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1.  Liver transcriptome analysis in gilthead sea bream upon exposure to low temperature 
BMC Genomics  2014;15(1):765.
Background
Water temperature greatly influences the physiology and behaviour of teleost fish as other aquatic organisms. While fish are able to cope with seasonal temperature variations, thermal excursions outside their normal thermal range might exceed their ability to respond leading to severe diseases and death.
Profound differences exist in thermal tolerance across fish species living in the same geographical areas, promoting for investigating the molecular mechanisms involved in susceptibility and resistance to low and high temperatures toward a better understanding of adaptation to environmental challenges. The gilthead sea bream, Sparus aurata, is particularly sensitive to cold and the prolonged exposure to low temperatures may lead to the "winter disease", a metabolic disorder that significantly affects the aquaculture productions along the Northern Mediterranean coasts during winter-spring season. While sea bream susceptibility to low temperatures has been extensively investigated, the cascade of molecular events under such stressful condition is not fully elucidated.
Results
In the present study two groups of wild sea bream were exposed for 21 days to two temperature regimes: 16 ± 0.3°C (control group) and 6.8 ± 0.3°C (cold-exposed group) and DNA microarray analysis of liver transcriptome was carried out at different time points during cold exposure.
A large set of genes was found to be differentially expressed upon cold-exposure with increasingly relevant effects being observed after three weeks at low temperature. All major known responses to cold (i.e. anti-oxidant response, increased mitochondrial function, membrane compositional changes) were found to be conserved in the gilthead sea bream, while, evidence for a key role of unfolded protein response (UPR) to endoplasmic reticulum (ER) stress, during short- and long-term exposure to cold is reported here for the first time.
Conclusions
Transcriptome data suggest a scenario where oxidative stress, altered lipid metabolism, ATP depletion and protein denaturation converge to induce ER stress. The resulting UPR activation further promotes conditions for cell damage, and the inability to resolve ER stress leads to severe liver dysfunction and potentially to death.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-765) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-765
PMCID: PMC4167152  PMID: 25194679
Gilthead sea bream; Winter syndrome; Cold stress; Microarray; Gene expression; Liver
2.  Analysis of Differential miRNA Expression in Primary Tumor and Stroma of Colorectal Cancer Patients 
BioMed Research International  2014;2014:840921.
Microarray technology was used to profile miRNA expression in primary tumor and stromal tissue from paraffin embedded material of 51 patients with colorectal cancer. 26 miRNAs resulted differentially expressed with at least 2-fold change in tumor tissue with respect to stroma (16 more expressed in the tumor and 10 more expressed in the stroma). 10/26 were confirmed as differentially expressed at qRTPCR: miR-200c-3p, miR-141-3p, miR-200b-3p, miR-200a-3p, miR-1246, miR-92a-3p, miR-194-5p, miR-192-5p, miR-3651-5p, and miR-574-3p. No significant association was found between miRNA expressions and stage at diagnosis, site of primary tumor, first site of metastasis, progression-free, or overall survival.
doi:10.1155/2014/840921
PMCID: PMC4128171  PMID: 25143946
3.  Wiring miRNAs to pathways: a topological approach to integrate miRNA and mRNA expression profiles 
Nucleic Acids Research  2014;42(11):e96.
The production rate of gene expression data is nothing less than astounding. However, with the benefit of hindsight we can assert that, since we completely ignored the non-coding part of the transcriptome, we spent the last decade to study cell mechanisms having few data in our hands. In this scenario, microRNAs, which are key post-trascriptional regulators, deserve special attention. Given the state of knowledge about their biogenesis, mechanisms of action and the numerous experimentally validated target genes, miRNAs are also gradually appearing in the formal pathway representations such as KEGG and Reactome maps. However, the number of miRNAs annotated in pathway maps are very few and pathway analyses exploiting this new regulatory layer are still lacking. To fill these gaps, we present ‘micrographite’ a new pipeline to perform topological pathway analysis integrating gene and miRNA expression profiles. Here, micrographite is used to study and dissect the epithelial ovarian cancer gene and miRNA transcriptome defining and validating a new regulatory circuit related to ovarian cancer histotype specificity.
doi:10.1093/nar/gku354
PMCID: PMC4066781  PMID: 24803669
4.  timeClip: pathway analysis for time course data without replicates 
BMC Bioinformatics  2014;15(Suppl 5):S3.
