RESUMO
Objective To construct a ciaR gene-knockout (ΔciaR) mutant of Streptococcus pneu-moniae ( S. pneumoniae) and to investigate the effects of CiaR in CiaH/CiaR, a streptococcal two-component signal-transducing system, on the expression of genes encoding penicillin-binding proteins ( pbps genes) and cia-dependent small RNAs (csRNAs). Methods Electrophoretic mobility shift assay (ESMA) was per-formed to detect the recombinant CiaR (rCiaR)-binding pbps genes. A suicide plasmid pEVP3ciaR for ciaR gene knockout was constructed and then aΔciaR mutant was obtained through homologous recombination and insertion inactivation of the suicide plasmid, and screening with chloromycin. The mutant was identified using PCR and sequencing analysis. E-test was used to detect the minimal inhibitory concentrations ( MIC) of penicillin ( PCN) and cefotaxime ( CTX) against S. pneumoniae strains. Changes and differences in the expression of pbps genes and csRNAs in theΔciaR mutant and its wild-type strain before and after treatment with 1/4 MIC of PCN or CTX were detected using real-time quantitative RT-PCR. Results The rCiaR could bind to the promoter regions in pbp1a, pbp1b and pbp2b genes of S. pneumoniae. The ciaR gene in ΔciaR mutant was inactivated by insertion according to the results of PCR and sequencing analysis. After treatment with 1/4 MIC of PCN or CTX, the expression of pbps genes at mRNA level ( pbps-mRNAs) in theΔciaR mu-tant was significantly increased (P<0. 05), but the levels of csRNAs were significantly decreased (P<0. 05);whereas a significantly decreased pbps-mRNAs (P<0. 05) and increased csRNAs (P<0. 05) were observed in its wild-type strain. The result of E-test showed that the MICs of PCN and CTX against ΔciaR mutant were increased by 250-fold as compared with those against its wild-type strain. Conclusion The CiaR can enhance the drug resistance of S. pneumoniae to PCN and CTX through down-regulating the expres-sion of PBP1a, PBP1b and PBP2b and up-regulating the expression of csRNAs to inhibit the expression of PBPs.
RESUMO
Objective To longitudinally analyze microstructural white matter changes in high risk mild cognitive impairment (MCI) patients with DTI.Methods Structural MRI,DTI and psychometric analyses were performed in 102 individuals with 1 year follow-up.At the end of the follow-up,11 participants were diagnosed with MCI (CN-MCI group),while 91 participants were classified as cognitively stable (CN-stable group).The differences of fractional anisotropy (FA) and mean diffusivity (MD) between the two groups were analyzed,and the relationship with MCIs and cognitive ability was observed with multivariate logistic regression analysis.Results At the baseline assessment,MD of CN-MCI group increased in fornix and left parahippocampal gyrus white matter compared with those of CN-stable group (P＜0.05).For 1-year follow-up reassessment,the MD of CN-MCI group increased in the fornix,left parahippocampal gyrus white matter,left eingulum and splenium,while FA of CN-MCI group reduced in fornix and left parahippocampal gyrus white matter compared with those of CN-stable group (P＜0.05).Multivariate Logistic regression analysis showed that MD of the fornix could be a predictor of conversion from a high risk MCI to MCI,and MD of left parahippocampus gyrus white matter was a risk factor for increased CDR scores.Conclusion For high-risk MCI individuals,microstructural white matter changes may be used as potential imaging biomarkers in the early phase of AD.