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1.
Biophys Chem ; 256: 106270, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706136

RESUMO

DNA strands can be designed to assemble into stable three-dimensional structures, based on Watson-Crick base pairing rules. The simplest of these is the DNA tetrahedron that is composed of four oligonucleotides. We have re-designed the sequence of a DNA tetrahedron so that it contains a single (AATT) binding site for the minor groove binding ligand Hoechst 33258. We examined the stability of this structure by placing fluorescent groups within each of its edges and have shown that all the edges melt at the same temperature in the absence of the ligand. The minor groove ligand still binds to its recognition sequence within the tetrahedron and increases the melting temperature of the folded complex. This ligand-induced stabilisation is propagated into the adjacent helical arms and the tetrahedron melts as a single entity in a cooperative fashion.


Assuntos
DNA/química , Ligantes , Sequência de Bases , Sítios de Ligação , Bisbenzimidazol/química , Bisbenzimidazol/metabolismo , Conformação de Ácido Nucleico , Transição de Fase/efeitos da radiação , Espectrometria de Fluorescência , Temperatura de Transição , Raios Ultravioleta
2.
Biosci Biotechnol Biochem ; 84(1): 53-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31483222

RESUMO

Large numbers of miRNAs are found in biofluid exosomes. We isolated ~50-200 nm diameter exosomes from four types of porcine biofluid (urine, plasma, semen, and bile) using serial centrifugation and ultracentrifugation procedures. A total of 42.15 M raw data were generated from four small RNA libraries. This produced 40.17 M map-able sequences, of which we identified 204 conserved miRNAs, and 190 novel candidate miRNAs. Furthermore, we identified 34 miRNAs specifically expressed in only one library, all with well-characterized immune-related functions. A set of five universally abundant miRNAs (miR-148a-3p, miR-21-5p, let-7f-5p, let-7i-5p, and miR-99a-5p) across all four biofluids was also found. Function enrichment analysis revealed that the target genes of the five ubiquitous miRNAs are primarily involved in immune and RNA metabolic processes. In summary, our findings suggest that porcine biofluid exosomes contain a large number of miRNAs, many of which may be crucial regulators of the immune system.


Assuntos
Secreções Corporais , Líquidos Corporais , Exossomos/genética , Imunidade Inata , MicroRNAs/genética , MicroRNAs/imunologia , Suínos/genética , Animais , Sequência de Bases/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia de Força Atômica , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
3.
Biosci Biotechnol Biochem ; 84(1): 43-52, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31495297

RESUMO

To date, studies on the application of loop-mediated isothermal amplification (LAMP) in the detection of genetically modified organisms (GMOs) are stably increasing and demonstrates LAMP is a potential and promising method for on spot identification of GMOs. However, little information is known for detection of GM potato events by LAMP. In this report, we developed an optimized and visual LAMP assay with high specificity and sensitivity to rapidly amplify genomic DNA of potato EH92-527-1 within 45 min. The limit of detection of LAMP in our study is 10-fold higher than the conventional PCR. Furthermore, LAMP products can be directly observed via naked eyes by addition of SYBR Green I without gel electrophoresis analysis and PCR-based equipment. Therefore, the LAMP assay developed in this paper provides an efficient, convenient and cost-effective tool for the detection of GM potato EH92-527-1.


Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/genética , Sequência de Bases/genética , Percepção de Cores , Primers do DNA/genética , Enzimas de Restrição do DNA/genética , Eletroforese em Gel de Ágar , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Amplificação de Genes , Limite de Detecção , Compostos Orgânicos/química , Reação em Cadeia da Polimerase/economia , Sensibilidade e Especificidade , Temperatura Ambiente , Tempo
4.
Insect Sci ; 27(1): 143-158, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29873880

RESUMO

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Primers do DNA/análise , Tephritidae/classificação , Animais , Ásia Sudeste , Austrália , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/análise , Proteínas de Insetos/análise , Masculino , Ilhas do Pacífico , Filogenia , Tephritidae/genética
5.
Zootaxa ; 4695(6): zootaxa.4695.6.1, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31719322