Background
Time-course gene expression experiments are useful tools for exploring biological processes. In this type of experiments, gene expression changes are monitored along time. Unfortunately, replication of time series is still costly and usually long time course do not have replicates. Many approaches have been proposed to deal with this data structure, but none of them in the field of pathway analysis. Pathway analyses have acquired great relevance for helping the interpretation of gene expression data. Several methods have been proposed to this aim: from the classical enrichment to the more complex topological analysis that gains power from the topology of the pathway. None of them were devised to identify temporal variations in time course data.
Results
Here we present timeClip, a topology based pathway analysis specifically tailored to long time series without replicates. timeClip combines dimension reduction techniques and graph decomposition theory to explore and identify the portion of pathways that is most time-dependent. In the first step, timeClip selects the time-dependent pathways; in the second step, the most time dependent portions of these pathways are highlighted. We used timeClip on simulated data and on a benchmark dataset regarding mouse muscle regeneration model. Our approach shows good performance on different simulated settings. On the real dataset, we identify 76 time-dependent pathways, most of which known to be involved in the regeneration process. Focusing on the 'mTOR signaling pathway' we highlight the timing of key processes of the muscle regeneration: from the early pathway activation through growth factor signals to the late burst of protein production needed for the fiber regeneration.
Conclusions
timeClip represents a new improvement in the field of time-dependent pathway analysis. It allows to isolate and dissect pathways characterized by time-dependent components. Furthermore, using timeClip on a mouse muscle regeneration dataset we were able to characterize the process of muscle fiber regeneration with its correct timing.
doi:10.1186/1471-2105-15-S5-S3
PMCID: PMC4095003  PMID: 25077979
5.  Tissue-Specific Expression and Regulatory Networks of Pig MicroRNAome 
PLoS ONE  2014;9(4):e89755.
Background
Despite the economic and medical importance of the pig, knowledge about its genome organization, gene expression regulation, and molecular mechanisms involved in physiological processes is far from that achieved for mouse and rat, the two most used model organisms in biomedical research. MicroRNAs (miRNAs) are a wide class of molecules that exert a recognized role in gene expression modulation, but only 280 miRNAs in pig have been characterized to date.
Results
We applied a novel computational approach to predict species-specific and conserved miRNAs in the pig genome, which were then subjected to experimental validation. We experimentally identified candidate miRNAs sequences grouped in high-confidence (424) and medium-confidence (353) miRNAs according to RNA-seq results. A group of miRNAs was also validated by PCR experiments. We established the subtle variability in expression of isomiRs and miRNA-miRNA star couples supporting a biological function for these molecules. Finally, miRNA and mRNA expression profiles produced from the same sample of 20 different tissue of the animal were combined, using a correlation threshold to filter miRNA-target predictions, to identify tissue-specific regulatory networks.
Conclusions
Our data represent a significant progress in the current understanding of miRNAome in pig. The identification of miRNAs, their target mRNAs, and the construction of regulatory circuits will provide new insights into the complex biological networks in several tissues of this important animal model.
doi:10.1371/journal.pone.0089755
PMCID: PMC3974652  PMID: 24699212
6.  Systems Biology Approach to the Dissection of the Complexity of Regulatory Networks in the S. scrofa Cardiocirculatory System 
Genome-wide experiments are routinely used to increase the understanding of the biological processes involved in the development and maintenance of a variety of pathologies. Although the technical feasibility of this type of experiment has improved in recent years, data analysis remains challenging. In this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. Here, we review strategies used in the gene set approach, and using datasets for the pig cardiocirculatory system as a case study, we demonstrate how the use of a combination of these strategies can enhance the interpretation of results. Gene set analyses are able to distinguish vessels from the heart and arteries from veins in a manner that is consistent with the different cellular composition of smooth muscle cells. By integrating microRNA elements in the regulatory circuits identified, we find that vessel specificity is maintained through specific miRNAs, such as miR-133a and miR-143, which show anti-correlated expression with their mRNA targets.
doi:10.3390/ijms141123160
PMCID: PMC3856112  PMID: 24284405
pathway analysis; miRNA; cardiocirculatory; network reconstruction; integrative analysis; pig; artery; vein; vessel
7.  Revertant Fibers in the mdx Murine Model of Duchenne Muscular Dystrophy: An Age- and Muscle-Related Reappraisal 
PLoS ONE  2013;8(8):e72147.
