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INTRODUCTION: Species A rotavirus (RVA) infections are a major cause of severe gastroenteritis in children of <5 years worldwide. In Brazil, before vaccination, RVA was associated with 3.5 million episodes of acute diarrheal disease per year. Due to the segmented nature of their genomes, rotaviruses can exchange genes during co-infections, and generate new virus strains and new reinfections. OBJECTIVE: To evaluate the genomic diversity of RVA isolated in Brazil in 30 years, between 1986 to 2016, to investigate possible changes in the frequency of genotype constellations before and after the implementation of the vaccine. METHODS: In total, 4,474 nucleotide sequences were obtained from the Virus Variation Database. Genomic constellation was compared, and the proportion of rotavirus genotypes was analyzed by time and geographic region. RESULTS: Our results showed that major known genotypes were circulating in the country during the period under analysis, with a prevalence of the G1P[8] Wa-like genotype, decreasing only in the period immediately after the introduction of the vaccine. Regarding the geographical distribution, most of our constellations, 62 (39.2%), and 50 (31.6%) were concentrated in the North and Northeast regions. Our analysis also showed the circulation of multiple strains during the periods when the DS-1-like and AU-1-like genotypes were co-circulating with the Wa-like genotype. CONCLUSION: Therefore, it is likely that inter-genogroup reassortments are still occurring in Brazil and so it is important to establish an efficient surveillance system to follow the emergence of novel reassorted strains that might not be targeted by the vaccine.
INTRODUÇÃO: As infecções por rotavírus A (RVA) são uma das principais causas de gastroenterite grave em crianças <5 anos em todo o mundo. No Brasil, antes da vacinação, o RVA estava associado a 3,5 milhões de episódios de diarreia aguda por ano. Devido à natureza segmentada de seus genomas, os rotavírus podem trocar genes durante as coinfecções, gerar novas cepas de vírus e novas reinfecções. OBJETIVO: Avaliar a diversidade genômica de RVA isolados no Brasil entre 1986 a 2016 para investigar possíveis alterações na frequência das constelações de genótipos antes e após a implantação da vacina. MÉTODOS: No total, 4.474 sequências de nucleotídeos foram obtidas do Banco de Dados de Variação de Vírus. A constelação genômica foi comparada e a proporção dos genótipos de rotavírus foi analisada por tempo e região geográfica. RESULTADOS: Nossos resultados mostraram que os principais genótipos conhecidos circulavam no país no período em análise, com prevalência do genótipo G1P[8] Wa-like, diminuindo apenas no período imediatamente após a introdução da vacina. Em relação à distribuição geográfica, a maioria das nossas constelações, 62 (39,2%) e 50 (31,6%), concentrava-se nas regiões Norte e Nordeste. Nossa análise também mostrou a circulação de cepas múltiplas durante os períodos em que os genótipos DS-1-like e AU-1-like estavam co-circulando com o genótipo Wa-like. CONCLUSÃO: Portanto, é provável que rearranjos inter-genogrupos ainda estejam ocorrendo no Brasil e por isso é importante estabelecer um sistema de vigilância eficiente para acompanhar o surgimento de novas cepas rearranjadas que podem não ser protegidas pela vacina.
Subject(s)
Phylogeny , Gene Rearrangement , Genome , Rotavirus/genetics , Rotavirus VaccinesABSTRACT
Thirteen microsatellite loci were isolated and characterized in Microglanis cottoides. Of these, two were monomorphic and 11 were polymorphic. These polymorphic loci tested on 24 individuals from a wild population produced a total of 108 different alleles, with levels of variability high, ranging from 2 to 20, with an average of 8.3 alleles per locus. The observed and expected heterozygosity ranged from 0.125 to 0.958 and from 0.119 to 0.931, respectively. A high combined probability of paternity exclusion value and a low probability combined genetic identity value obtained show that the set of loci described herein displays good suitability for paternity studies and differentiation of M. cottoides. Additionally, all thirteen microsatellite primers developed for M. cottoides were tested in four other Pseudopimelodidae species and successful cross-species amplification was achieved for the majority of loci.
Treze loci microssatélites foram isolados e caracterizados em Microglanis cottoides. Destes, dois foram monomórficos e 11 foram polimórficos. Estes loci polimórficos foram testados em 24 indivíduos de uma população selvagem e produziram um total de 108 alelos diferentes, com níveis de variabilidade alta, variando de 2 a 20, com uma média de 8,3 alelos por locus. A heterozigosidade observada e esperada variou de 0,125 a 0,958 e 0,119 a 0,931, respectivamente. Um elevado valor de exclusão de paternidade e um baixo valor de identidade genética foram obtidos, demostrando que o conjunto de loci descritos no presente trabalho exibe boa aplicabilidade no estudo de parentesco e diferenciação populacional em M. cottoides. Adicionalmente, os treze primers de microssatélites desenvolvidos para M. cottoides foram testados em outras quatro espécies de Pseudopimelodidae e a transferabilidade foi obtida para a maioria dos loci.
