Mapping novel QTL and fine mapping of previously identified QTL associated with glucose tolerance using the collaborative cross mice.

A chronic metabolic illness, type 2 diabetes (T2D) is a polygenic and multifactorial complicated disease. With an estimated 463 million persons aged 20 to 79 having diabetes, the number is expected to rise to 700 million by 2045, creating a significant worldwide health burden. Polygenic variants of diabetes are influenced by environmental variables. T2D is regarded as a silent illness that can advance for years before being diagnosed. Finding genetic markers for T2D and metabolic syndrome in groups with similar environmental exposure is therefore essential to understanding the mechanism of such complex characteristic illnesses. So herein, we demonstrated the exclusive use of the collaborative cross (CC) mouse reference population to identify novel quantitative trait loci (QTL) and, subsequently, suggested genes associated with host glucose tolerance in response to a high-fat diet. In this study, we used 539 mice from 60 different CC lines. The diabetogenic effect in response to high-fat dietary challenge was measured by the three-hour intraperitoneal glucose tolerance test (IPGTT) test after 12 weeks of dietary challenge. Data analysis was performed using a statistical software package IBM SPSS Statistic 23. Afterward, blood glucose concentration at the specific and between different time points during the IPGTT assay and the total area under the curve (AUC0-180) of the glucose clearance was computed and utilized as a marker for the presence and severity of diabetes. The observed AUC0-180 averages for males and females were 51,267.5 and 36,537.5 mg/dL, respectively, representing a 1.4-fold difference in favor of females with lower AUC0-180 indicating adequate glucose clearance. The AUC0-180 mean differences between the sexes within each specific CC line varied widely within the CC population. A total of 46 QTL associated with the different studied phenotypes, designated as T2DSL and its number, for Type 2 Diabetes Specific Locus and its number, were identified during our study, among which 19 QTL were not previously mapped. The genomic interval of the remaining 27 QTL previously reported, were fine mapped in our study. The genomic positions of 40 of the mapped QTL overlapped (clustered) on 11 different peaks or close genomic positions, while the remaining 6 QTL were unique. Further, our study showed a complex pattern of haplotype effects of the founders, with the wild-derived strains (mainly PWK) playing a significant role in the increase of AUC values.

A Comprehensive Analysis of Cutaneous Melanoma Patients in Greece Based on Multi-Omic Data.

Cutaneous melanoma (CM) is the most aggressive type of skin cancer, and it is characterised by high mutational load and heterogeneity. In this study, we aimed to analyse the genomic and transcriptomic profile of primary melanomas from forty-six Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from Greek patients. Molecular analysis for both germline and somatic variations was performed in genomic DNA from peripheral blood and melanoma samples, respectively, exploiting whole exome and targeted sequencing, and transcriptomic analysis. Detailed clinicopathological data were also included in our analyses and previously reported associations with specific mutations were recognised. Most analysed samples (43/46) were found to harbour at least one clinically actionable somatic variant. A subset of samples was profiled at the transcriptomic level, and it was shown that specific melanoma phenotypic states could be inferred from bulk RNA isolated from FFPE primary melanoma tissue. Integrative bioinformatics analyses, including variant prioritisation, differential gene expression analysis, and functional and gene set enrichment analysis by group and per sample, were conducted and molecular circuits that are implicated in melanoma cell programmes were highlighted. Integration of mutational and transcriptomic data in CM characterisation could shed light on genes and pathways that support the maintenance of phenotypic states encrypted into heterogeneous primary tumours.

ERß1 Sensitizes and ERß2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERß1 and ERß2 (isoforms of ERß), the second ER isotype. At present, the impact of ERß isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERß1 or ERß2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERß1 and MCF7-ERß2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERß1 cells and cancer-promoting effects in MCF7-ERß2 cells. Our data are favorable to ERß1 being a marker of responsiveness and ERß2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.

Comparative Evaluation of Reproducibility of Phage-Displayed Peptide Selections and NGS Data, through High-Fidelity Mapping of Massive Peptide Repertoires.