Muscles in Duchenne dystrophy patients are characterized by the absence of dystrophin, yet transverse sections show a small percentage of fibers (termed “revertant fibers”) positive for dystrophin expression. This phenomenon, whose biological bases have not been fully elucidated, is present also in the murine and canine models of DMD and can confound the evaluation of therapeutic approaches. We analyzed 11 different muscles in a cohort of 40 mdx mice, the most commonly model used in pre-clinical studies, belonging to four age groups; such number of animals allowed us to perform solid ANOVA statistical analysis. We assessed the average number of dystrophin-positive fibers, both absolute and normalized for muscle size, and the correlation between their formation and the ageing process. Our results indicate that various muscles develop different numbers of revertant fibers, with different time trends; besides, they suggest that the biological mechanism(s) behind dystrophin re-expression might not be limited to the early development phases but could actually continue during adulthood. Importantly, such finding was seen also in cardiac muscle, a fact that does not fit into the current hypothesis of the clonal origin of “revertant” myonuclei from satellite cells. This work represents the largest, statistically significant analysis of revertant fibers in mdx mice so far, which can now be used as a reference point for improving the evaluation of therapeutic approaches for DMD. At the same time, it provides new clues about the formation of revertant fibers/cardiomyocytes in dystrophic skeletal and cardiac muscle.
doi:10.1371/journal.pone.0072147
PMCID: PMC3756063  PMID: 24015212
8.  Graphite Web: web tool for gene set analysis exploiting pathway topology 
Nucleic Acids Research  2013;41(Web Server issue):W89-W97.
Graphite web is a novel web tool for pathway analyses and network visualization for gene expression data of both microarray and RNA-seq experiments. Several pathway analyses have been proposed either in the univariate or in the global and multivariate context to tackle the complexity and the interpretation of expression results. These methods can be further divided into ‘topological’ and ‘non-topological’ methods according to their ability to gain power from pathway topology. Biological pathways are, in fact, not only gene lists but can be represented through a network where genes and connections are, respectively, nodes and edges. To this day, the most used approaches are non-topological and univariate although they miss the relationship among genes. On the contrary, topological and multivariate approaches are more powerful, but difficult to be used by researchers without bioinformatic skills. Here we present Graphite web, the first public web server for pathway analysis on gene expression data that combines topological and multivariate pathway analyses with an efficient system of interactive network visualizations for easy results interpretation. Specifically, Graphite web implements five different gene set analyses on three model organisms and two pathway databases. Graphite Web is freely available at http://graphiteweb.bio.unipd.it/.
doi:10.1093/nar/gkt386
PMCID: PMC3977659  PMID: 23666626
9.  Synthesis of Mitochondrial DNA Precursors during Myogenesis, an Analysis in Purified C2C12 Myotubes* 
The Journal of Biological Chemistry  2013;288(8):5624-5635.
Background: In developing muscle, stimulation of mitochondrial biogenesis and mtDNA expansion occur with down-regulation of deoxynucleotide synthesis.
Results: siRNA silencing of mitochondrial thymidine or deoxyguanosine kinase impacts myotube differentiation causing depletion of mtDNA and of all four deoxynucleotides.
Conclusion: Shortage of even a single deoxynucleotide may upset the regulation of all DNA precursors.
Significance: Deoxynucleotide analysis in myotubes unveils unexpected outcomes of synthetic enzyme deficiencies.
During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.
doi:10.1074/jbc.M112.441147
PMCID: PMC3581417  PMID: 23297407
Mitochondrial Diseases; Mitochondrial DNA; Nucleoside Nucleotide Metabolism; Ribonucleotide Reductase; Skeletal Muscle; Deoxyguanosine Kinase; mtDNA Depletion; siRNA Silencing; Thymidine Kinase 2
10.  Along signal paths: an empirical gene set approach exploiting pathway topology 
Nucleic Acids Research  2012;41(1):e19.
Gene set analysis using biological pathways has become a widely used statistical approach for gene expression analysis. A biological pathway can be represented through a graph where genes and their interactions are, respectively, nodes and edges of the graph. From a biological point of view only some portions of a pathway are expected to be altered; however, few methods using pathway topology have been proposed and none of them tries to identify the signal paths, within a pathway, mostly involved in the biological problem. Here, we present a novel algorithm for pathway analysis clipper, that tries to fill in this gap. clipper implements a two-step empirical approach based on the exploitation of graph decomposition into a junction tree to reconstruct the most relevant signal path. In the first step clipper selects significant pathways according to statistical tests on the means and the concentration matrices of the graphs derived from pathway topologies. Then, it identifies within these pathways the signal paths having the greatest association with a specific phenotype. We test our approach on simulated and two real expression datasets. Our results demonstrate the efficacy of clipper in the identification of signal transduction paths totally coherent with the biological problem.
doi:10.1093/nar/gks866
PMCID: PMC3592432  PMID: 23002139
11.  MAGIA2: from miRNA and genes expression data integrative analysis to microRNA–transcription factor mixed regulatory circuits (2012 update) 
Nucleic Acids Research  2012;40(Web Server issue):W13-W21.
