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1.
J Med Microbiol ; 69(5): 712-720, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32368996

RESUMO

Introduction. Given the limited number of candidaemia studies in Iran, the profile of yeast species causing bloodstream infections (BSIs), especially in adults, remains limited. Although biochemical assays are widely used in developing countries, they produce erroneous results, especially for rare yeast species.Aim. We aimed to assess the profile of yeast species causing BSIs and to compare the accuracy of the Vitek 2 system and 21-plex PCR.Methodology. Yeast blood isolates were retrospectively collected from patients recruited from two tertiary care training hospitals in Tehran from 2015 to 2017. Relevant clinical data were mined. Identification was performed by automated Vitek 2, 21-plex PCR and sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2).Results. In total, 137 yeast isolates were recovered from 107 patients. The overall all-cause 30-day mortality rate was 47.7 %. Fluconazole was the most widely used systemic antifungal. Candida albicans (58/137, 42.3 %), Candida glabrata (30/137, 21.9 %), Candida parapsilosis sensu stricto (23/137, 16.8 %), Candida tropicalis (10/137, 7.3 %) and Pichia kudriavzevii (Candida krusei) (4/137, 2.9 %) constituted almost 90 % of the isolates and 10 % of the species detected were rare yeast species (12/137; 8.7 %). The 21-plex PCR method correctly identified 97.1 % of the isolates, a higher percentage than the Vitek 2 showed (87.6 %).Conclusion. C. albicans was the main cause of yeast-derived fungaemia in this study. Future prospective studies are warranted to closely monitor the epidemiological landscape of yeast species causing BSIs in Iran. The superiority of 21-plex PCR over automated Vitek 2 indicates its potential clinical utility as an alternative identification tool use in developing countries.


Assuntos
Fungemia/diagnóstico , Fungemia/epidemiologia , Fungemia/microbiologia , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência de DNA , Leveduras/classificação , Leveduras/genética , Idoso , Idoso de 80 Anos ou mais , DNA Intergênico , Feminino , Fungemia/história , História do Século XXI , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
2.
Int J Syst Evol Microbiol ; 70(5): 3433-3439, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375982

RESUMO

During a survey of endophytic fungi in aquatic plants collected from Tibet, PR China, a novel species, Ramichloridium endophyticum, was isolated from Potamogeton pectinatus. This novel species differs from other species of the genus Ramichloridium by its finely verrucose, obovoid, ellipsoidal-obovoid and occasionally subglobose conidia. Phylogenetic analysis of the combined sequences of the internal transcribed spacers (ITS) and the translation elongation factor 1-alpha gene (tef1-α) confirmed that the isolated strain represents a member of the genus Ramichloridium. A full description, illustrations and a phylogenetic tree showing the position of R. endophyticum are provided.


Assuntos
Ascomicetos/classificação , Filogenia , Potamogetonaceae/microbiologia , Ascomicetos/isolamento & purificação , DNA Fúngico/genética , DNA Intergênico/genética , Genes Fúngicos , Técnicas de Tipagem Micológica , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Esporos Fúngicos , Tibet
3.
Nat Commun ; 11(1): 1528, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251294

RESUMO

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.


Assuntos
Técnicas de Cultura de Células/métodos , Cromossomos Humanos X/genética , DNA Intergênico/genética , Taxa de Mutação , Células-Tronco Pluripotentes/fisiologia , Linhagem Celular , Meios de Cultura/farmacologia , Metilação de DNA , Análise Mutacional de DNA , Epigênese Genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Oxigênio/química , Oxigênio/farmacologia , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
4.
PLoS One ; 15(4): e0231436, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298321