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer.

IRE1α is constitutively active in several cancers and can contribute to cancer progression. Activated IRE1α cleaves XBP1 mRNA, a key step in production of the transcription factor XBP1s. In addition, IRE1α cleaves select mRNAs through regulated IRE1α-dependent decay (RIDD). Accumulating evidence implicates IRE1α in the regulation of lipid metabolism. However, the roles of XBP1s and RIDD in this process remain ill-defined. In this study, transcriptome and lipidome profiling of triple negative breast cancer cells subjected to pharmacological inhibition of IRE1α reveals changes in lipid metabolism genes associated with accumulation of triacylglycerols (TAGs). We identify DGAT2 mRNA, encoding the rate-limiting enzyme in TAG biosynthesis, as a RIDD target. Inhibition of IRE1α, leads to DGAT2-dependent accumulation of TAGs in lipid droplets and sensitizes cells to nutritional stress, which is rescued by treatment with the DGAT2 inhibitor PF-06424439. Our results highlight the importance of IRE1α RIDD activity in reprograming cellular lipid metabolism.

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models.

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

Integration of Raman spectra with transcriptome data in glioblastoma multiforme defines tumour subtypes and predicts patient outcome.

Raman spectroscopy is an imaging technique that has been applied to assess molecular compositions of living cells to characterize cell types and states. However, owing to the diverse molecular species in cells and challenges of assigning peaks to specific molecules, it has not been clear how to interpret cellular Raman spectra. Here, we provide firm evidence that cellular Raman spectra (RS) and transcriptomic profiles of glioblastoma can be computationally connected and thus interpreted. We find that the dimensions of high-dimensional RS and transcriptomes can be reduced and connected linearly through a shared low-dimensional subspace. Accordingly, we were able to predict global gene expression profiles by applying the calculated transformation matrix to Raman spectra and vice versa. From these analyses, we extract a minimal gene expression signature associated with specific RS profiles and predictive of disease outcome.

Epigenetics in Breast Cancer Therapy-New Strategies and Future Nanomedicine Perspectives.

Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identification, allowing for patient stratification and tailored management. In combination with new-generation epi-drugs, nanomedicine can help to overcome low therapeutic efficacy in treatment-resistant tumors. This review provides an overview of ongoing clinical trials focusing on combination therapies employing epi-drugs for breast cancer treatment and summarizes the latest nano-based targeted delivery approaches for epi-drugs. Moreover, it highlights the current limitations and obstacles associated with applying these experimental strategies in the clinics.

Container-aided integrative QTL and RNA-seq analysis of Collaborative Cross mice supports distinct sex-oriented molecular modes of response in obesity.

BACKGROUND: The Collaborative Cross (CC) mouse population is a valuable resource to study the genetic basis of complex traits, such as obesity. Although the development of obesity is influenced by environmental factors, underlying genetic mechanisms play a crucial role in the response to these factors. The interplay between the genetic background and the gene expression pattern can provide further insight into this response, but we lack robust and easily reproducible workflows to integrate genomic and transcriptomic information in the CC mouse population. RESULTS: We established an automated and reproducible integrative workflow to analyse complex traits in the CC mouse genetic reference panel at the genomic and transcriptomic levels. We implemented the analytical workflow to assess the underlying genetic mechanisms of host susceptibility to diet induced obesity and integrated these results with diet induced changes in the hepatic gene expression of susceptible and resistant mice. Hepatic gene expression differs significantly between obese and non-obese mice, with a significant sex effect, where male and female mice exhibit different responses and coping mechanisms. CONCLUSION: Integration of the data showed that different genes but similar pathways are involved in the genetic susceptibility and disturbed in diet induced obesity. Genetic mechanisms underlying susceptibility to high-fat diet induced obesity are different in female and male mice. The clear distinction we observed in the systemic response to the high-fat diet challenge and to obesity between male and female mice points to the need for further research into distinct sex-related mechanisms in metabolic disease.