RESUMO

Sparsorythus is a genus of Tricorythidae from the Oriental Region. Sparsorythus multilabeculatus Sroka Soldán, 2008 was described based on a male imago from Vietnam. Unknown nymphs and female subimagines of Sparsorythus and male imagines of S. multilabeculatus were collected from Thap Lan National Park, Khon Buri District, Nakhon Ratchasima Province, Thailand. Nymphs and female subimagines of Sparsorythus were associated with male imagines of S. multilabeculatus by analyzing sequences from the DNA barcoding region of the mitochrondrial gene cytochrome oxidase I. Phylogenetic analysis based on Maximum Likelihood indicated that all unknown specimens are conspecific with male imagines of S. multilabeculatus (bootstrap 100% and genetic distance 0-0.004). Male and female nymphs, female subimago and egg are described for the first time. Nymphs each bear a medial emargination on the hypopharynx, one bristle-like process at the base of the left prostheca, and a bifurcate rudimentary gill on abdominal segment VII. The male usually has smudges and light blotches on its forewings; the penis extends to the basal segment of the forceps and reaches to approximately 1/3 of the second segment of the forceps. Forewings of the female subimago have dark colour over more than half of the basal area, and the distal portion of each wing is translucent. The egg has a rounded pole; the polar cap covers approximately 1/4 of the surface; and the surface is covered with hexagonal structures.


Assuntos
Ephemeroptera , Distribuição Animal , Estruturas Animais , Animais , Sequência de Bases , Feminino , Masculino , Filogenia , Tailândia , Vietnã
6.
Zootaxa ; 4642(1): zootaxa.4642.1.1, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31712451

RESUMO

Phylogenetic relationships of snailfishes of the family Liparidae were analyzed on the basis of two sets of molecular sequence data: one from the mitochondrial DNA cytochrome c oxidase subunit one gene (COI) and another from restriction-site associated genome-wide sequences (RADseq). The analysis of COI sequence data from at least 122 species of 18 genera from the Pacific, Atlantic, and Southern oceans resulted in a moderately well-resolved phylogeny among the major clades, albeit with significant polytomy among central clades. Nectoliparis was the sister of all other members of the family, followed by Liparis. Liparis, Careproctus, and Paraliparis were paraphyletic. Liparis was recovered in two closely related clades, with L. fucensis sister of all other liparids except Nectoliparis, and both Careproctus and Paraliparis were each recovered among at least three widely separated clades. The RADseq analysis of 26 species of 11 genera from the eastern North Pacific strongly confirmed the overall results of the COI analysis, with the exception of the paraphyly of Liparis due to the absence of L. fucensis. Our results show that the pelvic disc has been independently lost multiple times and the pectoral-fin girdle has been independently reduced in multiple lineages.


Assuntos
DNA Mitocondrial , Genômica , Perciformes , Animais , Sequência de Bases , Perciformes/genética , Filogenia , Análise de Sequência de DNA
7.
Zootaxa ; 4586(1): zootaxa.4586.1.2, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-31716141

RESUMO

Intra- and interspecific variability, being at the very core of alpha taxonomy, has been a long-standing topic of debate among tardigrade taxonomists. Early studies tended to assume that tardigrades exhibit wide intraspecific variation. However, with more careful morphological studies, especially those incorporating molecular tools that allow for an independent verification of species identifications based on phenotypic traits, we now recognise that ranges of tardigrade intraspecific variability are narrower, and that differences between species may be more subtle than previously assumed. The taxonomic history of the genus Milnesium, and more specifically that of the nominal species, M. tardigradum described by Doyère in 1840, is a good illustration of the evolution of views on intraspecific variability in tardigrades. The assumption of wide intraspecific variability in claw morphology led Marcus (1928) to synonymise two species with different claw configurations, M. alpigenum and M. quadrifidum, with M. tardigradum. Currently claw configuration is recognised as one of the key diagnostic traits in the genus Milnesium, and the two species suppressed by Marcus have recently been suggested to be valid. In this study, we clarify the taxonomic status of M. alpigenum, a species that for nearly a century was considered invalid. We redescribe M. alpigenum, using a population collected from the locus typicus, by the means of integrative taxonomy, i.e. including light microscopy, scanning electron microscopy, ontogenetic observations, and genetic barcoding. Moreover, the redescription of M. alpigenum allowed us to verify the uncertain taxonomic status of two popular laboratory models that were originally considered to be M. tardigradum; though one was recently reidentified as M. cf. alpigenum. Our analysis showed that both laboratory strains, despite being morphologically and morphometrically nearly identical to M. alpigenum, in fact represent a new species, M. inceptum sp. nov.  The two species, being disnguishable only by statistical morphometry and/or DNA sequences, are the first example of pseudocryptic species in tardigrades.