RESUMO

Molecular-based taxonomy, specifically DNA barcoding, has streamlined organism identification. For land plants, the recommended 2-locus barcode of rbcL and matK is not suitable for all groups, thus the second subunit of the nuclear internal transcribed spacer (ITS2) has received attention as a possible alternative. To date, evaluations of ITS2 have mostly been limited in scope to specific plant orders/families and single source material. Prior to using ITS2 to routinely characterize land plants present in environmental samples (i.e., DNA metabarcoding), a wet lab protocol optimized for bulk sample types is needed. To address this gap, in this study we determined the broad recoverability across land plants when using published ITS2 primer pairs, and subsequently optimized the PCR reaction constituents and cycling conditions for the best two performing primer pairs (ITS2F/ITSp4 and ITSp3/ITSu4). Using these conditions, both primer pairs were used to characterize land plants present in 17 diverse soils collected from across the US. The resulting PCR amplicons were prepared into libraries and pooled for sequencing on an Illumina® MiniSeq. Our existing bioinformatics workflow was used to process raw sequencing data and taxonomically assign unique ITS2 plant sequences by comparison to GenBank. Given strict quality criteria were imposed on sequences for inclusion in data analysis, only 43.6% and 7.5% of sequences from ITS2F/ITSp4 and ITSp3/ITSu4 respectively remained for taxonomic comparisons; ~7-11% of sequences originated from fungal co-amplification. The number of orders and families recovered did differ between primer pairs, with ITS2F/ITSp4 consistently outperforming ITSp3/ITSu4 by >15%. Primer pair bias was observed in the recovery of certain taxonomic groups; ITS2F/ITSp4 preferentially recovered flowering plants and grasses, whereas ITSp3/ITSu4 recovered more moss taxa. To maximize data recovery and reduce potential bias, we advocate that studies using ITS2 to characterize land plants from environmental samples such as soil use a multiple primer pair approach.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA Intergênico/genética , DNA de Plantas/genética , Metagenômica/métodos , Briófitas/classificação , Briófitas/genética , Código de Barras de DNA Taxonômico/normas , DNA Intergênico/química , DNA de Plantas/química , Gleiquênias/classificação , Gleiquênias/genética , Magnoliopsida/classificação , Magnoliopsida/genética , Metagenômica/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Solo/química
5.
Int J Food Microbiol ; 319: 108496, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-31911209

RESUMO

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.


Assuntos
Cinnamomum aromaticum/microbiologia , Fungos/classificação , Fungos/genética , Pólen/microbiologia , Aspergillus/genética , Candida/genética , Cladosporium/genética , DNA Intergênico/genética , Contaminação de Alimentos/análise , Inocuidade dos Alimentos/métodos , Fungos/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Medicina Tradicional Chinesa , Micobioma , Micotoxinas/análise , Penicillium/genética , Chá/microbiologia
6.
BMC Res Notes ; 13(1): 35, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952556

RESUMO

OBJECTIVE: Grasslands are widespread ecosystems that fulfil many functions. Plant species richness (PSR) is known to have beneficial effects on such functions and monitoring PSR is crucial for tracking the effects of land use and agricultural management on these ecosystems. Unfortunately, traditional morphology-based methods are labor-intensive and cannot be adapted for high-throughput assessments. DNA barcoding could aid increasing the throughput of PSR assessments in grasslands. In this proof-of-concept work, we aimed at determining which of three plant DNA barcodes (rbcLa, matK and trnH-psbA) best discriminates 16 key grass and legume species common in temperate sub-alpine grasslands. RESULTS: Barcode trnH-psbA had a 100% correct assignment rate (CAR) in the five analyzed legumes, followed by rbcLa (93.3%) and matK (55.6%). Barcode trnH-psbA had a 100% CAR in the grasses Cynosurus cristatus, Dactylis glomerata and Trisetum flavescens. However, the closely related Festuca, Lolium and Poa species were not always correctly identified, which led to an overall CAR in grasses of 66.7%, 50.0% and 46.4% for trnH-psbA, matK and rbcLa, respectively. Barcode trnH-psbA is thus the most promising candidate for PSR assessments in permanent grasslands and could greatly support plant biodiversity monitoring on a larger scale.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA Intergênico/genética , Fabaceae/genética , Poaceae/genética , Biodiversidade , DNA de Plantas , Ecossistema , Filogenia , Análise de Sequência de DNA , Especificidade da Espécie
7.
PLoS Genet ; 16(1): e1008544, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978080

