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Background: RNA post-transcriptional modification is an exciting field of research that has evidenced this editing process as a sophisticated epigenetic mechanism to fine tune the ribosome function and to control gene expression. Although tRNA modifications seem to be more relevant for the ribosome function and cell physiology as a whole, some rRNA modifications have also been seen to play pivotal roles, essentially those located in central ribosome regions. RNA methylation at nucleobases and ribose moieties of nucleotides appear to frequently modulate its chemistry and structure. RNA methyltransferases comprise a superfamily of highly specialized enzymes that accomplish a wide variety of modifications. These enzymes exhibit a poor degree of sequence similarity in spite of using a common reaction cofactor and modifying the same substrate type. Results: Relationships and lineages of RNA methyltransferases have been extensively discussed, but no consensus has been reached. To shed light on this topic, we performed amino acid and codon-based sequence analyses to determine phylogenetic relationships and molecular evolution. We found that most Class I RNA MTases are evolutionarily related to protein and cofactor/vitamin biosynthesis methyltransferases. Additionally, we found that at least nine lineages explain the diversity of RNA MTases. We evidenced that RNA methyltransferases have high content of polar and positively charged amino acid, which coincides with the electrochemistry of their substrates. Conclusions: After studying almost 12,000 bacterial genomes and 2,000 patho-pangenomes, we revealed that molecular evolution of Class I methyltransferases matches the different rates of synonymous and non-synonymous substitutions along the coding region. Consequently, evolution on Class I methyltransferases selects against amino acid changes affecting the structure conformation COL0014064

Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104 microconidia/mL at 37°C over expressed many more proteins than other treatments.

Background: Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. Results: Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. Conclusions: The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health.

Isobaric tags for relative and absolute quantification of protein expression (iTRAQ®) is a powerful tool which is combined with the accuracy of Mass Spectrometry for protein identification. This tool was approached to detect proteins associated to the innate immune system of Galleria mellonella in response to pathogenesis caused by Fusarium oxysporum. After experimental approaches, iTRAQ data was used to set up computational analysis based on identification and quantification of peptides and proteins against different protein databases by using ProteinPilotTM and Mascot Distiller search engines. iTRAQ battery was able to identify more than 340 peptides corresponding to 39 putative proteins from G. mellonella and close related species. Despite the low level of genomic and proteomic information available for G. mellonella, iTRAQ demonstrated to be reliable strategy to determine changes in protein expression as a consequence of the infection process induced in G. mellonella. Consequently, it was found differential expression in proteins directly involved in innate immune response such as cecropin-D-like peptide, lysozyme, and hemolin, indicating an active response of G. mellonella in early stages of fungal infection.

Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m 7 G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m 7 G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning.After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG , we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance.To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains.We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance.We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function.

Objective: The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemia following dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing. Methods: The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venous blood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples were analysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well as pyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene. Results: Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at 30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five blood samples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods, a single microbial species was detected in six patients, and Streptococcus viridans was the most frequently cultured identified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dental extractions was 13.4 +/- 1.7 bacterial families and 22.8 +/- 1.1 genera per sample. Conclusion: The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemia following dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However, this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterial diversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of an iceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied.

Objective:The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemiafollowing dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing.Methods:The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venousblood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples wereanalysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well aspyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene.Results:Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five bloodsamples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods,a single microbial species was detected in six patients, andStreptococcus viridanswas the most frequently culturedidentified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dentalextractions was 13.461.7 bacterial families and 22.861.1 genera per sample.Conclusion:The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemiafollowing dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However,this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterialdiversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of aniceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied. This work was supported by projects SAF2009-13032-C02-02, Consolider-Ingenio CSD2009-00006 from the Spanish Ministry of Science and Innovation and by project FIS 2011/PF004 from Spanish Institute of Health “Carlos III” SI

A Colombian population was typed for 12 STRs by silver stain method. Initial routine was extended from 9 to 12 STR loci for increasing forensic values in paternity testing, new multiplex developed decreased significantly Probability of Match (PM) and increased power of exclusion. Kodak 1D Image Analysis Software was used for assigning allele out-ladders with analysis of their correct molecular sizes.