RA-map: building a state-of-the-art interactive knowledge base for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a progressive, inflammatory autoimmune disease of unknown aetiology. The complex mechanism of aetiopathogenesis, progress and chronicity of the disease involves genetic, epigenetic and environmental factors. To understand the molecular mechanisms underlying disease phenotypes, one has to place implicated factors in their functional context. However, integration and organization of such data in a systematic manner remains a challenging task. Molecular maps are widely used in biology to provide a useful and intuitive way of depicting a variety of biological processes and disease mechanisms. Recent large-scale collaborative efforts such as the Disease Maps Project demonstrate the utility of such maps as versatile tools to organize and formalize disease-specific knowledge in a comprehensive way, both human and machine-readable. We present a systematic effort to construct a fully annotated, expert validated, state-of-the-art knowledge base for RA in the form of a molecular map. The RA map illustrates molecular and signalling pathways implicated in the disease. Signal transduction is depicted from receptors to the nucleus using the Systems Biology Graphical Notation (SBGN) standard representation. High-quality manual curation, use of only human-specific studies and focus on small-scale experiments aim to limit false positives in the map. The state-of-the-art molecular map for RA, using information from 353 peer-reviewed scientific publications, comprises 506 species, 446 reactions and 8 phenotypes. The species in the map are classified to 303 proteins, 61 complexes, 106 genes, 106 RNA entities, 2 ions and 7 simple molecules. The RA map is available online at ramap.elixir-luxembourg.org as an open-access knowledge base allowing for easy navigation and search of molecular pathways implicated in the disease. Furthermore, the RA map can serve as a template for omics data visualization.

A Computational Pipeline for the Extraction of Actionable Biological Information From NGS-Phage Display Experiments.

Phage Display is a powerful method for the identification of peptide binding to targets of variable complexities and tissues, from unique molecules to the internal surfaces of vessels of living organisms. Particularly for in vivo screenings, the resulting repertoires can be very complex and difficult to study with traditional approaches. Next Generation Sequencing (NGS) opened the possibility to acquire high resolution overviews of such repertoires and thus facilitates the identification of binders of interest. Additionally, the ever-increasing amount of available genome/proteome information became satisfactory regarding the identification of putative mimicked proteins, due to the large scale on which partial sequence homology is assessed. However, the subsequent production of massive data stresses the need for high-performance computational approaches in order to perform standardized and insightful molecular network analysis. Systems-level analysis is essential for efficient resolution of the underlying molecular complexity and the extraction of actionable interpretation, in terms of systemic biological processes and pathways that are systematically perturbed. In this work we introduce PepSimili, an integrated workflow tool, which performs mapping of massive peptide repertoires on whole proteomes and delivers a streamlined, systems-level biological interpretation. The tool employs modules for modeling and filtering of background noise due to random mappings and amplifies the biologically meaningful signal through coupling with BioInfoMiner, a systems interpretation tool that employs graph-theoretic methods for prioritization of systemic processes and corresponding driver genes. The current implementation exploits the Galaxy environment and is available online. A case study using public data is presented, with and without a control selection.

ANASTASIA: An Automated Metagenomic Analysis Pipeline for Novel Enzyme Discovery Exploiting Next Generation Sequencing Data.

Metagenomic analysis of environmental samples provides deep insight into the enzymatic mixture of the corresponding niches, capable of revealing peptide sequences with novel functional properties exploiting the high performance of next-generation sequencing (NGS) technologies. At the same time due to their ever increasing complexity, there is a compelling need for ever larger computational configurations to ensure proper bioinformatic analysis, and fine annotation. With the aiming to address the challenges of such an endeavor, we have developed a novel web-based application named ANASTASIA (automated nucleotide aminoacid sequences translational plAtform for systemic interpretation and analysis). ANASTASIA provides a rich environment of bioinformatic tools, either publicly available or novel, proprietary algorithms, integrated within numerous automated algorithmic workflows, and which enables versatile data processing tasks for (meta)genomic sequence datasets. ANASTASIA was initially developed in the framework of the European FP7 project HotZyme, whose aim was to perform exhaustive analysis of metagenomes derived from thermal springs around the globe and to discover new enzymes of industrial interest. ANASTASIA has evolved to become a stable and extensible environment for diversified, metagenomic, functional analyses for a range of applications overarching industrial biotechnology to biomedicine, within the frames of the ELIXIR-GR project. As a showcase, we report the successful in silico mining of a novel thermostable esterase termed "EstDZ4" from a metagenomic sample collected from a hot spring located in Krisuvik, Iceland.

Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation.

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.

Radiogenomic Analysis of F-18-Fluorodeoxyglucose Positron Emission Tomography and Gene Expression Data Elucidates the Epidemiological Complexity of Colorectal Cancer Landscape.

PURPOSE: Transcriptomic profiling has enabled the neater genomic characterization of several cancers, among them colorectal cancer (CRC), through the derivation of genes with enhanced causal role and informative gene sets. However, the identification of small-sized gene signatures, which can serve as potential biomarkers in CRC, remains challenging, mainly due to the great genetic heterogeneity of the disease. METHODS: We developed and exploited an analytical framework for the integrative analysis of CRC datasets, encompassing transcriptomic data and positron emission tomography (PET) measurements. Profiling data comprised two microarray datasets, pertaining biopsy specimen from 30 untreated patients with primary CRC, coupled by their F-18-Fluorodeoxyglucose (FDG) PET values, using tracer kinetic analysis measurements. The computational framework incorporates algorithms for semantic processing, multivariate analysis, data mining and dimensionality reduction. RESULTS: Transcriptomic and PET data feature sets, were evaluated for their discrimination performance between primary colorectal adenocarcinomas and adjacent normal mucosa. A composite signature was derived, pertaining 12 features: 7 genes and 5 PET variables. This compact signature manifests superior performance in classification accuracy, through the integration of gene expression and PET data. CONCLUSIONS: This work represents an effort for the integrative, multilayered, signature-oriented analysis of CRC, in the context of radio-genomics, inferring a composite signature with promising results for patient stratification.

Integrative Bioinformatic Analysis of Transcriptomic Data Identifies Conserved Molecular Pathways Underlying Ionizing Radiation-Induced Bystander Effects (RIBE).

Ionizing radiation-induced bystander effects (RIBE) encompass a number of effects with potential for a plethora of damages in adjacent non-irradiated tissue. The cascade of molecular events is initiated in response to the exposure to ionizing radiation (IR), something that may occur during diagnostic or therapeutic medical applications. In order to better investigate these complex response mechanisms, we employed a unified framework integrating statistical microarray analysis, signal normalization, and translational bioinformatics functional analysis techniques. This approach was applied to several microarray datasets from Gene Expression Omnibus (GEO) related to RIBE. The analysis produced lists of differentially expressed genes, contrasting bystander and irradiated samples versus sham-irradiated controls. Furthermore, comparative molecular analysis through BioInfoMiner, which integrates advanced statistical enrichment and prioritization methodologies, revealed discrete biological processes, at the cellular level. For example, the negative regulation of growth, cellular response to Zn2+-Cd2+, and Wnt and NIK/NF-kappaB signaling, thus refining the description of the phenotypic landscape of RIBE. Our results provide a more solid understanding of RIBE cell-specific response patterns, especially in the case of high-LET radiations, like α-particles and carbon-ions.

The Development of an Angiogenic Protein "Signature" in Ovarian Cancer Ascites as a Tool for Biologic and Prognostic Profiling.

