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1.  Transcriptional analysis of South African cassava mosaic virus-infected susceptible and tolerant landraces of cassava highlights differences in resistance, basal defense and cell wall associated genes during infection 
BMC Genomics  2014;15(1):1006.
Background
Cassava mosaic disease is caused by several distinct geminivirus species, including South African cassava mosaic virus-[South Africa:99] (SACMV). To date, there is limited gene regulation information on viral stress responses in cassava, and global transcriptome profiling in SACMV-infected cassava represents an important step towards understanding natural host responses to plant geminiviruses.
Results
A RNA-seq time course (12, 32 and 67 dpi) study, monitoring gene expression in SACMV-challenged susceptible (T200) and tolerant (TME3) cassava landraces, was performed using the Applied Biosystems (ABI) SOLiD next-generation sequencing platform. The multiplexed paired end sequencing run produced a total of 523 MB and 693 MB of paired-end reads for SACMV-infected susceptible and tolerant cDNA libraries, respectively. Of these, approximately 50.7% of the T200 reads and 55.06% of TME3 reads mapped to the cassava reference genome available in phytozome. Using a log2 fold cut-off (p <0.05), comparative analysis between the six normalized cDNA libraries showed that 4181 and 1008 transcripts in total were differentially expressed in T200 and TME3, respectively, across 12, 32 and 67 days post infection, compared to mock-inoculated. The number of responsive transcripts increased dramatically from 12 to 32 dpi in both cultivars, but in contrast, in T200 the levels did not change significantly at 67 dpi, while in TME3 they declined. GOslim functional groups illustrated that differentially expressed genes in T200 and TME3 were overrepresented in the cellular component category for stress-related genes, plasma membrane and nucleus. Alterations in the expression of other interesting genes such as transcription factors, resistance (R) genes, and histone/DNA methylation-associated genes, were observed. KEGG pathway analysis uncovered important altered metabolic pathways, including phenylpropanoid biosynthesis, sucrose and starch metabolism, and plant hormone signalling.
Conclusions
Molecular mechanisms for TME3 tolerance are proposed, and differences in patterns and levels of transcriptome profiling between T200 and TME3 with susceptible and tolerant phenotypes, respectively, support the hypothesis that viruses rearrange their molecular interactions in adapting to hosts with different genetic backgrounds.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1006) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1006
PMCID: PMC4253015  PMID: 25412561
Transcriptome profiling; Cassava; Next-generation sequencing; Geminivirus; South African cassava mosaic virus; Tolerance; Susceptibility
2.  A comprehensive look at transcription factor gene expression changes in colorectal adenomas 
BMC Cancer  2014;14:46.
Background
Biological processes are controlled by transcription networks. Expression changes of transcription factor (TF) genes in precancerous lesions are therefore crucial events in tumorigenesis. Our aim was to obtain a comprehensive picture of these changes in colorectal adenomas.
Methods
Using a 3-pronged selection procedure, we analyzed transcriptomic data on 34 human tissue samples (17 adenomas and paired samples of normal mucosa, all collected with ethics committee approval and written, informed patient consent) to identify TFs with highly significant tumor-associated gene expression changes whose potential roles in colorectal tumorigenesis have been under-researched. Microarray data were subjected to stringent statistical analysis of TF expression in tumor vs. normal tissues, MetaCore-mediated identification of TF networks displaying enrichment for genes that were differentially expressed in tumors, and a novel quantitative analysis of the publications examining the TF genes’ roles in colorectal tumorigenesis.
Results
The 261 TF genes identified with this procedure included DACH1, which plays essential roles in the proper proliferation and differentiation of retinal and leg precursor cell populations in Drosophila melanogaster. Its possible roles in colorectal tumorigenesis are completely unknown, but it was found to be markedly overexpressed (mRNA and protein) in all colorectal adenomas and in most colorectal carcinomas. However, DACH1 expression was absent in some carcinomas, most of which were DNA mismatch-repair deficient. When networks were built using the set of TF genes identified by all three selection procedures, as well as the entire set of transcriptomic changes in adenomas, five hub genes (TGFB1, BIRC5, MYB, NR3C1, and TERT) where identified as putatively crucial components of the adenomatous transformation process.
Conclusion
The transcription-regulating network of colorectal adenomas (compared with that of normal colorectal mucosa) is characterized by significantly altered expression of over 250 TF genes, many of which have never been investigated in relation to colorectal tumorigenesis.
doi:10.1186/1471-2407-14-46
PMCID: PMC4078005  PMID: 24472434
Transcription factors; Gene expression; Colorectal adenomas; DACH1
3.  Early Insights into the Function of KIAA1199, a Markedly Overexpressed Protein in Human Colorectal Tumors 
PLoS ONE  2013;8(7):e69473.
