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Sci China Life Sci ; 63(2): 251-258, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31250189


RtcB, a highly conserved RNA ligase, is found in all three domains of life, and demonstrated to be an essential tRNA splicing component in archaea and metazoans. However, the biological functions of RtcB in bacteria, where there is no splicing, remains to be clarified. We first performed bioinformatics analysis which revealed highly conserved structures and presumably conserved functions of RtcB in bacteria. However, its orthologs only occur in ∼ 0.5% of bacterial species across diverse phyla with significant signals of frequent horizontal transfer, highlighting its non-essential role in bacteria. Next, by constructing an rtcB-knockout strain, we find that the removal of antibiotic stress induces a significant impact on rtcB expression in wild-type strain, and furthermore the depletion of RtcB (ARtcB strain) delays the recovery process. Our transcriptomic analysis, comprising the 3'-end labeling of RNAs, highlights a significant increase in unmapped reads and cleaved rRNAs in the Δ RtcB strain, particularly during recovery. Our observations suggest that the conserved RNA ligase RtcB, repairs damaged rRNAs following stress, which potentially saves energy and accelerates recovery of its host. We propose that acquisition of RtcB by diverse bacterial taxa provides a competitive advantage under stressful conditions.

mSystems ; 4(4)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117025


Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for microbes, whereas its application is rather limited in airways due to host DNA contaminants that overwhelm a minute amount of microbial content. Here, we describe a microfluidics-based enrichment device and an emulsion-based whole-genome amplification procedure (MEEA) for the preparation of DNA from sputa for shotgun sequencing in a metagenomics study. The two protocols coupled in MEEA are first separately assayed with mock samples and are both promising in efficiency and bias. The efficiency and consistency of MEEA are further evaluated in six clinical sputum samples against direct sequencing without enrichment, and MEEA enables 2 to 14 times enrichment for microbial reads, which take 14.68% to 33.52% of total reads. The dominant pathogens detected in MEEA are in excellent agreement with those from clinical etiological tests. Meanwhile, MEEA presents much more microbiome complexity and genome information at a strain level than direct sequencing, exhibiting high sensitivity for identifying prophages and DNA viruses. MEEA provides better microbiome profiling than direct sequencing without a preference for specific microorganisms. The more detailed functional and taxonomic characterization of their species constituents, including both bacterium and virus, facilitates metagenomics studies on the pathogenesis of respiratory microbiomes.IMPORTANCE The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community-the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases.

Lab Chip ; 17(21): 3601-3608, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-28975175


The separation or purification of bacterial samples from a mixed cell suspension is critical in a variety of biomedical applications, such as sputum diagnostics and cell biology studies. We propose a streamline-based microfluidic filtration device for highly efficient purification of bacterial samples from a mixed cell suspension. The device is composed of tens of repeated streamline-based separation units that continuously filter the solution. By injecting a liquid sample such as liquefied human sputum solution through the device, approximately 50% of the injected sample solution can be collected from the filtration collection channels, which filter approximately 99.9% of the mammalian cells but retain approximately 60% to 90% of the bacteria. Different injection rates (0.2 ml h-1 to 30 ml h-1), different sample viscosities, and different initial bacterial densities were tested and confirmed that our separation method was robust. The easy operation, robustness and high efficiency indicate that our method may be useful for the separation or purification of bacterial samples from a mixed cell suspension, such as bacterial samples for sputum diagnostics.

Bactérias/isolamento & purificação , Técnicas Bacteriológicas , Separação Celular/instrumentação , Filtração/instrumentação , Técnicas Analíticas Microfluídicas , Escarro/microbiologia , Infecções Bacterianas/microbiologia , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Desenho de Equipamento , Células HeLa , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Viscosidade
Nat Genet ; 46(11): 1212-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25240282


The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

Carpas/genética , Evolução Molecular , Variação Genética , Genoma/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Componentes Genômicos/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Pele/metabolismo , Especificidade da Espécie
Zhonghua Zhong Liu Za Zhi ; 25(6): 535-7, 2003 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-14690555


OBJECTIVE: To investigate the heterogeneity of human breast cancer cells, their influence on biological behavior of tumor cells and clinical implications. METHODS: The subpopulations of MCF-7 breast cancer cells were isolated by Percoll gradient centrifugation. DNA content and cell cycle distribution were detected with flow cytometry. Tumor chemosensitivity analysis was performed with MTT assay. RESULTS: Heterogeneity was observed in DNA content and cell cycle distribution among four subpopulations of breast cancer cells, which were related to their proliferation ability and chemosensitivity results. CONCLUSION: Hereditary instability and intrinsic characteristics of most tumor cells, not only lead to tumor progression and heterogeneity but also cause the loss of monoclonality and the generation of subclones. Further study on some profiles of tumor heterogeneity such as DNA content, cell cycle distribution and their influence on tumor proliferation and chemosensitivity may very well improve the clinical treatment.

Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , DNA de Neoplasias/análise , Feminino , Humanos