RESUMO

The genetic architecture of the small and isolated Greenlandic population is advantageous for identification of novel genetic variants associated with cardio-metabolic traits. We aimed to identify genetic loci associated with body mass index (BMI), to expand the knowledge of the genetic and biological mechanisms underlying obesity. Stage 1 BMI-association analyses were performed in 4,626 Greenlanders. Stage 2 replication and meta-analysis were performed in additional cohorts comprising 1,058 Yup'ik Alaska Native people, and 1,529 Greenlanders. Obesity-related traits were assessed in the stage 1 study population. We identified a common variant on chromosome 11, rs4936356, where the derived G-allele had a frequency of 24% in the stage 1 study population. The derived allele was genome-wide significantly associated with lower BMI (beta (SE), -0.14 SD (0.03), p = 3.2x10-8), corresponding to 0.64 kg/m2 lower BMI per G allele in the stage 1 study population. We observed a similar effect in the Yup'ik cohort (-0.09 SD, p = 0.038), and a non-significant effect in the same direction in the independent Greenlandic stage 2 cohort (-0.03 SD, p = 0.514). The association remained genome-wide significant in meta-analysis of the Arctic cohorts (-0.10 SD (0.02), p = 4.7x10-8). Moreover, the variant was associated with a leaner body type (weight, -1.68 (0.37) kg; waist circumference, -1.52 (0.33) cm; hip circumference, -0.85 (0.24) cm; lean mass, -0.84 (0.19) kg; fat mass and percent, -1.66 (0.33) kg and -1.39 (0.27) %; visceral adipose tissue, -0.30 (0.07) cm; subcutaneous adipose tissue, -0.16 (0.05) cm, all p<0.0002), lower insulin resistance (HOMA-IR, -0.12 (0.04), p = 0.00021), and favorable lipid levels (triglyceride, -0.05 (0.02) mmol/l, p = 0.025; HDL-cholesterol, 0.04 (0.01) mmol/l, p = 0.0015). In conclusion, we identified a novel variant, where the derived G-allele possibly associated with lower BMI in Arctic populations, and as a consequence also leaner body type, lower insulin resistance, and a favorable lipid profile.


Assuntos
Índice de Massa Corporal , Cromossomos Humanos Par 11/genética , Inuítes/genética , Polimorfismo de Nucleotídeo Único , Adiposidade , Colesterol/sangue , DNA Intergênico/genética , Feminino , Groenlândia , Humanos , Resistência à Insulina , Masculino , Metaboloma , Circunferência da Cintura
8.
Parasitol Res ; 119(2): 473-481, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31897790

RESUMO

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species.


Assuntos
Oftalmopatias/veterinária , Filarioidea/isolamento & purificação , Doenças dos Cavalos/parasitologia , Cavalos/parasitologia , Setaria (Nematoide)/isolamento & purificação , Setaríase/parasitologia , Animais , DNA Intergênico/genética , DNA Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Oftalmopatias/parasitologia , Filarioidea/genética , Índia , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico/genética , Análise de Sequência de DNA , Setaria (Nematoide)/genética
9.
BMC Evol Biol ; 20(1): 2, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906844

RESUMO

BACKGROUND: Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. RESULTS: We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny. CONCLUSIONS: The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.


