Search

The achievements coming from genome analysis depend greatly on the quality of computational and processing methods. Tools for functional mRNA profiling and for gene information integration have become essential to this task. We have developed GeneBrowser as a novel approach that combines the advantages of mRNA profiling tools, at genome-scale experiments, with the features provided by data integration systems. For a given set of genes, GeneBrowser integrates bibliography information with functional annotations, using Gene Ontology, Entrez Gene, KEGG Orthology and KEGG Pathways. The result is a comprehensive and easy to use web application that helps researchers to extract knowledge from large data sets and to speed up the discovery process.

Background Helicobacter pylori infection induces cellular phenotypes relevant for cancer progression, namely cell motility and invasion. We hypothesized that the extracellular matrix (ECM) could be involved in these deleterious effects. Methods Microarrays were used to uncover ECM interactors in cells infected with H. pylori. LAMC2 , encoding laminin γ2, was selected as a candidate gene and its expression was assessed in vitro and in vivo. The role of LAMC2 was investigated by small interference RNA (siRNA) combined with a set of functional assays. Laminin γ2 and E-cadherin expression patterns were evaluated in gastric cancer cases. Results Laminin γ2 was found significantly overexpressed in gastric cancer cells infected with H. pylori. This finding was validated in vitro by infection with clinical isolates and in vivo by using gastric biopsies of infected and noninfected individuals. We showed that laminin γ2 overexpression is dependent on the bacterial type IV secretion system and on the CagA. Functionally, laminin γ2 promotes cell invasion and resistance to apoptosis, through modulation of Src, JNK, and AKT activity. These effects were abrogated in cells with functional E-cadherin. Conclusions These data highlight laminin γ2 and its downstream effectors as potential therapeutic targets, and the value of H. pylori eradication to delay gastric cancer onset and progression. [ABSTRACT FROM AUTHOR]

A study which was aimed at characterizing new Thermus strains and confirming the presence of glycerol-linked and diol-linked glycolipids within these strains was conducted. The methods used were extraction and purification of polar lipids, hydrolysis and analysis of glycolipids, preparation of fatty acid methyl esters (FAMEs) and derivatization. Results showed that partial structures of intact diol-containing lipids were present in the bacteria and some Thermus strains contain a mixture of diol- and glycerol-based glycolipids which were not present in the Thermomicrobium roseum.

The compositions of the primary glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11 and Thermus sp. strain CG-2 were studied. With the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was identified as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. The unique major glycolipid of T. aquaticus 15004 was established as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-glucosyl diacylglycerol.

Abstract Background Burkholderia pseudomallei is a facultative intracellular pathogen of phagocytic and non-phagocytic cells. How the bacterium interacts with host macrophage cells is still not well understood and is critical to appreciate the strategies used by this bacterium to survive and how intracellular survival leads to disease manifestation. Results Here we report the expression profile of intracellular B. pseudomallei following infection of human macrophage-like U937 cells. During intracellular growth over the 6 h infection period, approximately 22 % of the B. pseudomallei genome showed significant transcriptional adaptation. B. pseudomallei adapted rapidly to the intracellular environment by down-regulating numerous genes involved in metabolism, cell envelope, motility, replication, amino acid and ion transport system and regulatory function pathways. Reduced expression in catabolic and housekeeping genes suggested lower energy requirement and growth arrest during macrophage infection, while expression of genes encoding anaerobic metabolism functions were up regulated. However, whilst the type VI secretion system was up regulated, expression of many known virulence factors was not significantly modulated over the 6hours of infection. Conclusions The transcriptome profile described here provides the first comprehensive view of how B. pseudomallei survives within host cells and will help identify potential virulence factors and proteins that are important for the survival and growth of B. pseudomallei within human cells.

Abstract Background Organisms use highly accurate molecular processes to transcribe their genes and a variety of mRNA quality control and ribosome proofreading mechanisms to maintain intact the fidelity of genetic information flow. Despite this, low level gene translational errors induced by mutations and environmental factors cause neurodegeneration and premature death in mice and mitochondrial disorders in humans. Paradoxically, such errors can generate advantageous phenotypic diversity in fungi and bacteria through poorly understood molecular processes. Results In order to clarify the biological relevance of gene translational errors we have engineered codon misreading in yeast and used profiling of total and polysome-associated mRNAs, molecular and biochemical tools to characterize the recombinant cells. We demonstrate here that gene translational errors, which have negligible impact on yeast growth rate down-regulate protein synthesis, activate the unfolded protein response and environmental stress response pathways, and down-regulate chaperones linked to ribosomes. Conclusions We provide the first global view of transcriptional and post-transcriptional responses to global gene translational errors and we postulate that they cause gradual cell degeneration through synergistic effects of overloading protein quality control systems and deregulation of protein synthesis, but generate adaptive phenotypes in unicellular organisms through activation of stress cross-protection. We conclude that these genome wide gene translational infidelities can be degenerative or adaptive depending on cellular context and physiological condition.

