RNA interference: a new and powerful tool for functional genomic analysis.

In many species, the introduction of double-stranded RNA induces potent and specific gene silencing, referred to as RNA interference. This phenomenon, which is based on targeted degradation of mRNAs and occurs in almost any eukaryote, from trypanosomes to mice including plants and fungi, has sparked general interest from both applied and fundamental standpoints. RNA interference, which is currently used to investigate gene function in a variety of systems, is linked to natural resistance to viruses and transposon silencing, as if it were a primitive immune system involved in genome surveillance. Here, we review the mechanism of RNA interference in post-transcriptional gene silencing, its function in nature, its value for functional genomic analysis, and the modifications and improvements that may make it more efficient and inheritable. We also discuss the future directions of this versatile technique in both fundamental and applied science.

Bacterial membrane injuries induced by lactacin F and nisin

The combined action of nisin and lactacin F, two bacteriocins produced by lactic acid bacteria, is additive. In this report, the basis of this effect is examined. Channels formed by lactacin F were studied by experiments using planar lipid bilayers, and bactericidal effects were analyzed by flow cytometry. Lactacin F produced pores with a conductance of 1 ns in black lipid bilayers in 1 mM KClat 10 mV at 20°C. Pore formation was strongly dependent on voltage. Although lactacin F formed pores at very low potential (10 mV), the dependence was exponentialabov e 40 mV. The injuries induced by nisin and lactacin F in the membranes of Lactobacillus helveticus produced different flow cytometric profiles. Probably, when both bacteriocins are present, each acts separately; their cooperation may be due to an increase in the number of single membrane injuries

Enrichment cultures should be performed in the detection of bacterial oral human pathogens in DUWLs

Water delivered by dental units during routine dental practice is densely contaminated by bacteria. The aim of this study was to determine actual isolation of the microorganisms sprayed from Dental Unit Water Lines (DUWLs) when enrichment cultures are performed and to compare frequencies with those obtained without enrichment cultures. Moreover, the antimicrobial susceptibilities of the microorganisms isolated were also studied. Water samples were collected from one hundred dental equipments in use at Dental Hospital of our University in order to evaluate the presence/absence of microorganisms and to perform their presumptive identification. Aliquots from all of the samples were inoculated in eight different media including both enrichment and selective media. Minimal inhibitory concentrations (MIC) were determined by the broth dilution method. The results herein reported demonstrate that most of the DUWLs were colonized by bacteria from human oral cavity; when enrichment procedures were applied the percentage of DUWLs with detectable human bacteria was one hundred percent. The results showed that in order to evaluate the actual risk of infections spread by DUWLs the inclusion of a step of pre-enrichment should be performed. The need for devices preventing bacterial contamination of DUWLs is a goal to be achieved in the near future that would contribute to maintain safety in dental medical assistance

T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension

This article introduces a new interface for T-Coffee, a consistency-based multiple sequence alignment program. This interface provides an easy and intuitive access to the most popular functionality of the package. These include the default T-Coffee mode for protein and nucleic acid sequences, the M-Coffee mode that allows combining the output of any other aligners, and template-based modes of T-Coffee that deliver high accuracy alignments while using structural or homology derived templates. These three available template modes are Expresso for the alignment of protein with a known 3D-Structure, R-Coffee to align RNA sequences with conserved secondary structures and PSI-Coffee to accurately align distantly related sequences using homology extension. The new server benefits from recent improvements of the T-Coffee algorithm and can align up to 150 sequences as long as 10 000 residues and is available from both http://www.tcoffee.org and its main mirror http://tcoffee.crg.cat.

The UlaG protein familly defines novel structural and functional motifs grafted on an ancient RNase fold

Background: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-b-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent)metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gramnegative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. Conclusions: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events.

Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli.

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli.

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

Bacterial adhesion efficiency on implant abutments: a comparative study

The attachment of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 28213 onto six different materials used to manufacture dental implant abutments was quantitatively determined after 2 and 24 h of contact between the materials and the bacterial cultures. The materials were topographically characterized and their wettability determined, with both parameters subsequently related to bacterial adhesion. Atomic force microscopy, interferometry, and contact angle measurement were used to characterize the materials" surfaces. The results showed that neither roughness nor nano-roughness greatly influenced bacterial attachment whereas wettability strongly correlated with adhesion. After 2 h the degree of E. coli attachment markedly differed depending on the material whereas similar differences were not observed for S. aureus, which yielded consistently higher counts of adhered cells. Nevertheless, after 24 h the adhesion of the two species to the different test materials no longer significantly differed, although on all surfaces the numbers of finally adhered E. coli were higher than those of S. aureus

A highly expressed miR-101 isomiR is a functional silencing small RNA

Background MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5" or 3" of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5"-trimming variant (5"-isomiR-101). Results The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5"-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5"- isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5"-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5"-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5"-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5"-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5"-isomiR-101. Conclusions These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Identification of molecular subtypes and gene expression patterns of breast cancer analysing RNA-seq data

Breast cancer is the most common diagnosed cancer and the leading cause of cancer death among females worldwide. It is considered a highly heterogeneous disease and it must be classified into more homogeneous groups. Hence, the purpose of this study was to classify breast tumors based on variations in gene expression patterns derived from RNA sequencing by using different class discovery methods. 42 breast tumors paired-samples were sequenced by Illumine Genome Analyzer and the data was analyzed and prepared by TopHat2 and htseq-count. As reported previously, breast cancer could be grouped into five main groups known as basal epithelial-like group, HER2 group, normal breast-like group and two Luminal groups with a distinctive expression profile. Classifying breast tumor samples by using PAM50 method, the most common subtype was Luminal B and was significantly associated with ESR1 and ERBB2 high expression. Luminal A subtype had ESR1 and SLC39A6 significant high expression, whereas HER2 subtype had a high expression of ERBB2 and CNNE1 genes and low luminal epithelial gene expression. Basal-like and normal-like subtypes were associated with low expression of ESR1, PgR and HER2, and had significant high expression of cytokeratins 5 and 17. Our results were similar compared with TGCA breast cancer data results and with known studies related with breast cancer classification. Classifying breast tumors could add significant prognostic and predictive information to standard parameters, and moreover, identify marker genes for each subtype to find a better therapy for patients with breast cancer.

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis

Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era

Ralstonia solanacearum is a soil-borne bacterium causing the widespread disease known as bacterial wilt. Ralstonia solanacearum is also the causal agent of Moko disease of banana and brown rot of potato. Since the last R. solanacearum pathogen profile was published 10 years ago, studies concerning this plant pathogen have taken a genomic and post-genomic direction. This was pioneered by the first sequenced and annotated genome for a major plant bacterial pathogen and followed by many more genomes in subsequent years. All molecular features studied now have a genomic flavour. In the future, this will help in connecting the classical field of pathology and diversity studies with the gene content of specific strains. In this review, we summarize the recent research on this bacterial pathogen, including strain classification, host range, pathogenicity determinants, regulation of virulence genes, type III effector repertoire, effector-triggered immunity, plant signalling in response to R. solanacearum, as well as a review of different new pathosystems.