Modifying RNA enzymes are highly specific for substrate—rRNA or tRNA—and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m2A synthesis at purine 37 in a tRNA set still remains unknown. m2A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m2A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m2A37 synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m2A37 from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m2A from 23S rRNA and, accordingly, that the m2A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2′-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeuCmAA and tRNALeucmnm5UmAA. Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2′-O-methylation at position 34 in both tRNALeuCmAA and tRNALeucmnm5UmAA. Loss of YibK methylation reduces the efficiency of codon–wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2′-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N6-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.

La bioinformática, como la conocemos hoy en día, es una de las ciencias con mayor proyección en la adquisición de conocimiento científico. Contrario a lo que se piensa, esta ciencia tuvo sus inicios en los años 50, pero no fue sino hasta hace un par de décadas cuando tuvo su verdadero auge con la creación de las primeras bases de datos y el desarrollo de algoritmos computacionales diseñados para el análisis de secuencia. El desarrollo científico y tecnológico alcanzado a nivel mundial, constantemente nos lleva a evaluar las posibilidades de transferencia de esos logros a nuestra sociedad científica. En este punto, la bioinformática se ha sumado a la larga lista de retrasos científicos de los cuales padece constantemente nuestra sociedad. No obstante, las nuevas proyecciones y políticas de investigación y desarrollo logradas recientemente en nuestro país, han abierto definitivamente el camino para la aplicación y desarrollo de la bioinformática, la cual durante años ha sido mantenida tímidamente como objeto de estudio por pocos grupos de investigación de nuestro país. Una vez fueron propuestos los actuales modelos de sostenimiento y desarrollo con base biotecnológica, se ha dado un salto generacional en la investigación computacional en Colombia y nuestros objetivos científicos, aunque a largo plazo, están trazados al mismo nivel de países como Chile, Argentina, Brasil y México, que son los referentes inmediatos para la región. En este breve ensayo queremos resaltar la situación actual en la investigación bioinformática llevada a cabo en nuestro país, así como también, plantear las perspectivas que se esperan para esta ciencia de gran impacto a nivel mundial. Bioinformatics emerged about 50 years ago, but it was developed greatly during the early 1980’s by robust databases such as GenBank, EMBL, and DNA Database of Japan (DDBJ). Bioinformatic routines were rapidly adapted once the main algorithms for sequence analysis became available worldwide. As in other science fields, bioinformatics had minimal impact in low-income countries of Latin America until the last decade. We revised the bioinformatics state of art in Colombia and found a few bioinformatics groups carrying out basic computational biology research. Nowadays, bioinformatics in Colombia has a hopeful scenario thanks to recent science policies adopted by the Colombian Government. Such policies have been adopted in order to establish a new model of sustainable scientific research. In this brief report we revise the bioinformatics state of the art in Colombia. Finally, we conclude with some considerations for the proposed science model and we describe different perspectives of interest for the Colombian scientific community.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

Bioinformatics emerged about 50 years ago, but it was developed greatly during the early 1980s by robust databases such as GenBank, EMBL, and DNA Database of Japan (DDBJ). Bioinformatic routines were rapidly adapted once the main algorithms for sequence analysis became available worldwide. As in other science fields, bioinformatics had minimal impact in low-income countries of Latin America until the last decade. We revised the bioinformatics state of art in Colombia and found a few bioinformatics groups carrying out basic computational biology research. Nowadays, bioinformatics in Colombia has a hopeful scenario thanks to recent science policies adopted by the Colombian Government. Such policies have been adopted in order to establish a new model of sustainable scientific research. In this brief report we revise the bioinformatics state of the art in Colombia. Finally, we conclude with some considerations for the proposed science model and we describe different perspectives of interest for the Colombian scientific community.

Type I and type-II functional divergences have been stated to highlight specific residues carrying out differential functions in evolutionary-divergent protein clusters from a single common ancestor. Briefly, type I analysis is based on residue constraints reflecting a gain of function just in one cluster of an entire family of proteins; while the type-II approach is based on residue constraints showing a different chemical nature in every cluster of a protein family. This last evidence is understood as differential functionality among clusters. The Receptor Activity-Modifying Proteins constitute a family characterized by its paralogous distribution in vertebrates. They are known as G-Protein Coupled Receptor modulators. Although several studies have determined their involvement in ligand binding, specificity, and enhancement of signal transduction, the responsible residues supporting those functions are unclear. Using different bioinformatic approaches, we predicted residues involved in different RAMP functional tasks. Many residues localized in an extracellular coil of RAMP proteins were predicted to be under functional divergence suggesting a gain of function in their respective proteins. Interestingly, the transmembrane region also showed important results for residues playing relevant roles where most of them showed a biased distribution on the structure. A relevant role was conferred by the enrichment of type-II residues observed in their sequences. We show a collection of residues explaining possible gain of function and differential functionality in RAMP proteins. These residues are still experimentally unexplored with regards to functionality. Finally, an evolutionary history could be discerned. Mainly, the RAMP2 cluster has evolved in a higher manner than other RAMP clusters. However, a deacceleration in the aminoacid substitution rate of RAMP2 was observed in mammals. Such effect could be caused by the co-evolution of ligands and receptors interacting with RAMP2 through evolution and/or the specialization of this cluster in GPCR modulation.