Abstract Background Saccharomyces cerevisiae (Baker's yeast) is found in diverse ecological niches and is characterized by high adaptive potential under challenging environments. In spite of recent advances on the study of yeast genome diversity, little is known about the underlying gene expression plasticity. In order to shed new light onto this biological question, we have compared transcriptome profiles of five environmental isolates, clinical and laboratorial strains at different time points of fermentation in synthetic must medium, during exponential and stationary growth phases. Results Our data unveiled diversity in both intensity and timing of gene expression. Genes involved in glucose metabolism and in the stress response elicited during fermentation were among the most variable. This gene expression diversity increased at the onset of stationary phase (diauxic shift). Environmental isolates showed lower average transcript abundance of genes involved in the stress response, assimilation of nitrogen and vitamins, and sulphur metabolism, than other strains. Nitrogen metabolism genes showed significant variation in expression among the environmental isolates. Conclusions Wild type yeast strains respond differentially to the stress imposed by nutrient depletion, ethanol accumulation and cell density increase, during fermentation of glucose in synthetic must medium. Our results support previous data showing that gene expression variability is a source of phenotypic diversity among closely related organisms.

Abstract Background Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate. Results In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates. Conclusion We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in sub-telomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome.

Abstract Background The evolutionary forces that determine the arrangement of synonymous codons within open reading frames and fine tune mRNA translation efficiency are not yet understood. In order to tackle this question we have carried out a large scale study of codon-triplet contexts in 11 fungal species to unravel associations or relationships between codons present at the ribosome A-, P- and E-sites during each decoding cycle. Results Our analysis unveiled high bias within the context of codon-triplets, in particular strong preference for triplets of identical codons. We have also identified a surprisingly large number of codon-triplet combinations that vanished from fungal ORFeomes. Candida albicans exacerbated these features, showed an unbalanced tRNA population for decoding its pool of codons and used near-cognate decoding for a large set of codons, suggesting that unique evolutionary forces shaped the evolution of its ORFeome. Conclusion We have developed bioinformatics tools for large-scale analysis of codon-triplet contexts. These algorithms identified codon-triplets context biases, allowed for large scale comparative codon-triplet analysis, and identified rules governing codon-triplet context. They could also detect alterations to the standard genetic code.

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)--VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases. [ABSTRACT FROM AUTHOR]

A double compartment membrane system was constructed in order to systematically study possible microbial interactions between yeasts Saccharomyces cerevisiae and Dekkera bruxellensis and their impact on wine aroma. The presence of D. bruxellensis induced 77 transcripts of S. cerevisiae. These were mostly of unknown function; however, some were involved in thiamine biosynthesis and in amino acid and polyamine transport, suggesting a competitive relationship between the two yeast species. Among the transcripts with no biological function, 14 of them were found to be the members of the PAU gene family that is associated with response to anaerobiosis stress. In separated cultures, S. cerevisiae produced glycerol which was subsequently consumed by D. bruxellensis. The concentration of ethylphenols was reduced and we assume that they were absorbed onto the surfaces of S. cerevisiae yeast walls. Also in separated cultures, D. bruxellensis formed a typical profile of aromatic esters with decreased levels of acetate esters and increased level of ethyl esters. [ABSTRACT FROM AUTHOR]

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia. [ABSTRACT FROM AUTHOR]

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources. [ABSTRACT FROM AUTHOR]

Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress. [ABSTRACT FROM AUTHOR]

The choroid plexus (CP) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF), the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis. [ABSTRACT FROM AUTHOR]

Background: Quercetin is a naturally occurring flavonol with antioxidant, anticancer and anti-ageing properties. In this study we aimed to identify genes differentially expressed in yeast cells treated with quercetin and its role in oxidative stress protection. Methods: A microarray analysis was performed to characterize changes in the transcriptome and the expression of selected genes was validated by RT-qPCR. Biological processes significantly affected were identified by using the FUNSPEC software and their relevance in H2O2 resistance induced by quercetin was assessed. Results: Genes associated with RNA metabolism and ribosome biogenesis were down regulated in cells treated with quercetin, whereas genes associated with carbohydrate metabolism, endocytosis and vacuolar proteolysis were up regulated. The induction of genes related to the metabolism of energy reserves, leading to the accumulation of the stress protectant disaccharide trehalose, and the activation of the cell wall integrity pathway play a key role in oxidative stress resistance induced by quercetin. Conclusions: These results suggest that quercetin may act as a modulator of cell signaling pathways related to carbohydrate metabolism and cell integrity to exert its protective effects against oxidative stress. [ABSTRACT FROM AUTHOR]

