Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

Mutation screening of the sodium iodide symporter gene in a cohort of 105 China patients with congenital hypothyroidism / Análise de mutações no gene simportador sódio/iodeto em coorte de 105 pacientes chineses com hipotireoidismo congênito

Objective Dyshormonogenetic congenital hypothyroidism (CH) was reported to be associated with a mutation in the sodium iodide symporter (NIS) gene. The present study was undertaken in the Guangxi Zhuang Autonomous Region of China, to determine the nature and frequency of NIS gene mutations among patients with CH due to dyshormonogenesis. Subjects and methods: Blood samples were collected from 105 dyshormonogenetic CH patients in Guangxi Zhuang Autonomous Region, China, and genomic DNA was extracted from peripheral blood leukocytes. All exons of the NIS gene together with their exon-intron boundaries were screened by next-generation sequencing. Results Two silent variations (T221T and T557T) and one missense variation (M435L), as well as two polymorphisms (rs200587561 and rs117626343) were found. Conclusions Our results indicate that the NIS mutation rate is very low in the Guangxi Zhuang Autonomous Region, China, and it is necessary to study mutations of other genes that have major effects on thyroid dyshormonogenesis and have not as yet been studied in this population. .

Objetivo O hipotireoidismo congênito disormonogenético (CH) foi relatado como associado a uma mutação no gene simportador sódio/iodeto (NIS). O presente estudo foi feito na região autônoma de Guangxi Zhuang na China para se determinar a natureza e a frequência das mutações no gene NIS entre pacientes com CH causado por disormonogênese. Sujeitos e métodos: Amostras de sangue foram coletadas de 105 pacientes com CH disormonogenéticos e o DNA genômico foi extraído de leucócitos do sangue periférico. Todos os éxons do gene NIS, junto com seus limites éxon-íntron, foram analisados por sequenciamento de nova geração. Resultados Foram encontradas duas variações silenciosas (T221T e T557T) e uma variação missense (M435L), assim como dois polimorfismos (rs200587561 e rs117626343). Conclusões Nossos resultados indicam que a taxa de mutação em NIS é muito baixa na região de Guangxi Zhuang. É necessário estudar mutações de outros genes que tenham efeitos maiores na disormonogênese da tiroide e que ainda não tenham sido estudados nesta população. .

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Molecular cloning, expression and characterization of a serine proteinase from Japanese edible mushroom, Grifola frondosa: solving the structure - function anomaly of a reported aminopeptidase

The N-terminal amino acid sequence of an aminopeptidase from Japanese edible mushroom, Grifola frondosa, was reported to have high similarity with that of a serine proteinase from basidiomycete, Agaricus bisporous (Nishiwaki and Hayashi, 2001). The full-length cDNA and the corresponding genomic DNA of the enzyme were cloned, based on the reported N-terminal amino acid sequence. The predicted open reading frame (ORF) of the cloned cDNA, encoding a product of 379 amino acids, was expressed in E. coli using pET expression vector. The expressed pro-enzyme (40 kDa) underwent autolysis to produce the mature protein (30 kDa) and a pro-peptide (10 kDa). The mature protein and the pro-peptide remained tightly bound to each other and could not be separated by Ni-NTA metal affinity chromatography or Q-Sepharose ion-exchange chromatography. The enzyme was inactive in the bound form. Upon treatment with subtilisin, the bound pro-peptide was further hydrolyzed and a high serine proteinase activity was recovered. No aminopeptidase activity was detected at any stage of the protein processing. These results clearly indicated that the N-terminal amino acid sequence and the function of the reported aminopeptidase were not derived from the same protein entity and hence caused the structure-function anomaly.

Influencia de las variables preanalíticas en la determinación de proteína S / Influence of preanalytical variables in S protein determination

La proteína S (PS) regula el sistema de coagulación mostrando actividad de cofactor de la Proteína C activada (PCa) con la cual forma un complejo equimolecular. En presencia de iones calcio este complejo inactiva por proteólisis los factores V y VIII activados por trombina. La proteína S plasmática circula 40% libre (fracción que presenta actividad de cofactor de la PCa) y 60% unida al C4-BP (proteína ligante de la fracción C4 del complemento). El objetivo fue comparar el dosaje de PS realizado por método coagulable e inmunoturbidimétrico e investigar cómo las variables preanalíticas afectan los niveles de PS determinados. Se obtuvieron los siguientes resultados: método coagulable: CV intra ensayo: (n=20): 4%, CV interensayo (n=20, 3 días): 3,4%. Método inmunoturbidimétrico: CV intraensayo (n=20): 3,7%.CV Inter. ensayo (n=20,3 días): 4,5%. Existe buena correlación (R2=0,94) entre ambos métodos, cuando la calibración por el método coagulable se realiza en la misma corrida analítica que las muestras. Cuando se realizó el estudio de Bland y Altman los dos métodos mostraron ser comparables en todos los niveles de PS estudiados. No se observaron diferencias significativas entre las muestras determinadas frescas y conservadas a -20 y -80 °C descongeladas solo una vez.