Assuntos
Tardígrados , Animais , Sequência de Bases , Microscopia Eletrônica de Varredura
8.
Zootaxa ; 4563(3): zootaxa.4563.3.8, 2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-31716534

RESUMO

The long-tailed parakeets of the genus Psittacula Cuvier, 1800 have thus far been regarded as a homogeneous and monophyletic group of parrots. We used nucleotide sequences of two genetic markers (mitochondrial CYTB, nuclear RAG-1) to reconstruct the phylogenetic relationships of Psittacula and closely related species. We found that the Asian genus Psittacula is apparently paraphyletic because two genera of short-tailed parrots, Psittinus Blyth, 1842 and Tanygnathus Wagler, 1832, cluster within Psittacula, as does †Mascarinus Lesson, 1830. To create monophyletic genera, we propose recognition of the following genera: Himalayapsitta Braun, 2016 for P. himalayana, P. finschii, P. roseata, and P. cyanocephala; Nicopsitta Braun, 2016 for P. columboides and P. calthrapae; Belocercus S. Müller, 1847 for P. longicauda; Psittacula Cuvier, 1800 for P. alexandri and P. derbiana; Palaeornis Vigors, 1825 for †P. wardi and P. eupatria; and Alexandrinus Braun, 2016 for P. krameri, †P. exsul, and P. (eques) echo. Additionally, Psittacula krameri and P. alexandri are paraphyletic species, which should be split to form monophyletic species.


Assuntos
Papagaios , Psittaciformes , Psittacula , Animais , Sequência de Bases , Mitocôndrias , Filogenia
9.
Zootaxa ; 4651(1): zootaxa.4651.1.11, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31716929

RESUMO

Development of next generation sequencing rapidly increased the number of total mitogenome in data bases. However, the documented number of total mitogenome from species of Tettigoniinae is still limited and a comparison at subfamily level has not been made sufficiently. This paper aims to describe total mitogenome of A. babadaghi (Orthoptera, Tettigoniidae) by comparing to the known mitogenomes of other Tettigoniinae. The total mitogenome of A. babadaghi is 15882-15883 bp, AT skewed with 70.2% AT percentage, and consists of 13 protein coding genes (PCG), 22 tRNA genes, two rRNA genes and an AT rich control region. The genes are ordered as in pancrustacean. The comparative description of mitogenomes in Tettigoniinae showed that total length varies between 15766-16788 bp, the start codon for protein coding genes almost always fits to the ATN pattern, the stop codons are incomplete T-- / TA- and rarely complete TAA, intergenic spacers (IGS) and overlapping regions (OR) in species of the subfamily are similar in number, location, length and nucleotide sequence. We arrived to following conclusion from comparative data: (i) A. babadaghi has a typical orthopteran mitogenome by general features; (ii) this generalisation seems valid for Tettigoniinae as gene content, gene location, gene order, average AT content, anticodons and secondary structure of the tRNA genes, the start and stop codons of the protein coding genes, and several IGSs/ORs are similar to other orthopteran and hexapopods, (iii) variation range in total mitogenome length is narrow in Tettigoniinae and mainly determined by the lengths of control region and total IGSs, (iv) mitogenome of the subfamily exhibits conserved patterns especially in overlapping regions, but conserved features are mostly plesiomorphic.


Assuntos
Genoma Mitocondrial , Ortópteros , Animais , Sequência de Bases , Ortópteros/genética , Filogenia , RNA de Transferência
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(11): 1133-1135, 2019 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-31703144

RESUMO

OBJECTIVE: To identify a novel human leukocyte antigen (HLA) B allele in a Chinese Han individual and construct its three-dimensional structure. METHODS: The initial HLA genotyping was performed by PCR-sequence-based typing (PCR-SBT). The ambiguous allele was confirmed with single-strand DNA sequencing. The DNA sequence was analyzed to identify the difference between the novel allele and its closest matching allele. Finally, the three-dimensional molecular structure of the novel allele was constructed using a Swiss-Model. RESULTS: One allele of the subject at the HLA-B locus was B*44:03:01, whilst the other was a novel allele which differed from the closest matching allele B*51:01:01:01 by nucleotide (nt) 329 A to C in exon 2, resulting in an amino acid change at codon 86 (p.Asn86Thr). CONCLUSION: A novel HLA-B allele has been identified and officially named as HLA-B*51:159 by the WHO Nomenclature Committee for Factors of the HLA System. The three-dimensional structure of B*51:159 was simulated.