MAGIA2 (http://gencomp.bio.unipd.it/magia2) is an update, extension and evolution of the MAGIA web tool. It is dedicated to the integrated analysis of in silico target prediction, microRNA (miRNA) and gene expression data for the reconstruction of post-transcriptional regulatory networks. miRNAs are fundamental post-transcriptional regulators of several key biological and pathological processes. As miRNAs act prevalently through target degradation, their expression profiles are expected to be inversely correlated to those of the target genes. Low specificity of target prediction algorithms makes integration approaches an interesting solution for target prediction refinement. MAGIA2 performs this integrative approach supporting different association measures, multiple organisms and almost all target predictions algorithms. Nevertheless, miRNAs activity should be viewed as part of a more complex scenario where regulatory elements and their interactors generate a highly connected network and where gene expression profiles are the result of different levels of regulation. The updated MAGIA2 tries to dissect this complexity by reconstructing mixed regulatory circuits involving either miRNA or transcription factor (TF) as regulators. Two types of circuits are identified: (i) a TF that regulates both a miRNA and its target and (ii) a miRNA that regulates both a TF and its target.
doi:10.1093/nar/gks460
PMCID: PMC3394337  PMID: 22618880
12.  A Systems Biology Approach to Characterize the Regulatory Networks Leading to Trabectedin Resistance in an In Vitro Model of Myxoid Liposarcoma 
PLoS ONE  2012;7(4):e35423.
Trabectedin, a new antitumor compound originally derived from a marine tunicate, is clinically effective in soft tissue sarcoma. The drug has shown a high selectivity for myxoid liposarcoma, characterized by the translocation t(12;16)(q13; p11) leading to the expression of FUS-CHOP fusion gene. Trabectedin appears to act interfering with mechanisms of transcription regulation. In particular, the transactivating activity of FUS-CHOP was found to be impaired by trabectedin treatment. Even after prolonged response resistance occurs and thus it is important to elucidate the mechanisms of resistance to trabectedin. To this end we developed and characterized a myxoid liposarcoma cell line resistant to trabectedin (402-91/ET), obtained by exposing the parental 402-91 cell line to stepwise increases in drug concentration. The aim of this study was to compare mRNAs, miRNAs and proteins profiles of 402-91 and 402-91/ET cells through a systems biology approach. We identified 3,083 genes, 47 miRNAs and 336 proteins differentially expressed between 402-91 and 402-91/ET cell lines. Interestingly three miRNAs among those differentially expressed, miR-130a, miR-21 and miR-7, harbored CHOP binding sites in their promoter region. We used computational approaches to integrate the three regulatory layers and to generate a molecular map describing the altered circuits in sensitive and resistant cell lines. By combining transcriptomic and proteomic data, we reconstructed two different networks, i.e. apoptosis and cell cycle regulation, that could play a key role in modulating trabectedin resistance. This approach highlights the central role of genes such as CCDN1, RB1, E2F4, TNF, CDKN1C and ABL1 in both pre- and post-transcriptional regulatory network. The validation of these results in in vivo models might be clinically relevant to stratify myxoid liposarcoma patients with different sensitivity to trabectedin treatment.
doi:10.1371/journal.pone.0035423
PMCID: PMC3327679  PMID: 22523595
13.  graphite - a Bioconductor package to convert pathway topology to gene network 
BMC Bioinformatics  2012;13:20.
Background
Gene set analysis is moving towards considering pathway topology as a crucial feature. Pathway elements are complex entities such as protein complexes, gene family members and chemical compounds. The conversion of pathway topology to a gene/protein networks (where nodes are a simple element like a gene/protein) is a critical and challenging task that enables topology-based gene set analyses.
Unfortunately, currently available R/Bioconductor packages provide pathway networks only from single databases. They do not propagate signals through chemical compounds and do not differentiate between complexes and gene families.