We previously reported that the expression of KIAA1199 in human colorectal tumors (benign and malignant) is markedly higher than that in the normal colonic mucosa. In this study, we investigated the functions of the protein encoded by this gene, which are thus far unknown. Immunostaining studies were used to reveal its subcellular localization, and proteomic and gene expression experiments were conducted to identify proteins that might interact with KIAA1199 and molecular pathways in which it might play roles. Using colon cancer cell lines, we showed that both endogenous and ectopically expressed KIAA1199 is secreted into the extracellular environment. In the cells, it was found mainly in the perinuclear space (probably the ER) and cell membrane. Both cellular compartments were also over-represented in lists of proteins identified by mass spectrometry as putative KIAA1199 interactors and/or proteins encoded by genes whose transcription was significantly changed by KIAA1199 expression. These proteomic and transcriptomic datasets concordantly link KIAA1199 to several genes/proteins and molecular pathways, including ER processes like protein binding, transport, and folding; and Ca2+, G-protein, ephrin, and Wnt signaling. Immunoprecipitation experiments confirmed KIAA1199’s interaction with the cell-membrane receptor ephrin A2 and with the ER receptor ITPR3, a key player in Ca2+ signaling. By modulating Ca2+ signaling, KIAA1199 could affect different branches of the Wnt network. Our findings suggest it may negatively regulate the Wnt/CTNNB1 signaling, and its expression is associated with decreased cell proliferation and invasiveness.
doi:10.1371/journal.pone.0069473
PMCID: PMC3720655  PMID: 23936024
4.  Cross-Species Genome Wide Expression Analysis during Pluripotent Cell Determination in Mouse and Rat Preimplantation Embryos 
PLoS ONE  2012;7(10):e47107.
The transition between morula and blastocyst stage during preimplantation development represents the first differentiation event of embryogenesis. Morula cells undergo the first cellular specialization and produce two well-defined populations of cells, the trophoblast and the inner cell mass (ICM). Embryonic stem cells (ESCs) with unlimited self-renewal capacity are believed to represent the in vitro counterpart of the ICM. Both mouse and rat ESCs can be derived from the ICM cells, but their in vitro stability differs. In this study we performed a microarray analysis in which we compared the transcriptome of mouse and rat morula, blastocyst, and ICM. This cross-species comparison represents a good model for understanding the differences in derivation and cultivation of ESCs observed in the two species. In order to identify alternative regulation of important molecular mechanisms the investigation of differential gene expression between the two species was extended at the level of signaling pathways, gene families, and single selected genes of interest. Some of the genes differentially expressed between the two species are already known to be important factors in the maintenance of pluripotency in ESCs, like for example Sox2 or Stat3, or play a role in reprogramming somatic cells to pluripotency like c-Myc, Klf4 and p53 and therefore represent interesting candidates to further analyze in vitro in the rat ESCs. This is the first study investigating the gene expression changes during the transition from morula to blastocyst in the rat preimplantation development. Our data show that in the pluripotent pool of cells of the rat and mouse preimplantation embryo substantial differential regulation of genes is present, which might explain the difficulties observed for the derivation and culture of rat ESCs using mouse conditions.
doi:10.1371/journal.pone.0047107
PMCID: PMC3471948  PMID: 23077551
5.  PGC-1α Determines Light Damage Susceptibility of the Murine Retina 
PLoS ONE  2012;7(2):e31272.
The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases.
doi:10.1371/journal.pone.0031272
PMCID: PMC3278422  PMID: 22348062
6.  The removal of multiplicative, systematic bias allows integration of breast cancer gene expression datasets – improving meta-analysis and prediction of prognosis 
BMC Medical Genomics  2008;1:42.
Background
The number of gene expression studies in the public domain is rapidly increasing, representing a highly valuable resource. However, dataset-specific bias precludes meta-analysis at the raw transcript level, even when the RNA is from comparable sources and has been processed on the same microarray platform using similar protocols. Here, we demonstrate, using Affymetrix data, that much of this bias can be removed, allowing multiple datasets to be legitimately combined for meaningful meta-analyses.
Results
A series of validation datasets comparing breast cancer and normal breast cell lines (MCF7 and MCF10A) were generated to examine the variability between datasets generated using different amounts of starting RNA, alternative protocols, different generations of Affymetrix GeneChip or scanning hardware. We demonstrate that systematic, multiplicative biases are introduced at the RNA, hybridization and image-capture stages of a microarray experiment. Simple batch mean-centering was found to significantly reduce the level of inter-experimental variation, allowing raw transcript levels to be compared across datasets with confidence. By accounting for dataset-specific bias, we were able to assemble the largest gene expression dataset of primary breast tumours to-date (1107), from six previously published studies. Using this meta-dataset, we demonstrate that combining greater numbers of datasets or tumours leads to a greater overlap in differentially expressed genes and more accurate prognostic predictions. However, this is highly dependent upon the composition of the datasets and patient characteristics.
Conclusion
Multiplicative, systematic biases are introduced at many stages of microarray experiments. When these are reconciled, raw data can be directly integrated from different gene expression datasets leading to new biological findings with increased statistical power.
doi:10.1186/1755-8794-1-42
PMCID: PMC2563019  PMID: 18803878

Results 1-6 (6)