Protein S has an essential anticoagulant function acting as activated Protein C cofactor and forming an equimolecular complex with it. In the presence of calcium this complex regulates the coagulation process inactivating thrombin activated factors V and VIII by proteolysis. In plasma there are two different forms: a) free Protein S which acts as the cofactor of activated protein C (representing about 40% of total Protein S) and b) C4-BP(C4 binding protein) bound protein S which exhibits no activity as cofactor of activated Protein C (representing about 60% of total PS). The objetive was to compare the PS dosage determination by two methods: immunoturbidimetric and clotting, and to investigate how pre-analytical variables affect the results. The following results were obtained: Clotting method: CV intra assay: (n=20): 4%, CV interassay (n=20, 3 days): 3.4%; immunoturbidimetric method CV intra assay (n=20): 3.7%: CV inter assay (n=20.3 days): 4.5%. There is a good correlation (R2 = 0.94) between both methods; when the clotting method is calibrated in batch with the samples. There is significant difference between fresh and frozen ( -20 °C and -80 °C) samples when the latter have been desfrozen only once.

Protein local conformations arise from a mixture of Gaussian distributions.

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures,which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF's corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end,we have analyzed the correlation between sequence features and the conformational classes.

La proteína de estrés, heat shock cognate (HSC70)se sobreexpresa en osteoblastos sometidos a centrifugación / Overexpression heat shock cognate(HSC70)protein in osteoblasts submited to centrifugal forces

El osteoblasto tiene la capacidad de estimular la neoformación del tejido óseo, mediante la síntesis de proteínas tales como las proteínas morfogenéticas, factores de crecimiento y proteínas colágenas y no colágenas. A su vez, indirectamente controla los procesos de reabsorción sintetizando otra serie de proteínas estimuladoras de la actividad osteoclastogénica

PDB-Metrics: a web tool for exploring the PDB contents

PDB-Metrics (http://sms.cbi.cnptia.embrapa.br/SMS/pdb_metrics/index.html) is a component of the Diamond STING suite of programs for the analysis of protein sequence, structure and function. It summarizes the characteristics of the collection of protein structure descriptions deposited in the Protein Data Bank (PDB) and provides a Web interface to search and browse the PDB, using a variety of alternative criteria. PDB-Metrics is a powerful tool for bioinformaticians to examine the data span in the PDB from several perspectives. Although other Web sites offer some similar resources to explore the PDB contents, PDB-Metrics is among those with the most complete set of such facilities, integrated into a single Web site. This program has been developed using SQLite, a C library that provides all the query facilities of a database management system

A distributed computation of Interpro Pfam, PROSITE and ProDom for protein annotation

Interpro is a widely used tool for protein annotation in genome sequencing projects, demanding a large amount of computation and representing a huge time-consuming step. We present a strategy to execute programs using databases Pfam, PROSITE and ProDom of Interpro in a distributed environment using a Java-based messaging system. We developed a two-layer scheduling architecture of the distributed infrastructure. Then, we made experiments and analyzed the results. Our distributed system gave much better results than Interpro Pfam, PROSITE and ProDom running in a centralized platform. This approach seems to be appropriate and promising for highly demanding computational tools used for biological applications.

Identification of sequence similarities between Mycobacterium leprae and the myelin proteolipid protein by computational analysis.

This study was undertaken under the assumption that antigenic mimicry plays a role in the pathogenesis of neuropathy in leprosy, a unique feature among mycobacterial diseases. The SWISS-PROT protein sequence databank was scanned using a computer programme based on an identity matrix algorithm, to identify common amino acid regions between human myelin and mycobacterial proteins. The highlighted motifs were back-tested against a database of MHC-binding peptides (MHCPEP). Of the 28 common sequences between mycobacterial and human myelin proteins, only two were found to yield some matches with MHC-presenting peptides. Both motifs were from M.leprae. The myelin proteolipid protein was the human protein containing the identified similarities. We believe that this theoretical approach can provide a way to predict potentially "mimetic" motifs by search for antigenic regions in protein sequence databases without screening a large number of synthetic peptides.