The budding yeast Saccharomyces cerevisiae has developed several mechanisms to avoid either the drastic consequences of iron deprivation or the toxic effects of iron excess. In this work, we analysed the global gene expression changes occurring in yeast cells undergoing iron overload. Several genes directly or indirectly involved in iron homeostasis showed altered expression and the relevance of these changes are discussed. Microarray analyses were also performed to identify new targets of the iron responsive factor Yap5. Besides the iron vacuolar transporter CCC1, Yap5 also controls the expression of glutaredoxin GRX4, previously known to be involved in the regulation of Aft1 nuclear localization. Consistently, we show that in the absence of Yap5 Aft1 nuclear exclusion is slightly impaired. These studies provide further evidence that cells control iron homeostasis by using multiple pathways. [ABSTRACT FROM AUTHOR]

Background: Codon pair usage (codon context) is a species specific gene primary structure feature whose evolutionary and functional roles are poorly understood. The data available show that codon-context has direct impact on both translation accuracy and efficiency, but one does not yet understand how it affects these two translation variables or whether context biases shape gene evolution. Methodologies/Principal Findings: Here we study codon-context biases using a set of 72 orthologous highly conserved genes from bacteria, archaea, fungi and high eukaryotes to identify 7 distinct groups of codon context rules. We show that synonymous mutations, i.e., neutral mutations that occur in synonymous codons of codon-pairs, are selected to maintain context biases and that non-synonymous mutations, i.e., non-neutral mutations that alter protein amino acid sequences, are also under selective pressure to preserve codon-context biases. Conclusions: Since in vivo studies provide evidence for a role of codon context on decoding fidelity in E. coli and for decoding efficiency in mammalian cells, our data support the hypothesis that, like codon usage, codon context modulates the evolution of gene primary structure and fine tunes the structure of open reading frames for high genome translational fidelity and efficiency in the 3 domains of life. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Deregulation of miRNA expression has been discovered in a wide variety of tumours and it is now clear that they contribute to cancer development and progression. Cervical cancer is one of the most common cancers in women worldwide and there is a strong need for a non-invasive, fast and efficient method to diagnose the disease. We investigated miRNA expression profiles in cervical cancer using a microarray platform containing probes for mature miRNAs. We have evaluated miRNA expression profiles of a heterogeneous set of cervical tissues from 25 different patients. This set included 19 normal cervical tissues, 4 squamous cell carcinoma, 5 high-grade squamous intraepithelial lesion (HSIL) and 9 low-grade squamous intraepithelial lesion (LSIL) samples. We observed high variability in miRNA expression especially among normal cervical samples, which prevented us from obtaining a unique miRNA expression signature for this tumour type. However, deregulated miRNAs were identified in malignant and pre-malignant cervical tissues after tackling the high expression variability observed. We were also able to identify putative target genes of relevant candidate miRNAs. Our results show that miRNA expression shows natural variability among human samples, which complicates miRNA data profiling analysis. However, such expression noise can be filtered and does not prevent the identification of deregulated miRNAs that play a role in the malignant transformation of cervical squamous cells. Deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of this tumour type. [ABSTRACT FROM AUTHOR]

Examines the accumulation of intracellular organic solutes in six species of the order `Thermotogales' by nuclear magnetic resonance spectroscopy. Identification of organic solutes in `T. maritima'; Organic solutes in other members of the order `Thermotogales.'

An abstract of the article "Co-expression of E- and P-cadherin in breast cancer: role as an invasion suppressor or as an invasion promoter?," by Ana S. Ribeiro, Laura C. Carreto, and Fernanda Milanezi is presented.