Assuntos
Grupo com Ancestrais do Continente Asiático , Antígenos HLA-B/química , Antígenos HLA-B/genética , Alelos , Sequência de Bases , Humanos , Conformação Molecular , Análise de Sequência de DNA
11.
Artigo em Inglês | MEDLINE | ID: mdl-31618377

RESUMO

Chikungunya virus (CHIKV) is an arbovirus that emerged in the Americas in 2013. Infection with CHIKV is symptomatic in most of the cases and patients can develop chronic arthralgia that lasts from months to years in over 40% of the cases. The East-Central-South Africa (ECSA) genotype was introduced in Brazil in 2014, in Bahia State. Here we report the circulation of the CHIKV ECSA genotype in Piaui State, Northeast Brazil, during the years 2016-2017. The phylogenetic analysis revealed a single introduction of this lineage probably in 2015 and its maintenance at least until 2017. This analysis has also demonstrated the proximity of this genotype with isolates from neighboring States, and its partial nucleotide sequence of the viral E1 gene revealed a synapomorphy synonyms. This finding highlights the spread of the ECSA genotype in Brazil and supports its circulation in the Brazilian Northeast.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Genoma Viral/genética , Sequência de Bases , Brasil/epidemiologia , Febre de Chikungunya/epidemiologia , Surtos de Doenças , Genótipo , Humanos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , África do Sul
12.
Arch Virol ; 164(12): 3137-3140, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31598842

RESUMO

Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar "Ilzer Rose" in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named "apple chlorotic fruit spot viroid" should be considered a putative new member of the genus Apscaviroid.


Assuntos
Malus/virologia , Doenças das Plantas/virologia , Viroides/isolamento & purificação , Sequência de Bases , Frutas/virologia , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Viroides/química , Viroides/classificação , Viroides/genética
13.
Arch Virol ; 164(12): 3141-3144, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31598844

RESUMO

In this study, we report a novel double-stranded RNA (dsRNA) virus, Beauveria bassiana partitivirus 3 (BbPV-3), derived from the entomogenous fungus Beauveria bassiana isolate RCEF5853 from China. The genome of BbPV-3, whose sequence was determined by metagenomic sequencing, RT-PCR, and RACE cloning, comprises two dsRNA genome segments that are 1,856 and 1,719 bp long. The first segment contains a single ORF (ORF-1) encoding a 584-amino-acid-long protein (66.05 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif. The second segment also has a single ORF (ORF-2) encoding a 500-amino-acid-long coat protein (CP) (55.9 kDa). The CP and RdRp sequences showed highest identity of 43.4% and 60.2%, respectively, to those of Colletotrichum eremochloae partitivirus 1. Phylogenetic analysis of the RdRp domain of the polyprotein revealed that BbPV-3 grouped together with the members of the genus Epsilonpartitivirus. Hence, we proposed that Beauveria bassiana partitivirus 3 is a novel member of the proposed genus Epsilonpartitivirus.


Assuntos
Beauveria/virologia , Micovírus/isolamento & purificação , Genoma Viral , Vírus de RNA/isolamento & purificação , Sequência de Bases , China , Micovírus/classificação , Micovírus/genética , Filogenia , RNA Replicase/genética , Vírus de RNA/classificação , Vírus de RNA/genética , RNA Viral/genética , Proteínas Virais/genética , Sequenciamento Completo do Genoma
14.
Arch Virol ; 164(12): 3089-3093, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31595357

RESUMO

The phage vB_BthS-HD29phi infecting Bacillus thuringiensis strain HD29 was isolated and purified. The morphology of the phage showed that it belongs to the family Siphoviridae. The phage genome was 32,181 bp in length, comprised linear double-stranded DNA with an average G + C content of 34.9%, and exhibited low similarity to known phage genomes. Genomic and phylogenetic analysis revealed that vB_BthS-HD29phi is a novel phage. In total, 50 putative ORFs were predicted in the phage genome, and only 18 ORFs encoded proteins with known functions. This article reports the genome sequence of a new tailed phage and increases the known genetic diversity of tailed phages.