Results
Here we present graphite, a Bioconductor package addressing these issues. Pathway information from four different databases is interpreted following specific biologically-driven rules that allow the reconstruction of gene-gene networks taking into account protein complexes, gene families and sensibly removing chemical compounds from the final graphs. The resulting networks represent a uniform resource for pathway analyses. Indeed, graphite provides easy access to three recently proposed topological methods. The graphite package is available as part of the Bioconductor software suite.
Conclusions
graphite is an innovative package able to gather and make easily available the contents of the four major pathway databases. In the field of topological analysis graphite acts as a provider of biological information by reducing the pathway complexity considering the biological meaning of the pathway elements.
doi:10.1186/1471-2105-13-20
PMCID: PMC3296647  PMID: 22292714
14.  Impact of Host Genes and Strand Selection on miRNA and miRNA* Expression 
PLoS ONE  2011;6(8):e23854.
Dysregulation of miRNAs expression plays a critical role in the pathogenesis of genetic, multifactorial disorders and in human cancers. We exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection regulation and expression relationships between intragenic miRNAs and host genes. We considered miRNAs expression profiles, measured in five sizeable microarray datasets, including samples from different normal cell types and tissues, as well as different tumours and disease states. First, the study of expression profiles of “sister” miRNA pairs (miRNA/miRNA*, 5′ and 3′ strands of the same hairpin precursor) showed that the strand selection is highly regulated since it shows tissue-/cell-/condition-specific modulation. We used information about the direction and the strength of the strand selection bias to perform an unsupervised cluster analysis for the sample classification evidencing that is able to distinguish among different tissues, and sometimes between normal and malignant cells. Then, considering a minimum expression threshold, in few miRNA pairs only one mature miRNA is always present in all considered cell types, whereas the majority of pairs were concurrently expressed in some cell types and alternatively in others. In a significant fraction of concurrently expressed pairs, the major and the minor forms found at comparable levels may contribute to post-transcriptional gene silencing, possibly in a coordinate way. In the second part of the study, the behaved tendency to co-expression of intragenic miRNAs and their “host” mRNA genes was confuted by expression profiles examination, suggesting that the expression profile of a given host gene can hardly be a good estimator of co-transcribed miRNA(s) for post-transcriptional regulatory networks inference. Our results point out the regulatory importance of post-transcriptional phases of miRNAs biogenesis, reinforcing the role of such layer of miRNA biogenesis in miRNA-based regulation of cell activities.
doi:10.1371/journal.pone.0023854
PMCID: PMC3166117  PMID: 21909367
15.  Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate 
BMC Genomics  2011;12:310.
Background
Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate.
Results
Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization.
Conclusions
Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation.
doi:10.1186/1471-2164-12-310
PMCID: PMC3141667  PMID: 21668972
16.  The Biological Connection Markup Language: a SBGN-compliant format for visualization, filtering and analysis of biological pathways 
Bioinformatics  2011;27(15):2127-2133.
Motivation: Many models and analysis of signaling pathways have been proposed. However, neither of them takes into account that a biological pathway is not a fixed system, but instead it depends on the organism, tissue and cell type as well as on physiological, pathological and experimental conditions.
Results: The Biological Connection Markup Language (BCML) is a format to describe, annotate and visualize pathways. BCML is able to store multiple information, permitting a selective view of the pathway as it exists and/or behave in specific organisms, tissues and cells. Furthermore, BCML can be automatically converted into data formats suitable for analysis and into a fully SBGN-compliant graphical representation, making it an important tool that can be used by both computational biologists and ‘wet lab’ scientists.
Availability and implementation: The XML schema and the BCML software suite are freely available under the LGPL for download at http://bcml.dc-atlas.net. They are implemented in Java and supported on MS Windows, Linux and OS X.
Contact: duccio.cavalieri@unifi.it; sorin@wayne.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr339
PMCID: PMC3137220  PMID: 21653523
17.  Statistical Test of Expression Pattern (STEPath): a new strategy to integrate gene expression data with genomic information in individual and meta-analysis studies 
BMC Bioinformatics  2011;12:92.
Background
In the last decades, microarray technology has spread, leading to a dramatic increase of publicly available datasets. The first statistical tools developed were focused on the identification of significant differentially expressed genes. Later, researchers moved toward the systematic integration of gene expression profiles with additional biological information, such as chromosomal location, ontological annotations or sequence features. The analysis of gene expression linked to physical location of genes on chromosomes allows the identification of transcriptionally imbalanced regions, while, Gene Set Analysis focuses on the detection of coordinated changes in transcriptional levels among sets of biologically related genes.
