pairs of samples hybridised together on a slide) were between 7h and 14h samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, etc.), including comparisons between samples collected at 39 DAS and at 19 DAS, and different arbitrarily labelled biological replicates. These 88 comparisons formed a single loop connecting all 88 treatments, therefore ensuring that the design was fully connected (allowing each sample to be compared with every other sample).
Project description:Arabidopsis thaliana shows hybrid vigour (heterosis) in progeny of crosses between Col and C24 accessions (1). Hybrid vigour was evident as early as the mature seeds and in the seedlings 3 days after sowing (DAS). At 3 DAS genes encoding chloroplast-located proteins were significantly overrepresented (187) among the 724 genes which have greater than mid parent values of expression in the hybrid. Many of these genes are involved in chlorophyll biosynthesis and photosynthesis. The rate of photosynthesis was constant per unit leaf area in parents and hybrids. Larger cell sizes in the hybrids were associated with more chloroplasts per cell, more total chlorophyll and more photosynthesis. The increased transcription of the chloroplast-targeted genes was restricted to the 3 to 7 DAS period. At 10 DAS only 118 genes had expression levels different from the expected mid parent value in the hybrid and only 12 of these genes were differentially expressed at 3 DAS. The early increase in activity of genes involved in photosynthesis and the associated phenomena of increase in cell size and number through development, leading to larger leaf areas of all leaves in the hybrid, suggest a central role for increased photosynthesis in the production of the heterotic biomass. In support of this correlation we found that an inhibitor of photosynthesis eliminated heterosis and higher light intensities enhanced both photosynthesis and heterosis. In hybrids with low level heterosis (Ler x Col) chloroplast-targeted genes were not upregulated and leaf areas were only marginally increased.
Project description:We obtained early-senescence (ES)- and late-senescence (LS)-type F7 plants from a cross between a hybrid (Glycine max × Glycine soja) and the Glycine max cultivar. The ES-type plants presented the reproductive (R2) growth stage at 50 days after sowing (DAS) and the R7 growth stage at 95 DAS, whereas the LS-type plants presented the beginning of the R1 and R6 growth stages at 50 and 95 DAS, respectively. To understand the molecular mechanisms underlying this senescence, we performed transcriptome analysis of leaves from 50 and 95 DAS of ES- and LS-type plants. A total of 2,414 and 2,471 genes at 50 and 95 DAS, respectively, were differentially expressed between ES-type and LS-type plants. Twenty-three candidate genes associated with the circadian clock, chlorophyll biosynthesis, phytohormones, and protein kinases were identified, and their expression levels were analyzed. In addition, we analyzed the expression patterns of circadian clock-related genes such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), CONSTANS-LIKE 9 (COL9), EARLY FLOWERING 3 (ELF3) and pseudo response regulator 5 (PRR5) in ES- and LS-type plants under light and dark conditions. The expression patterns of circadian clock-related genes were similar in the ES- and LS-type plants. However, the transcription levels of these genes were compared between ES- and LS-type plants, and the expression of these genes was greater than that in LS-type plants during the period when expression increased. Therefore, each set of candidate genes regulated senescence in each plant by regulating their expression level.