Assuntos
Ascomicetos/genética , Código de Barras de DNA Taxonômico , Líquens/genética , Ascomicetos/classificação , Núcleo Celular/genética , Código de Barras de DNA Taxonômico/métodos , DNA Fúngico/genética , DNA Intergênico , DNA Ribossômico , DNA Espaçador Ribossômico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Líquens/classificação , Filogenia , Simbiose , Sequências de Repetição em Tandem
10.
J Nat Med ; 74(1): 282-293, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31587135

RESUMO

The dried fruits of Terminalia plant (Combretaceae) called "Samo" have been used as herbal medicine in Thai traditional medicine. Four "Samo" crude drugs, namely, Samo thai, Samo thed, Samo dee-ngu, and Samo phiphek, are used as the main ingredients in Triphala and Trisamo recipes. Their commercial products are available in processed and powdered form, but are difficult to authenticate by conventional methods. In this study, we aimed to discriminate species of genus Terminalia for the identification of their crude drugs by a DNA barcoding technique. A total of 208 closely related nucleotide sequences were obtained from nine Terminalia species collected from Thailand and the DDBJ/EMBL/GenBank database. An effective DNA barcode marker was selected from six DNA loci (matK, rbcL, psbA-trnH, ITS, ITS1, and ITS2) and their two-locus combination. All sequences were analyzed by three major methods: (1) BLAST search; (2) the genetic divergence method using Kimura 2-parameter (K2P) distance matrices; and (3) tree topology analysis based on the neighbor-joining method. Comparison of the six candidate DNA loci indicated that ITS identified Terminalia with 100% accuracy at the species and genus levels in the BLAST1 method. ITS2 showed the highest K2P variability. The data from the single markers and the two-locus combinations revealed that only the two-locus combinations, namely, the combinations of rbcL, ITS, ITS1, and ITS2 with psbA-trnH, clearly discriminated all the species. From the results of DNA sequence analysis and the three methods, ITS2 is recommended for the identification of Terminalia species to supplement psbA-trnH.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA Intergênico/genética , Complexo de Proteína do Fotossistema II/genética , Terminalia/classificação , Terminalia/genética , Sequência de Bases , DNA de Plantas/genética , Marcadores Genéticos/genética , Fitoterapia , Extratos Vegetais/química , Plantas Medicinais/genética , Análise de Sequência de DNA , Tailândia
11.
J Nat Med ; 74(1): 106-118, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31377923

RESUMO

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.


Assuntos
Preparações de Plantas/farmacologia , Raízes de Plantas/química , Pueraria/química , Pueraria/classificação , DNA Intergênico/genética , Fitoestrógenos/análise , Preparações de Plantas/análise , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Pueraria/genética , Tailândia
12.
Int J Food Microbiol ; 312: 108373, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31654841

RESUMO

The use of non-Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non-Saccharomyces, through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non-Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania, Aureobasidium, Meyerozyma, Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H2S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds.


Assuntos
Saccharomycetales/genética , Saccharomycetales/metabolismo , Vitis/metabolismo , Vinho/microbiologia , Leveduras/metabolismo , Austrália , DNA Intergênico/genética , Fazendas , Fermentação , RNA Ribossômico 5,8S/genética , Saccharomycetales/classificação , Saccharomycetales/isolamento & purificação , Austrália do Sul , Vinho/análise
13.
Parasitol Int ; 74: 101976, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31513918

RESUMO

Species in the genus Notocotylus (Trematoda, Notocotylidae) are cosmopolitan parasites of mainly aquatic birds and, to a lesser extent, mammals. In this paper we describe Notocotylus primulus n. sp. parasitizing the Crested Duck, Lophonetta specularioides, from the Patagonian coast, southwestern Atlantic Ocean, based on morphological and molecular data. The new species could be distinguished from its congeners by the combination of several features; among them, the position of the genital pore that is prebifurcal and located close to the posterior margin of the oral sucker, a greater number of both ventral papillae and uterine loops, and a large cirrus sac. Molecular results of the ITS2 sequence support the location of this new species in the genus Notocotylus due to a close relationship with Notocotylus malhamensis (the lower genetic distance of the ITS2 sequences).


Assuntos
Patos/parasitologia , Filogenia , Trematódeos/anatomia & histologia , Trematódeos/classificação , Animais , Oceano Atlântico , DNA Intergênico/genética , Feminino , Genitália/anatomia & histologia , Masculino , RNA Ribossômico 28S/genética
14.
BMC Genomics ; 20(1): 972, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830913

RESUMO

BACKGROUND: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03. RESULTS: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones. CONCLUSIONS: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.