In this field, meta-analysis offers the possibility to compare different studies, addressing the same biological question to fully exploit public gene expression datasets.
Results
We describe STEPath, a method that starts from gene expression profiles and integrates the analysis of imbalanced region as an a priori step before performing gene set analysis. The application of STEPath in individual studies produced gene set scores weighted by chromosomal activation. As a final step, we propose a way to compare these scores across different studies (meta-analysis) on related biological issues. One complication with meta-analysis is batch effects, which occur because molecular measurements are affected by laboratory conditions, reagent lots and personnel differences. Major problems occur when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. We evaluated the power of combining chromosome mapping and gene set enrichment analysis, performing the analysis on a dataset of leukaemia (example of individual study) and on a dataset of skeletal muscle diseases (meta-analysis approach).
In leukaemia, we identified the Hox gene set, a gene set closely related to the pathology that other algorithms of gene set analysis do not identify, while the meta-analysis approach on muscular disease discriminates between related pathologies and correlates similar ones from different studies.
Conclusions
STEPath is a new method that integrates gene expression profiles, genomic co-expressed regions and the information about the biological function of genes. The usage of the STEPath-computed gene set scores overcomes batch effects in the meta-analysis approaches allowing the direct comparison of different pathologies and different studies on a gene set activation level.
doi:10.1186/1471-2105-12-92
PMCID: PMC3094239  PMID: 21481242
18.  Gene set analysis exploiting the topology of a pathway 
BMC Systems Biology  2010;4:121.
Background
Recently, a great effort in microarray data analysis is directed towards the study of the so-called gene sets. A gene set is defined by genes that are, somehow, functionally related. For example, genes appearing in a known biological pathway naturally define a gene set. The gene sets are usually identified from a priori biological knowledge. Nowadays, many bioinformatics resources store such kind of knowledge (see, for example, the Kyoto Encyclopedia of Genes and Genomes, among others). Although pathways maps carry important information about the structure of correlation among genes that should not be neglected, the currently available multivariate methods for gene set analysis do not fully exploit it.
Results
We propose a novel gene set analysis specifically designed for gene sets defined by pathways. Such analysis, based on graphical models, explicitly incorporates the dependence structure among genes highlighted by the topology of pathways. The analysis is designed to be used for overall surveillance of changes in a pathway in different experimental conditions. In fact, under different circumstances, not only the expression of the genes in a pathway, but also the strength of their relations may change. The methods resulting from the proposal allow both to test for variations in the strength of the links, and to properly account for heteroschedasticity in the usual tests for differential expression.
Conclusions
The use of graphical models allows a deeper look at the components of the pathway that can be tested separately and compared marginally. In this way it is possible to test single components of the pathway and highlight only those involved in its deregulation.
doi:10.1186/1752-0509-4-121
PMCID: PMC2945950  PMID: 20809931
19.  MAGIA, a web-based tool for miRNA and Genes Integrated Analysis 
Nucleic Acids Research  2010;38(Web Server issue):W352-W359.
MAGIA (miRNA and genes integrated analysis) is a novel web tool for the integrative analysis of target predictions, miRNA and gene expression data. MAGIA is divided into two parts: the query section allows the user to retrieve and browse updated miRNA target predictions computed with a number of different algorithms (PITA, miRanda and Target Scan) and Boolean combinations thereof. The analysis section comprises a multistep procedure for (i) direct integration through different functional measures (parametric and non-parametric correlation indexes, a variational Bayesian model, mutual information and a meta-analysis approach based on P-value combination) of mRNA and miRNA expression data, (ii) construction of bipartite regulatory network of the best miRNA and mRNA putative interactions and (iii) retrieval of information available in several public databases of genes, miRNAs and diseases and via scientific literature text-mining. MAGIA is freely available for Academic users at http://gencomp.bio.unipd.it/magia.
doi:10.1093/nar/gkq423
PMCID: PMC2896126  PMID: 20484379
20.  Impact of probe annotation on the integration of miRNA–mRNA expression profiles for miRNA target detection 
Nucleic Acids Research  2010;38(7):e97.
MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional and translational levels by an imperfect binding to target mRNA 3′UTR regions. While the ab-initio computational prediction of miRNA–mRNA interactions still poses significant challenges, it is possible to overcome some of its limitations by carefully integrating into the analysis the paired expression profiles of miRNAs and mRNAs. In this work, we show how the choice of a proper probe annotation for microarray platforms is an essential requirement to achieve good sensitivity in the identification of miRNA–mRNA interactions. We compare the results obtained from the analysis of the same expression profiles using both gene and transcript based custom CDFs that we have developed for a number of different annotations (ENSEMBL, RefSeq, AceView). In all cases, transcript-based annotations clearly improve the effectiveness of data integration and thus provide a more reliable confirmation of computationally predicted miRNA–mRNA interactions.
doi:10.1093/nar/gkp1239
PMCID: PMC2853140  PMID: 20071740
21.  Filling gaps in PPAR-alpha signaling through comparative nutrigenomics analysis 
BMC Genomics  2009;10:596.
Background
The application of high-throughput genomic tools in nutrition research is a widespread practice. However, it is becoming increasingly clear that the outcome of individual expression studies is insufficient for the comprehensive understanding of such a complex field. Currently, the availability of the large amounts of expression data in public repositories has opened up new challenges on microarray data analyses. We have focused on PPARα, a ligand-activated transcription factor functioning as fatty acid sensor controlling the gene expression regulation of a large set of genes in various metabolic organs such as liver, small intestine or heart. The function of PPARα is strictly connected to the function of its target genes and, although many of these have already been identified, major elements of its physiological function remain to be uncovered. To further investigate the function of PPARα, we have applied a cross-species meta-analysis approach to integrate sixteen microarray datasets studying high fat diet and PPARα signal perturbations in different organisms.
Results
We identified 164 genes (MDEGs) that were differentially expressed in a constant way in response to a high fat diet or to perturbations in PPARs signalling. In particular, we found five genes in yeast which were highly conserved and homologous of PPARα targets in mammals, potential candidates to be used as models for the equivalent mammalian genes. Moreover, a screening of the MDEGs for all known transcription factor binding sites and the comparison with a human genome-wide screening of Peroxisome Proliferating Response Elements (PPRE), enabled us to identify, 20 new potential candidate genes that show, both binding site, both change in expression in the condition studied. Lastly, we found a non random localization of the differentially expressed genes in the genome.
Conclusion
The results presented are potentially of great interest to resume the currently available expression data, exploiting the power of in silico analysis filtered by evolutionary conservation. The analysis enabled us to indicate potential gene candidates that could fill in the gaps with regards to the signalling of PPARα and, moreover, the non-random localization of the differentially expressed genes in the genome, suggest that epigenetic mechanisms are of importance in the regulation of the transcription operated by PPARα.
doi:10.1186/1471-2164-10-596
PMCID: PMC2801700  PMID: 20003344
22.  A-MADMAN: Annotation-based microarray data meta-analysis tool 
BMC Bioinformatics  2009;10:201.
Background
Publicly available datasets of microarray gene expression signals represent an unprecedented opportunity for extracting genomic relevant information and validating biological hypotheses. However, the exploitation of this exceptionally rich mine of information is still hampered by the lack of appropriate computational tools, able to overcome the critical issues raised by meta-analysis.
Results
This work presents A-MADMAN, an open source web application which allows the retrieval, annotation, organization and meta-analysis of gene expression datasets obtained from Gene Expression Omnibus. A-MADMAN addresses and resolves several open issues in the meta-analysis of gene expression data.
Conclusion
A-MADMAN allows i) the batch retrieval from Gene Expression Omnibus and the local organization of raw data files and of any related meta-information, ii) the re-annotation of samples to fix incomplete, or otherwise inadequate, metadata and to create user-defined batches of data, iii) the integrative analysis of data obtained from different Affymetrix platforms through custom chip definition files and meta-normalization. Software and documentation are available on-line at .
doi:10.1186/1471-2105-10-201
PMCID: PMC2711946  PMID: 19563634
23.  A comparison on effects of normalisations in the detection of differentially expressed genes 
BMC Bioinformatics  2009;10:61.
Background
Various normalisation techniques have been developed in the context of microarray analysis to try to correct expression measurements for experimental bias and random fluctuations. Major techniques include: total intensity normalisation; intensity dependent normalisation; and variance stabilising normalisation. The aim of this paper is to discuss the impact of normalisation techniques for two-channel array technology on the process of identification of differentially expressed genes.