Brain-derived neurotrophic factor (BDNF) is an important mediator of long-term synaptic potentiation (LTP) in the hippocampus. The local effects of BDNF depend on the activation of translation activity, which requires the delivery of transcripts to the synapse. In this work, we found that neuronal activity regulates the dendritic localization of the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cultured rat hippocampal neurons by stimulating BDNF-Trk signaling. Microarray experiments identified a large number of transcripts that are coimmunoprecipitated with hnRNP K, and about 60% of these transcripts are dissociated from the protein upon stimulation of rat hippocampal neurons with BDNF. In vivo studies also showed a role for TrkB signaling in the dissociation of transcripts from hnRNP K upon high-frequency stimulation (HFS) of medial perforant path-granule cell synapses of male rat dentate gyrus (DG). Furthermore, treatment of rat hippocampal synaptoneurosomes with BDNF decreased the coimmunoprecipitation of hnRNP K with mRNAs coding for glutamate receptor subunits, Ca 2+ - and calmodulin-dependent protein kinase IIβ (CaMKIIβ) and BDNF. Downregulation of hnRNP K impaired the BDNF-induced enhancement of NMDA receptor (NMDAR)-mediated mEPSC, and similar results were obtained upon inhibition of protein synthesis with cycloheximide. The results demonstrate that BDNF regulates specific populations of hnRNP-associated mRNAs in neuronal dendrites and suggests an important role of hnRNP K in BDNF-dependent forms of synaptic plasticity.

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)-VEGF 165 , have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia.

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.

Background: Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate., Results: In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates., Conclusion: We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in sub-telomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome.

Background: The evolutionary forces that determine the arrangement of synonymous codons within open reading frames and fine tune mRNA translation efficiency are not yet understood. In order to tackle this question we have carried out a large scale study of codon-triplet contexts in 11 fungal species to unravel associations or relationships between codons present at the ribosome A-, P- and E-sites during each decoding cycle., Results: Our analysis unveiled high bias within the context of codon-triplets, in particular strong preference for triplets of identical codons. We have also identified a surprisingly large number of codon-triplet combinations that vanished from fungal ORFeomes. Candida albicans exacerbated these features, showed an unbalanced tRNA population for decoding its pool of codons and used near-cognate decoding for a large set of codons, suggesting that unique evolutionary forces shaped the evolution of its ORFeome., Conclusion: We have developed bioinformatics tools for large-scale analysis of codon-triplet contexts. These algorithms identified codon-triplets context biases, allowed for large scale comparative codon-triplet analysis, and identified rules governing codon-triplet context. They could also detect alterations to the standard genetic code.

Background: The discovery of genetic code alterations and expansions in both prokaryotes and eukaryotes abolished the hypothesis of a frozen and universal genetic code and exposed unanticipated flexibility in codon and amino acid assignments. It is now clear that codon identity alterations involve sense and non-sense codons and can occur in organisms with complex genomes and proteomes. However, the biological functions, the molecular mechanisms of evolution and the diversity of genetic code alterations remain largely unknown. In various species of the genus Candida, the leucine CUG codon is decoded as serine by a unique serine tRNA that contains a leucine 5'-CAG-3'anticodon (tRNA(CAG)(Ser)). We are using this codon identity redefinition as a model system to elucidate the evolution of genetic code alterations., Methodology/principal Findings: We have reconstructed the early stages of the Candida genetic code alteration by engineering tRNAs that partially reverted the identity of serine CUG codons back to their standard leucine meaning. Such genetic code manipulation had profound cellular consequences as it exposed important morphological variation, altered gene expression, re-arranged the karyotype, increased cell-cell adhesion and secretion of hydrolytic enzymes., Conclusion/significance: Our study provides the first experimental evidence for an important role of genetic code alterations as generators of phenotypic diversity of high selective potential and supports the hypothesis that they speed up evolution of new phenotypes.

Background: Codon usage and codon-pair context are important gene primary structure features that influence mRNA decoding fidelity. In order to identify general rules that shape codon-pair context and minimize mRNA decoding error, we have carried out a large scale comparative codon-pair context analysis of 119 fully sequenced genomes., Methodologies/principal Findings: We have developed mathematical and software tools for large scale comparative codon-pair context analysis. These methodologies unveiled general and species specific codon-pair context rules that govern evolution of mRNAs in the 3 domains of life. We show that evolution of bacterial and archeal mRNA primary structure is mainly dependent on constraints imposed by the translational machinery, while in eukaryotes DNA methylation and tri-nucleotide repeats impose strong biases on codon-pair context., Conclusions: The data highlight fundamental differences between prokaryotic and eukaryotic mRNA decoding rules, which are partially independent of codon usage.

Alterations to the standard genetic code have been found in both prokaryotes and eukaryotes. This finding demolished the central dogma of molecular biology, postulated by Crick in 1968, of an immutable and universal genetic code and raised the question of how organisms survive genetic code alterations? Recent studies suggest that genetic code alterations are driven by selection using a mechanism that requires translational ambiguity. In C. albicans, the leucine CUG codon is decoded as serine through structural alterations of the translational machinery, in particular, of a Ser-tRNACAG which has dual identity and novel decoding properties. Here, we review the molecular mechanism of CUG reassignment focusing on the structural change of the translational machinery and on the impact that such alteration had on the evolution of the Candida albicans genome.