Assuntos
Mutação , Vitis/genética , Sequenciamento Completo do Genoma/métodos , Evolução Clonal , DNA Intergênico , Genoma de Planta
15.
Molecules ; 25(1)2019 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-31877894

RESUMO

In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.


Assuntos
Edição de Genes/métodos , Mutação Puntual , Sistemas CRISPR-Cas , DNA Complementar/genética , DNA Intergênico/genética , Receptores ErbB/genética , Humanos
16.
Parasitol Res ; 118(12): 3371-3375, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31705288

RESUMO

Enterocytozoon bieneusi is a single-celled obligate pathogen that seriously threatens animal and public health. However, information on the prevalence and genotypes of E. bieneusi in alpacas in China is limited. In the present study, 366 fresh fecal samples from alpacas in Shanxi Province, northern China, were collected to detect E. bieneusi by nested PCR amplification of the internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA). The overall prevalence of E. bieneusi in alpacas was 4.4% (16/366), including 3.9% (12/305) in Yangqu County and 6.6% (4/61) in Dai county, respectively. Four known genotypes were identified, namely ALP1, ALP3, P, and SH11, all of which belong to the zoonotic group 1 by phylogenetic analysis. Moreover, ITS-positive samples were further characterized by PCR amplification of other four targets, including three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4). Multilocus sequence typing (MLST) showed that 5, 2, 3, and 3 types were identified at MS1, MS3, MS7, and MS4 loci, respectively, representing eight multilocus genotypes (MLGs). These findings contribute to the improved understanding of the prevalence and genotypes of E. bieneusi in alpacas in China and have important implications for controlling E. bieneusi infections in animals and humans.


Assuntos
Camelídeos Americanos/parasitologia , DNA Intergênico/genética , Enterocytozoon/genética , Repetições de Microssatélites/genética , Microsporidiose/veterinária , Doenças Parasitárias em Animais/parasitologia , Animais , China/epidemiologia , Enterocytozoon/classificação , Enterocytozoon/isolamento & purificação , Fezes/parasitologia , Genótipo , Humanos , Microsporidiose/epidemiologia , Tipagem de Sequências Multilocus , Filogenia , Reação em Cadeia da Polimerase , Prevalência
17.
Nat Commun ; 10(1): 4943, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666509

RESUMO

Mutations and variations in and around SCN5A, encoding the major cardiac sodium channel, influence impulse conduction and are associated with a broad spectrum of arrhythmia disorders. Here, we identify an evolutionary conserved regulatory cluster with super enhancer characteristics downstream of SCN5A, which drives localized cardiac expression and contains conduction velocity-associated variants. We use genome editing to create a series of deletions in the mouse genome and show that the enhancer cluster controls the conformation of a >0.5 Mb genomic region harboring multiple interacting gene promoters and enhancers. We find that this cluster and its individual components are selectively required for cardiac Scn5a expression, normal cardiac conduction and normal embryonic development. Our studies reveal physiological roles of an enhancer cluster in the SCN5A-SCN10A locus, show that it controls the chromatin architecture of the locus and Scn5a expression, and suggest that genetic variants affecting its activity may influence cardiac function.