Advanced ovarian cancer (AOC) is one of the leading lethal gynecological cancers in developed countries. Based on the important role of angiogenesis in ovarian cancer oncogenesis and expansion, we hypothesized that the development of an "angiogenic signature" might be helpful in prediction of prognosis and efficacy of anti-angiogenic therapies in this disease. Sixty-nine samples of ascitic fluid- 35 from platinum sensitive and 34 from platinum resistant patients managed with cytoreductive surgery and 1st-line carboplatin-based chemotherapy- were analyzed using the Proteome ProfilerTM Human Angiogenesis Array Kit, screening for the presence of 55 soluble angiogenesis-related factors. A protein profile based on the expression of a subset of 25 factors could accurately separate resistant from sensitive patients with a success rate of approximately 90%. The protein profile corresponding to the "sensitive" subset was associated with significantly longer PFS (8 [95% Confidence Interval {CI}: 8-9] vs. 20 months [95% CI: 15-28]; Hazard ratio {HR}: 8.3, p<0.001) and OS (20.5 months [95% CI: 13.5-30] vs. 74 months [95% CI: 36-not reached]; HR: 5.6 [95% CI: 2.8-11.2]; p<0.001). This prognostic performance was superior to that of stage, histology and residual disease after cytoreductive surgery and the levels of vascular endothelial growth factor (VEGF) in ascites. In conclusion, we developed an "angiogenic signature" for patients with AOC, which can be used, after appropriate validation, as a prognostic marker and a tool for selection for anti-angiogenic therapies.

KENeV: A web-application for the automated reconstruction and visualization of the enriched metabolic and signaling super-pathways deriving from genomic experiments.

Gene expression analysis, using high throughput genomic technologies,has become an indispensable step for the meaningful interpretation of the underlying molecular complexity, which shapes the phenotypic manifestation of the investigated biological mechanism. The modularity of the cellular response to different experimental conditions can be comprehended through the exploitation of molecular pathway databases, which offer a controlled, curated background for statistical enrichment analysis. Existing tools enable pathway analysis, visualization, or pathway merging but none integrates a fully automated workflow, combining all above-mentioned modules and destined to non-programmer users. We introduce an online web application, named KEGG Enriched Network Visualizer (KENeV), which enables a fully automated workflow starting from a list of differentially expressed genes and deriving the enriched KEGG metabolic and signaling pathways, merged into two respective, non-redundant super-networks. The final networks can be downloaded as SBML files, for further analysis, or instantly visualized through an interactive visualization module. In conclusion, KENeV (available online at http://www.grissom.gr/kenev) provides an integrative tool, suitable for users with no programming experience, for the functional interpretation, at both the metabolic and signaling level, of differentially expressed gene subsets deriving from genomic experiments.

Cancer Biomarkers from Genome-Scale DNA Methylation: Comparison of Evolutionary and Semantic Analysis Methods.

DNA methylation profiling exploits microarray technologies, thus yielding a wealth of high-volume data. Here, an intelligent framework is applied, encompassing epidemiological genome-scale DNA methylation data produced from the Illumina's Infinium Human Methylation 450K Bead Chip platform, in an effort to correlate interesting methylation patterns with cancer predisposition and, in particular, breast cancer and B-cell lymphoma. Feature selection and classification are employed in order to select, from an initial set of ~480,000 methylation measurements at CpG sites, predictive cancer epigenetic biomarkers and assess their classification power for discriminating healthy versus cancer related classes. Feature selection exploits evolutionary algorithms or a graph-theoretic methodology which makes use of the semantics information included in the Gene Ontology (GO) tree. The selected features, corresponding to methylation of CpG sites, attained moderate-to-high classification accuracies when imported to a series of classifiers evaluated by resampling or blindfold validation. The semantics-driven selection revealed sets of CpG sites performing similarly with evolutionary selection in the classification tasks. However, gene enrichment and pathway analysis showed that it additionally provides more descriptive sets of GO terms and KEGG pathways regarding the cancer phenotypes studied here. Results support the expediency of this methodology regarding its application in epidemiological studies.

Escherichia coli genome-wide promoter analysis: identification of additional AtoC binding target elements.

BACKGROUND: Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome. RESULTS: Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences. CONCLUSIONS: This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.