Results
Through three precise simulation plans, we quantify the impact of normalisations: (a) on the sensitivity and specificity of a specified test statistic for the identification of deregulated genes, (b) on the gene ranking induced by the statistic.
Conclusion
Although we found a limited difference of sensitivities and specificities for the test after each normalisation, the study highlights a strong impact in terms of gene ranking agreement, resulting in different levels of agreement between competing normalisations. However, we show that the combination of two normalisations, such as glog and lowess, that handle different aspects of microarray data, is able to outperform other individual techniques.
doi:10.1186/1471-2105-10-61
PMCID: PMC2680204  PMID: 19216778
24.  Meta-analysis of expression signatures of muscle atrophy: gene interaction networks in early and late stages 
BMC Genomics  2008;9:630.
Background
Skeletal muscle mass can be markedly reduced through a process called atrophy, as a consequence of many diseases or critical physiological and environmental situations. Atrophy is characterised by loss of contractile proteins and reduction of fiber volume. Although in the last decade the molecular aspects underlying muscle atrophy have received increased attention, the fine mechanisms controlling muscle degeneration are still incomplete. In this study we applied meta-analysis on gene expression signatures pertaining to different types of muscle atrophy for the identification of novel key regulatory signals implicated in these degenerative processes.
Results
We found a general down-regulation of genes involved in energy production and carbohydrate metabolism and up-regulation of genes for protein degradation and catabolism. Six functional pathways occupy central positions in the molecular network obtained by the integration of atrophy transcriptome and molecular interaction data. They are TGF-β pathway, apoptosis, membrane trafficking/cytoskeleton organization, NFKB pathways, inflammation and reorganization of the extracellular matrix. Protein degradation pathway is evident only in the network specific for muscle short-term response to atrophy. TGF-β pathway plays a central role with proteins SMAD3/4, MYC, MAX and CDKN1A in the general network, and JUN, MYC, GNB2L1/RACK1 in the short-term muscle response network.
Conclusion
Our study offers a general overview of the molecular pathways and cellular processes regulating the establishment and maintenance of atrophic state in skeletal muscle, showing also how the different pathways are interconnected. This analysis identifies novel key factors that could be further investigated as potential targets for the development of therapeutic treatments. We suggest that the transcription factors SMAD3/4, GNB2L1/RACK1, MYC, MAX and JUN, whose functions have been extensively studied in tumours but only marginally in muscle, appear instead to play important roles in regulating muscle response to atrophy.
doi:10.1186/1471-2164-9-630
PMCID: PMC2642825  PMID: 19108710
25.  Development and validation of a gene expression oligo microarray for the gilthead sea bream (Sparus aurata) 
BMC Genomics  2008;9:580.
Background
Aquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved. The application of genomic technologies offers much promise to rapidly increase our knowledge on biological processes in farmed species and overcome such bottlenecks. Here we present an integrated platform for mRNA expression profiling in the gilthead sea bream (Sparus aurata), a marine teleost of great importance for aquaculture.
Results
A public data base was constructed, consisting of 19,734 unique clusters (3,563 contigs and 16,171 singletons). Functional annotation was obtained for 8,021 clusters. Over 4,000 sequences were also associated with a GO entry. Two 60mer probes were designed for each gene and in-situ synthesized on glass slides using Agilent SurePrint™ technology. Platform reproducibility and accuracy were assessed on two early stages of sea bream development (one-day and four days old larvae). Correlation between technical replicates was always > 0.99, with strong positive correlation between paired probes. A two class SAM test identified 1,050 differentially expressed genes between the two developmental stages. Functional analysis suggested that down-regulated transcripts (407) in older larvae are mostly essential/housekeeping genes, whereas tissue-specific genes are up-regulated in parallel with the formation of key organs (eye, digestive system). Cross-validation of microarray data was carried out using quantitative qRT-PCR on 11 target genes, selected to reflect the whole range of fold-change and both up-regulated and down-regulated genes. A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates. Good concordance between qRT-PCR and microarray data was observed between 2- and 7-fold change, while fold-change compression in the microarray was present for differences greater than 10-fold in the qRT-PCR.
Conclusion
A highly reliable oligo-microarray platform was developed and validated for the gilthead sea bream despite the presently limited knowledge of the species transcriptome. Because of the flexible design this array will be able to accommodate additional probes as soon as novel unique transcripts are available.
doi:10.1186/1471-2164-9-580
PMCID: PMC2648989  PMID: 19055773

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