RESUMO
Helicobacter pylori (H. pylori) infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for H. pylori diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated H. pylori detection and genotyping. The system can achieve multi-target parallel detection of H. pylori nucleotide conservative genes (ureB) and virulence genes (cagA and vacA) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of H. pylori with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform.
Assuntos
Técnicas Biossensoriais , Técnicas de Genotipagem , Infecções por Helicobacter , Helicobacter pylori , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/microbiologia , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Técnicas de Genotipagem/instrumentação , Técnicas de Genotipagem/métodos , Genótipo , Proteínas de Bactérias/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Microfluídica/métodos , Antígenos de Bactérias/genética , Antígenos de Bactérias/análise , DNA Bacteriano/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Recombinases/metabolismoRESUMO
The COP9 signalosome (CSN) is a multiprotein complex which participates in diverse cellular and developmental processes. CSN1, one of the subunits of CSN, is essential for assembly of the multiprotein complex via PCI (proteasome, COP9 signalosome and initiation factor 3) domain in the C-terminal half of CSN1. However, the role of the N-terminal domain (NTD) of CSN1, which is critical for the function of CSN, is not completely understood. Using a yeast two-hybrid (Y2H) screen, we found that the NTD of CSN1 interacts with TSK-associating protein 1 (TSA1), a reported Ca(2+)-binding protein. The interaction between CSN1 and TSA1 was confirmed by co-immunoprecipitation in Arabidopsis. tsa1 mutants exhibited a short hypocotyl phenotype in darkness but were similar to wild-type Arabidopsis under white light, which suggested that TSA1 might regulate Arabidopsis hypocotyl development in the dark. Furthermore, the expression of TSA1 was significantly lower in a csn1 null mutant (fus6), while CSN1 expression did not change in a tsa1 mutant with weak TSA1 expression. Together, these findings suggest a functional relationship between TSA1 and CSN1 in seedling development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Ligação ao Cálcio/metabolismo , Plântula/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Complexo do Signalossomo COP9 , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Escuridão , Ligação Proteica , Estrutura Terciária de Proteína , Plântula/genética , Plântula/metabolismo , Plântula/efeitos da radiaçãoRESUMO
Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants.
Assuntos
Adaptação Fisiológica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/metabolismo , Oryza/genética , Temperatura Baixa , Secas , Genes de Plantas , Teste de Complementação Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucosiltransferases/genética , Oryza/enzimologia , Oryza/fisiologia , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Prolina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plantas Tolerantes a Sal/enzimologia , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/fisiologia , Estresse Fisiológico , Transformação Genética , Trealose/metabolismoRESUMO
Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development.
