Integrative omics analysis. A study based on Plasmodium falciparum mRNA and protein data

BACKGROUND: Technological improvements have shifted the focus from data generation to data analysis. The availability of large amounts of data from transcriptomics, protemics and metabolomics experiments raise new questions concerning suitable integrative analysis methods. We compare three integrative analysis techniques (co-inertia analysis, generalized singular value decomposition and integrative biclustering) by applying them to gene and protein abundance data from the six life cycle stages of Plasmodium falciparum. Co-inertia analysis is an analysis method used to visualize and explore gene and protein data. The generalized singular value decomposition has shown its potential in the analysis of two transcriptome data sets. Integrative Biclustering applies biclustering to gene and protein data. RESULTS: Using CIA, we visualize the six life cycle stages of Plasmodium falciparum, as well as GO terms in a 2D plane and interpret the spatial configuration. With GSVD, we decompose the transcriptomic and proteomic data sets into matrices with biologically meaningful interpretations and explore the processes captured by the data sets. IBC identifies groups of genes, proteins, GO Terms and life cycle stages of Plasmodium falciparum. We show method-specific results as well as a network view of the life cycle stages based on the results common to all three methods. Additionally, by combining the results of the three methods, we create a three-fold validated network of life cycle stage specific GO terms: Sporozoites are associated with transcription and transport; merozoites with entry into host cell as well as biosynthetic and metabolic processes; rings with oxidation-reduction processes; trophozoites with glycolysis and energy production; schizonts with antigenic variation and immune response; gametocyctes with DNA packaging and mitochondrial transport. Furthermore, the network connectivity underlines the separation of the intraerythrocytic cycle from the gametocyte and sporozoite stages. CONCLUSION: Using integrative analysis techniques, we can integrate knowledge from different levels and obtain a wider view of the system under study. The overlap between method-specific and common results is considerable, even if the basic mathematical assumptions are very different. The three-fold validated network of life cycle stage characteristics of Plasmodium falciparum could identify a large amount of the known associations from literature in only one study

Peroxidase gene discovery from the horseradish transcriptome

BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria

BACKGROUND: In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host. RESULTS: We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405(T) (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405(T). Instead, S. rhizophila DSM14405(T) possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405(T) is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism. CONCLUSIONS: While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-482) contains supplementary material, which is available to authorized users

QPCR: Application for real-time PCR data management and analysis

BACKGROUND: Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. RESULTS: QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. CONCLUSION: We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available a

TAMEE: data management and analysis for tissue microarrays

BACKGROUND: With the introduction of tissue microarrays (TMAs) researchers can investigate gene and protein expression in tissues on a high-throughput scale. TMAs generate a wealth of data calling for extended, high level data management. Enhanced data analysis and systematic data management are required for traceability and reproducibility of experiments and provision of results in a timely and reliable fashion. Robust and scalable applications have to be utilized, which allow secure data access, manipulation and evaluation for researchers from different laboratories. RESULTS: TAMEE (Tissue Array Management and Evaluation Environment) is a web-based database application for the management and analysis of data resulting from the production and application of TMAs. It facilitates storage of production and experimental parameters, of images generated throughout the TMA workflow, and of results from core evaluation. Database content consistency is achieved using structured classifications of parameters. This allows the extraction of high quality results for subsequent biologically-relevant data analyses. Tissue cores in the images of stained tissue sections are automatically located and extracted and can be evaluated using a set of predefined analysis algorithms. Additional evaluation algorithms can be easily integrated into the application via a plug-in interface. Downstream analysis of results is facilitated via a flexible query generator. CONCLUSION: We have developed an integrated system tailored to the specific needs of research projects using high density TMAs. It covers the complete workflow of TMA production, experimental use and subsequent analysis. The system is freely available for academic and non-profit institutions from

Translin and Trax differentially regulate telomere-associated transcript homeostasis

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1(+) (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion

MiR-199a and miR-497 are associated with better overall survival due to increased chemosensitivity in diffuse large b-cell lymphoma patients

Micro-RNAs (miRNAs) are short non-coding single-stranded RNA molecules regulating gene expression at the post-transcriptional level. miRNAs are involved in cell development, differentiation, apoptosis, and proliferation. miRNAs can either function as tumor suppressor genes or oncogenes in various important pathways. The expression of specific miRNAs has been identified to correlate with tumor prognosis. For miRNA expression analysis real-time PCR on 81 samples was performed, including 63 diffuse large B-cell lymphoma (DLBCL, 15 of germinal center B-cell like subtype, 17 non germinal center B-cell, 23 transformed, and eight unclassified) and 18 controls, including nine peripheral B-cells, 5 germinal-center B-cells, four lymphadenitis samples, and 4 lymphoma cell lines (RI-1, SUDHL4, Karpas, U2932). Expression levels of a panel of 11 miRNAs that have been previously involved in other types of cancer (miR-15b_2, miR-16_1*, miR-16_2, miR-16_2*, miR-27a, miR-27a*, miR-98-1, miR-103a, miR-185, miR-199a, and miR-497) were measured and correlated with clinical data. Furthermore, cell lines, lacking miR-199a and miR-497 expression, were electroporated with the two respective miRNAs and treated with standard immunochemotherapy routinely used in patients with DLBCL, followed by functional analyses including cell count and apoptosis assays. Seven miRNAs (miR-16_1*, miR-16_2*, miR-27a, miR-103, miR-185, miR-199, and miR-497) were statistically significantly up-regulated in DLBCL compared to normal germinal cells. However, high expression of miR-497 or miR-199a was associated with better overall survival (p = 0.042 and p = 0.007). Overexpression of miR-199a and miR-497 led to a statistically significant decrease in viable cells in a dose-dependent fashion after exposure to rituximab and various chemotherapeutics relevant in multi-agent lymphoma therapy. Our data indicate that elevated miR-199a and miR-497 levels are associated with improved survival in aggressive lymphoma patients most likely by modifying drug sensitivity to immunochemotherapy. This functional impairment may serve as a potential novel therapeutic target in future treatment of patients with DLBCL

Human germ/stem cell-specific gene TEX19 influences cancer cell proliferation and cancer prognosis

Table of cancer data sets analyzed. (DOCX 14 kb