Objective To assess the application value of REP-PCR in genotyping of candida glabrata strains in clinical pratice. MethodsFrom 2009 to 2010, thirty-eight candida glabrata strains were isolated from Shanghai Ruijin Hospitals, Shanghai Renji Hospital, Shanghai Huashan Hospital, Anhui Medical University Hospital, Shenzhen People's Hospital. Six loci in housekeeping genes (FKS, LEU2,NMT1, TRP1, UGP1 and URA3 ) were amplified and sequenced. The sequences were compared with the MIST database and allele profile and sequence type (ST) were obtained. With primers Ca21, Ca22 and Com21 used to amplify the adjacent variable gene regions,the amplicons were analyzed through electrophoresis to generate different REP-PCR types. Finally, the results of these two genotyping methods were compared. ResultsFor REP-PCR, Ca22-Com21 has the best genotyping effect. REP-PCR and MLST have the same genotyping results. Five REP-PCR types were found in 38 candida glabrsta isolates. Type A,B, C, D and E strains from REP-PCR were genotyped as ST 7, 3, 19, 45 and new type respectively byMIST. REP-PCR saves time compared with MIST. Conclusions REP-PCR offers a simple and rapid method for molecular typing, with similar discriminatory power with MIST. Therefore, REP-PCR can be the preferred choice in laboratory, especially for a large number of isolates.

Objective To investigate the mechanisms of fluconazole resistance in clinical and experimental induced isolates of C.glabrata.Methods Efflux of rhodamine 6G was performed to evaluate the effects of efflux pumps.The expression levels of transporter genes CDR1,CDR2,SNQ2 and ERG11 were examined by real-time RT-PCR.Meanwhile,sequence of PDR1 was determined by PCR based DNA sequencing.Results Efflux pumps of all fluconazole-resistant isolates had stronger effects than that of susceptible isolates,consistently with significant upregulation of CDR1,but no obvious difference was found in CDR2 or SNQ2.Also,no notable change in the expression level of ERG11 between susceptible and resistant isolates.PDR1 mutations existed in both clinical and experimental induced isolates of C.glabrata,among which P927S,L543P and S947L haven't been reported previously.Conclusion Mutations of PDR1 were induced by fluconazole both in vivo andin vitro,which will result in overexpression of CDR1 and strengthen the effect of efflux pump.

Objective To rapidly visualize and detect the extracellular vesicles of glial cells in a simulated space environment.Methods By using 2,5 Gy irradiation and 12,24 h microgravity treatment,a damage model of glial cells was established in a simulated space environment.Exosomes extracted from conditioned media with reagent kits were transferred to neurons to elucidate the impact of glial cells on neurons.By performing live-cell fluorescence labeling of exosomes,a visualization monitoring scheme based on fluorescence intensity analysis was developed to characterize the release patterns of extracellular vesicles.The release patterns of exosomes were represented by the fluorescence intensity of the conditioned media.The effects of different storage conditions and duration on the quantity and size of exosomes were investigated.Results The exosomes released from damaged glial cells in the simulated space environment could to some extent protect neurons,with exosomes playing a decisive role in this process,and the neurosystem exosome visualization detection scheme was consistent with the traditional exosome validation scheme.An empirical curve for exosome quantity and size was established for semi-quantitative analysis,providing a new approach for rapid detection of exosomes in cell culture media.Furthermore,the optimal storage conditions for culture medium samples were clarified,laying the foundation for ground/space-based online analysis of culture medium samples after spaceflight.Conclusion Exosome rapid detection and analysis can be achieved through fluorescence labeling and utilized to investigate glial cell injury in a simulated space environment.