Assuntos
Sistema de Condução Cardíaco/metabolismo , Coração/embriologia , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Animais , Sistemas CRISPR-Cas , Cromatina , DNA Intergênico/genética , Elementos Facilitadores Genéticos/genética , Edição de Genes , Regulação da Expressão Gênica , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Conformação de Ácido Nucleico , Elementos Reguladores de Transcrição
18.
BMC Genomics ; 20(1): 874, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747873

RESUMO

BACKGROUND: Several bioinformatics tools have been designed for assembly and annotation of chloroplast (cp) genomes, making it difficult to decide which is most useful and applicable to a specific case. The increasing number of plant genomes provide an opportunity to accurately obtain cp genomes from whole genome shotgun (WGS) sequences. Due to the limited genetic information available for European hazelnut (Corylus avellana L.) and as part of a genome sequencing project, we analyzed the complete chloroplast genome of the cultivar 'Tombul' with multiple annotation tools. RESULTS: Three different annotation strategies were tested, and the complete cp genome of C. avellana cv Tombul was constructed, which was 161,667 bp in length, and had a typical quadripartite structure. A large single copy (LSC) region of 90,198 bp and a small single copy (SSC) region of 18,733 bp were separated by a pair of inverted repeat (IR) regions of 26,368 bp. In total, 125 predicted functional genes were annotated, including 76 protein-coding, 25 tRNA, and 4 rRNA unique genes. Comparative genomics indicated that the cp genome sequences were relatively highly conserved in species belonging to the same order. However, there were still some variations, especially in intergenic regions, that could be used as molecular markers for analyses of phylogeny and plant identification. Simple sequence repeat (SSR) analysis showed that there were 83 SSRs in the cp genome of cv Tombul. Phylogenetic analysis suggested that C. avellana cv Tombul had a close affinity to the sister group of C. fargesii and C. chinensis, and then a closer evolutionary relationship with Betulaceae family than other species of Fagales. CONCLUSION: In this study, the complete cp genome of Corylus avellana cv Tombul, the most widely cultivated variety in Turkey, was obtained and annotated, and additionally phylogenetic relationships were predicted among Fagales species. Our results suggest a very accurate assembly of chloroplast genome from next generation whole genome shotgun (WGS) sequences. Enhancement of taxon sampling in Corylus species provide genomic insights into phylogenetic analyses. The nucleotide sequences of cv Tombul cp genomes can provide comprehensive genetic insight into the evolution of genus Corylus.


Assuntos
Cloroplastos/genética , Corylus/genética , Genoma de Cloroplastos , Filogenia , Mapeamento Cromossômico , Corylus/classificação , DNA Intergênico , Ontologia Genética , Tamanho do Genoma , Repetições de Microssatélites , Anotação de Sequência Molecular , Turquia , Sequenciamento Completo do Genoma
19.
J Helminthol ; 94: e105, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31735179

RESUMO

The superfamily Pronocephaloidea Looss, 1899 comprises digeneans occurring in the gut and respiratory organs of fishes, turtles, marine iguanas, birds and mammals. Although many life cycles are known for species of the Notocotylidae Lühe, 1909 maturing in birds and mammals, relatively few are known for the remaining pronocephaloid lineages. We report the cercariae of five pronocephaloid species from marine gastropods of the Queensland coast, Australia. From Lizard Island, northern Great Barrier Reef, we report three cercariae, two from Rhinoclavis vertagus (Cerithiidae) and one from Nassarius coronatus (Nassariidae). From Moreton Bay, southern Queensland, an additional two cercariae are reported from two genotypes of the gastropod worm shell Thylacodes sp. (Vermetidae). Phylogenetic analysis using 28S rRNA gene sequences shows all five species are nested within the Pronocephaloidea, but not matching or particularly close to any previously sequenced taxon. In combination, phylogenetic and ecological evidence suggests that most of these species will prove to be pronocephalids parasitic in marine turtles. The Vermetidae is a new host family for the Pronocephaloidea.


Assuntos
Gastrópodes/parasitologia , Filogenia , Trematódeos/anatomia & histologia , Trematódeos/classificação , Animais , Organismos Aquáticos/classificação , Organismos Aquáticos/parasitologia , Cercárias/anatomia & histologia , Cercárias/classificação , Cercárias/isolamento & purificação , DNA Intergênico/genética , Gastrópodes/classificação , Genótipo , Estágios do Ciclo de Vida , Queensland , RNA Ribossômico 28S/genética , Trematódeos/isolamento & purificação
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