Subject(s)
Animals , Animal Population Groups/classification , Loss of Heterozygosity , Polymorphism, Genetic , Fishes/classificationABSTRACT
Objective To screen down regulated genes and find down regulated novel genes in gastric cancer. Methods Genes mRNA expression were detected between gastric cancer and normal gastric mucous membrane of five patients using cDNA microarray. Genes mRNA expression signals on hybridization membranes were analysized with computer software. Down regulated genes in gastric cancer were screened. cDNA suppression subtraction library was established by counterpart normal gastric mucous membrane mRNA(Tester) subtracting gastric cancer tissues mRNA(Driver) of five patients. After identification of the subtraction library, positive clones choosen randomly were sequenced , and down regulated novel genes in gastric cancer were screened. Some of the genes were identified by RT PCR.Results Down regulated genes in gastric cancer consist of 60 genes including tumor suppressing genes, apoptosis related genes, DNA replication and transcript or translate related genes, cell cycle related genes, cell migration related genes, etc. Two unknown gene fragments in gastric cancer were cloned. Conclusions Sixty down regulated genes in gastric cancer are confirmed. They are involved in gastric tumorigenicity and metastasis. cDNA subtraction library of gastric cancer was constructed successfully. Two down regulated novel gene fragments in gastric cancer was found.
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To screen specific DNA probes by double hybridization from the constructed DNA li- brary of serotype A Cryptococcus neoformans.On the basis of the nucleotide sequence of vector pUC18, a pair of primers was synthesized.The insert fragments were amplified from the library on a PCR pro- cessor.The PCR products were spotted onto hybond-N~+ membranes.All inserts amplified from the ge- nomic library by PCR were screened by dot blot with 26 ~(32)P-labelled DNA from infectious agents for dif- ferentiation and from healthy persons.Twenty-eight candidate clones were obtained.The twenty-eight clone inserts got from low melting point agar were further characterized by dot blot with above 27 kinds of DNA for differentiation.Three specific DNA probes from the library of serotype A Cryptococcus neo- formans were obtained.Colony pCNIIA6 was serotype A-specific,which gave signals only with sertype A strain and did not cross hybridize with other DNAs.Colony pCNIIB5 was species-specific which gave signals only with DNA from Cryptococcus neoformans.Colony pCNIIIG1 was specific for var.neofor- mans,which gave signals only with serotype A and D strains.We can make a rapid diagnosis of Crypto- coccus neoformans infection at an early stage and distinguish the variants of C.neoformans and C.gattii using above specific probes.
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With specific primers designed,the aimed DNA fragment was amplified by PCR from aniline-degrading strain ANA5.A genomic library of strain ANA5 was constructed in the cosmid vector pLAFR3 using E.coli EPI100 as the host strain.Two recombinants were identified from the genomic library using in situ hybridization and Southern blotting probed with the PCR product.By the analysis of subclone sequencing,it was sure that the aniline dioxygenase gene of strain ANA5 was cloned.The sequence analysis and the deduced amino acid showed that they were different from the relative sequences registered in the GenBank and revealed that aniline dioxygenase gene was conserved through evolution.
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An acidic xylanase gene,named xyl3,was cloned from the genomic library of enviromental microbes constructed by using shotgun cloning strategy,and submitted to GeneBank with accession number of gb:AY300805.BLAST analysis indicated that the gene xyl3 has low similarity with other xylanase genes and the encoded xylanase,sorted as Glycosyl hydrolases family 10,has 77% similarity with the intra-cellular xylanase from Geobacillus stearothermophilus at amino acid level.Treated with T4 DNA polymerase,the gene xyl3 was ligated with the linearized Pichia pastoris expression vector pHBM905 produced by digestion of restriction endonuclease CpoI and NotI to generate the recombinant plasmid pHBM706.Then the plasmid pHBM706,digested by restriction endonuclease SalI,was transformed into P.pastoris GS115 to obtain the recombinant P.pastoris GS115(pHBM706),which was induced to produce the recombinant xylanase with 0.5% methanol at 28℃.At the 36th hours of induction,the porduced crude enzyme was detected to reach the higest enzyme activity of 0.177 IU/mL.The optimal pH and temperature of the enzyme activity is 5.5 and 50℃ respectively.
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The polymerase chain reaction was used to develop a method for the detection of Helicobacter pylori, a causative agent of gastritis, as well as for the elucidation of its mode of transmission. A genomic library of Helicobacter pylori DNA in Escherichia coli JM109 was constructed by cloning Hind III-digested DNA fragments into plasmid vector pUC18. The nucleotide sequences from seven recombinant clones were determined and five sets of oligonucleotide primers were synthesized on the basis of the sequences from five clones (B4, B9, B10, C15 and I22). The PCR amplifications with these primers were performed using DNA samples from five strains of Helicobacter pylori, two Campylobacter spp. and eleven species of enteric bacteria. Amplifications of the target DNA fragments in all of 5 strains of Helicobacter pylori were observed from the PCR with primers derived from clone B4, B9, C15 and I22. When the specificity was checked with the DNA samples from 13 other bacteria as template DNA for the PCR, specific amplification that produced the correct size of the target DNA of Helicobacter pylori was shown only in the PCR with primers derived from clone B9 and C15. The detection limit in the PCR amplification, determined by the heat-lysis method, was 500 cells of Helicobacter pylori.