Assuntos
Fagos Bacilares/genética , Bacillus thuringiensis/virologia , Genoma Viral , Siphoviridae/genética , Fagos Bacilares/classificação , Fagos Bacilares/isolamento & purificação , Composição de Bases , Sequência de Bases , DNA Viral/genética , Variação Genética , Filogenia , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Sequenciamento Completo do Genoma
15.
Analyst ; 144(22): 6641-6646, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31595888

RESUMO

The detection of the HPV L1 protein provides information about the infection status of the virus, reflects the replication status of the HPV virus in cervical cells, and helps understand the regression and progress of cervical lesions. Herein, we report a novel laser desorption ionization mass spectrometry (LDI MS) method for the sensitive detection of the HPV 16 L1 protein, based on non-covalent competitive adsorption between the HPV 16 L1 aptamer and melamine on gold nanoparticles (AuNPs). The intensity of the MS signal corresponding to the mass tag shows a linear relationship with the HPV 16 L1 concentration in the range 2-80 ng mL-1, with a limit of detection (LOD) of 58.8 pg mL-1. Using this method, the HPV 16 L1 protein is quantitatively analyzed in both clinical and vaccine samples. The described method is simple and has high sensitivity and good reliability.


Assuntos
Proteínas do Capsídeo/análise , Nanopartículas Metálicas/química , Proteínas Oncogênicas Virais/análise , Adsorção , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Proteínas do Capsídeo/química , Ouro/química , Papillomavirus Humano 16/química , Limite de Detecção , Espectrometria de Massas/métodos , Proteínas Oncogênicas Virais/química , Vacinas contra Papillomavirus/análise , RNA/química , Triazinas/química
16.
Anticancer Res ; 39(10): 5449-5459, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570439

RESUMO

BACKGROUND/AIM: Epigenetic abnormalities in microRNAs (miRNAs) have not been analyzed in samples other than pancreaticobiliary tissues in patients with pancreaticobiliary cancer (PBC). To identify miRNAs specific for PBC, the present study analyzed the methylation of tumor-suppressive miRNAs in bile from patients with pancreaticobiliary diseases. MATERIALS AND METHODS: Bile was collected endoscopically or percutaneously from 52 patients with pancreatic cancer, 26 with biliary tract cancer, and 20 with benign pancreaticobiliary diseases. Sequences encoding 16 tumor-suppressive miRNAs were amplified by polymerase chain reaction and sequenced, and their methylation rates were determined. RESULTS: The methylation rates of miR-1247 and miR-200a were significantly higher in patients with pancreatic cancer, and biliary tract cancer than in those with benign diseases, and the methylation rate of miR-200b was significantly higher in patients with pancreatic cancer than in those with benign diseases. CONCLUSION: Methylation of miR-1247, miR-200a, and miR-200b in bile may be useful for distinguishing PBC from benign diseases.


Assuntos
Neoplasias do Sistema Biliar/genética , Metilação de DNA/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Bile/metabolismo , Epigenômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade
17.
Exp Parasitol ; 206: 107771, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585116

RESUMO

A PCR targeting mitochondrial cytochrome oxidase subunit III (cox3) for molecular detection of Babesia gibsoni infection in dogs has been developed in this study. Fifty blood samples from suspected clinical cases from dogs, brought to the veterinary college clinics, were examined for presence of B. gibsoni using conventional diagnosis by microscopic examination of Giemsa stained thin blood smears. In addition, species specific PCRs targeting ITS-1 region (BgITS-1 PCR) and nested PCR targeting 18S ribosomal RNA gene (Bg18SnPCR) were carried out. A 634 bp PCR fragment of B. gibsoni cox3 gene was amplified in positive samples from three geographical locations of Satara, Wai and Pune in Maharashtra state of India. From analysis of the sequence of the B. gibsoni cox3 gene, we found that the Indian isolate had 96-98% similarity to the isolate from Japan and China. Post sequencing, de-novo diagnostic primer pair for species specific amplification of 164 bp fragment of B. gibsonicox3 was designed and the PCR was standardized. The diagnostic results of de-novo Bgcox3 PCR were compared with BgITS-1 PCR and Bg18S nPCR. Thin blood smears detected 22% (11/50) samples positive for small form of Babesia species. The BgITS-1 PCR detected 25% samples (15/50) as positive and Bg18S nPCR detected 80% (40/50) B. gibsoni positive samples. The de-novo Bgcox3 PCR detected 66% (33/50) samples positive for B. gibsoni (at 95% CI). The analytical sensitivity of cox3 PCR was evaluated as 0.000003% parasitaemia or 09 parasites in 100  µl of blood. The de-novo diagnostic cox3 PCR did not cross react with control positive DNA from other haemoprotozoa and rickettsia like B. vogeli, Hepatozoon canis, Trypanosoma evansi, Ehrlichia canis and Anaplasma platys. Statistically, cox3 PCR had better diagnostic efficiency than ITS-1 PCR in terms of sensitivity (p = 0.0006). No statistically significant difference between results of cox3 PCR and 18S nPCR was observed (p = 0.1760). Kappa values estimated for each test pair showed fair to moderate agreement between the observations. Specificity of Bgcox3 PCR was 100% when compared with microscopy or BgITS-1 PCR. Sensitivity of Bgcox3 PCR was 100% when compared with that of Bg18S nPCR.