The Receptor Activity-Modifying Proteins (RAMP) is a family constituted by a single N-terminal extracellular domain and a transmembrane region ending in a short cytoplasmic region. Due to their specific role in modulating the specificity of ligand binding in many class II G-Protein Coupled Receptors, these proteins are awaiting further characterization and elucidation of their structure. This was the aim of this study. We were able to find 13 new RAMP sequences including new protein sequences and predicted peptides from Expressed Sequence Tags and genomic DNA, all of them annotated in databases such as GeneBank, EMBL, Swissprot and ENSEMBL. The predicted peptides came from an array of different organisms including Teleostei and Elasmobranchii species, of which the latter was the most ancient RAMP sequence found. It was also possible to efficiently predict the 1D structure of the extracellular RAMP domain and its 3D conformation was inferred through a combination of bioinformatic approaches such as threading. The 1D structure of the extracellular RAMP domain was predicted as three alpha-helix domain. The most highly conserved residues in the RAMP family were found to be involved in critical functions. Bioinformatic data mining and multiple sequence alignment analysis were crucial for improving the characterization of RAMP proteins and prediction of their 1D and 3D configurations.

Three hundred and twenty-six to four hundred and fifty-one individuals were typed for 12 STR markers and revealed by silver stain method and assisted by a computer analysis. Forensic and Hardy-Weinberg values were found. The Kodak ds 1D Image Analysis Software V 2.0.3 EDAS DC 120 System was a useful tool for determining the correct size of pattern bands that were out allelic ladders, alleles 18 and 17 from F13A01, 15 from TPOX and 4 from TH01 were determined by this method and some corroborated by automatic analysis.

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2 (TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2 (TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems. © 2003 Elsevier Ireland Ltd. All rights reserved.

Introduction: Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m 7 G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m 7 G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Objectives: After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG , we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance. Materials and methods: To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains. Results: We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance. Conclusion: We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function. Introducción. Los aminoglucósidos son moléculas antibióticas capaces de inhibir la síntesis de proteínas bacterianas tras su unión al ribosoma procariota. La resistencia a aminoglucósidos está clásicamente asociada a mutaciones en genes estructurales del ribosoma bacteriano; sin embargo, varios estudios recientes han demostrado, de forma recurrente, la presencia de un nuevo mecanismo dependiente de mutación que no involucra genes estructurales. El gen rsmG es uno de ellos y se caracteriza por codificar una metiltransferasa que sintetiza el nucleósido m 7 G527 localizado en el loop 530 del ribosoma bacteriano, este último caracterizado como sitio preferencial al cual se une la estreptomicina. Objetivo. Partiendo de las recientes asociaciones clínicas entre las mutaciones en el gen rsmG y la resistencia a estreptomicina, este estudio se propuso la caracterización de nuevos puntos calientes de mutación en este gen que puedan causar resistencia a estreptomicina usando Escherichia coli como modelo de estudio. Materiales y métodos. Se indagó sobre el mecanismo genético y molecular por el cual se adquiere la resistencia a estreptomicina y su transición a la resistencia a altas dosis mediante mutagénesis dirigida del gen rsmG y genotipificación del gen rpsL . Resultados. Se encontró que la mutación N39A en rsmG inactiva la proteína y se reportó un nuevo conjunto de mutaciones en rpsL que confieren resistencia a altas dosis de estreptomicina. Conclusiones. Aunque los mecanismos genéticos subyacentes permanecen sin esclarecer, se concluyó que dichos patrones secuenciales de mutación podrían tener lugar en otros genes modificadores del ARN bacteriano debido a la conservación evolutiva y al papel crítico que juegan tales modificaciones en la síntesis de proteínas.