40 research outputs found

    Robust Conditional Independence maps of single-voxel Magnetic Resonance Spectra to elucidate associations between brain tumours and metabolites.

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    The aim of the paper is two-fold. First, we show that structure finding with the PC algorithm can be inherently unstable and requires further operational constraints in order to consistently obtain models that are faithful to the data. We propose a methodology to stabilise the structure finding process, minimising both false positive and false negative error rates. This is demonstrated with synthetic data. Second, to apply the proposed structure finding methodology to a data set comprising single-voxel Magnetic Resonance Spectra of normal brain and three classes of brain tumours, to elucidate the associations between brain tumour types and a range of observed metabolites that are known to be relevant for their characterisation. The data set is bootstrapped in order to maximise the robustness of feature selection for nominated target variables. Specifically, Conditional Independence maps (CI-maps) built from the data and their derived Bayesian networks have been used. A Directed Acyclic Graph (DAG) is built from CI-maps, being a major challenge the minimization of errors in the graph structure. This work presents empirical evidence on how to reduce false positive errors via the False Discovery Rate, and how to identify appropriate parameter settings to improve the False Negative Reduction. In addition, several node ordering policies are investigated that transform the graph into a DAG. The obtained results show that ordering nodes by strength of mutual information can recover a representative DAG in a reasonable time, although a more accurate graph can be recovered using a random order of samples at the expense of increasing the computation time

    Functional Analysis of the Phycomyces carRA Gene Encoding the Enzymes Phytoene Synthase and Lycopene Cyclase

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    Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism

    Two Origins for the Gene Encoding α-Isopropylmalate Synthase in Fungi

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    BACKGROUND: The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy. METHODOLOGY/PRINCIPAL FINDINGS: Here we identified the leuA gene encoding alpha-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently. CONCLUSIONS/SIGNIFICANCE: The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events

    The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

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    Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen

    Structure and function of the genes involved in the biosynthesis of carotenoids in the mucorales

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    Carotenoids are widely distributed natural pigments which are in an increasing demand by the market, due to their applications in the human food, animal feed, cosmetics, and pharmaceutical industries. Although more than 600 carotenoids have been identified in nature, only a few are industrially important (β-carotene, astaxanthin, lutein or lycopene). To date chemical processes manufacture most of the carotenoid production, but the interest for carotenoids of biological origin is growing since there is an increased public concern over the safety of artificial food colorants. Although much interest and effort has been devoted to the use of biological sources for industrially important carotenoids, only the production of biological β-carotene and astaxanthin has been reported. Among fungi, several Mucorales strains, particularly Blakeslea trispora, have been used to develop fermentation processes for the production of β-carotene on almost competitive cost-price levels. Similarly, the basidiomycetous yeast Xanthophyllomyces dendrorhous (the perfect state of Phaffia rhodozyma), has been proposed as a promising source of astaxanthin. This paper focuses on recent findings on the fungal pathways for carotenoid production, especially the structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. An outlook of the possibilities of an increased industrial production of carotenoids, based on metabolic engineering of fungi for carotenoid content and composition, is also discussed

    A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides.

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    Using functional analyses in Escherichia coli and Mucor circinelloides, it has been shown that a single M. circinelloides gene (carRP) codes for a protein with two different enzymatic activities, lycopene cyclase and phytoene synthase, which are encoded by independent genes in organisms other than fungi. This gene was identified using complementation tests among different classes of carotenoid mutants of M. circinelloides. The carRP gene product contains two domains: the R domain is located at the N-terminus and determines lycopene cyclase activity; the P domain is located at the C-terminus and displays phytoene synthase activity. The R domain is functional even in the absence of the P domain, while the latter needs the proper R domain conformation to carry out its function. The carRP gene is closely linked to the phytoene dehydrogenase (carB) gene, and the promoter regions of both genes are located within only 446 bp. Northern analyses show a co-ordinated regulation of the expression of both genes by blue light. Several motifs found in this promoter region suggest a bi-directional mode of transcription control

    Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides.

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    Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase

    A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides.

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    Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes
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