Assuntos
Babesia/isolamento & purificação , Babesiose/diagnóstico , Doenças do Cão/diagnóstico , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/enzimologia , Animais , Babesia/classificação , Babesia/genética , Babesiose/parasitologia , Sequência de Bases , Reações Cruzadas , DNA Espaçador Ribossômico/química , Doenças do Cão/parasitologia , Cães , Eritrócitos/parasitologia , Funções Verossimilhança , Filogenia , Reação em Cadeia da Polimerase/veterinária , Valor Preditivo dos Testes , RNA Ribossômico 18S/análise , Sensibilidade e Especificidade , Alinhamento de Sequência/veterinária
18.
Nucleic Acids Res ; 47(16): 8874-8887, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616952

RESUMO

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis ß-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.


Assuntos
Bacteriófago lambda/genética , Cromossomos Bacterianos/química , DNA Nucleotidiltransferases/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Proteínas Virais/química , Sítio Alostérico , Bacteriófago lambda/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparo de DNA por Recombinação , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismo
19.
J Med Microbiol ; 68(11): 1655-1663, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31573466

RESUMO

Purpose. Epidermophyton floccosum is an anthropophilic dermatophyte species, which is one of the common causative agents of dermatophytosis in different parts of the world. The aim of the present investigation was to evaluate the genetic diversity of E. floccosum strains isolated from different parts of Iran and to define the in vitro susceptibility profiles of seven antifungal drugs against these clinical isolates.Methodology. Forty clinical strains of E. floccosum isolated from 40 patients with dermatophytosis were subjected to DNA extraction and PCR amplification of the ITS rDNA region using universal primers ITS1 and ITS4. The in vitro activities of griseofulvin, itraconazole, voriconazole, posaconazole, caspofungin, ketoconazole and terbinafine were determined using a broth microdilution method according to the CLSI-M-38A2 protocol.Results. A mean genetic similarity of 99.5 % was found between E. floccosum strains, with intraspecies differences ranging from 0 to 3 nt. The geometric mean (GM) MICs and minimum effective concentrations (MECs) across all isolates were, in increasing order, as follows: terbinafine (GM=0.018 mg l-1), posaconazole (GM=0.022 mg l-1), itraconazole (GM=0.034 mg l-1) and voriconazole (GM=0.045 mg l-1), which had low MICs against all tested strains, whereas caspofungin (GM=0.22 mg l-1), ketoconazole (GM=0.41 mg l-1) and griseofulvin (GM=0.62 mg l-1) demonstrated higher MICs.Conclusion. Our study showed low intraspecies variation within strains of E. floccosum. Furthermore, terbinafine, posaconazole, itraconazole and voriconazole were shown to be the most potent antifungal drugs against E. floccosum strains.


Assuntos
Antifúngicos/farmacologia , Epidermophyton/efeitos dos fármacos , Tinha/microbiologia , Sequência de Bases , Epidermophyton/classificação , Epidermophyton/genética , Epidermophyton/isolamento & purificação , Humanos , Irã (Geográfico) , Itraconazol/farmacologia , Cetoconazol/farmacologia , Testes de Sensibilidade Microbiana , Terbinafina/farmacologia , Trichophyton/efeitos dos fármacos , Trichophyton/genética , Trichophyton/isolamento & purificação , Voriconazol/farmacologia
20.
Mem Inst Oswaldo Cruz ; 114: e190219, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31644704

RESUMO

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.


Assuntos
Genoma Viral , Bocavirus Humano/genética , Infecções por Parvoviridae/virologia , Sequência de Bases , Bocavirus Humano/isolamento & purificação , Humanos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Uruguai
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