Bimolecular fluorescence complementation (BiFC) assay is an innovative approach to investigate protein interactions, based on the reassembly of protein fragments of the fluorescent proteins which directly report interactions. The fluorescent proteins tolerate circular permutation and insertions of foreign proteins with maintenance of fluorescence. So when the proteins fused to the reporter fragments interact with each other, a direct readout of the association would be given from the facilitating reassembly of the active reporter protein. Moreover, with distinct spectra difference of the fluorescent protein family members, BiFC assay is expanded to multicolor BiFC assay which enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners. From it first reported in Molecular Cell in 2002 till now, this approach has been used on the networks of protein interaction in mammal cells, plant cells or even E.coli, and researches on transcription factors, G protein ?? dimmers, protein ubiqutination and so on.

Gene disruption by homologous recombination is a powerfu l tool for investigating gene function in yeast. Since 1980’s, it has been deve loped a lot. PCR-mediated gene disruption technique makes the manipulation easi er and it can be used to precisely delete genes in yeast. Multi-gene disruption technique can delete several genes successively in the same yeast strain. After the completion of the yeast Saccharomyces cerevisiae genome sequencing, the gene disruption technique for systematic analysis meets the need of the functio nal genomics in yeast. It also enlighten the study on human functional genomics.

Objective: To establish a highly effective transformation method of Cryptococcus neoformans. Methods: Special reagents was used to make C. neoformans take in external DNA under given condition. This chemical transformation result was compared with that of electrotransformation. The feasibility of this chemical transformation was tested by plasmid stability test. Results: The efficiency of this chemical transformation was more than 103 transformants/?g plasmid DNA, far more than that of the traditional electrotransformation. Conclusion: An appropriate transformation method is established for C. neoformans transformation, which has high transformation efficiency.

Objectives In order to develop a rapid microsatellite genotyping assay for inter-strain differentiation of Candida albicans isolates and understand the transmission characteristics of the infections. Methods DNA was extracted from C. albicans isolates from genitals, anal canals and oral cavities of 39 women and 27 men with genital candidiasis. The microsatellite sequences in stabel genes(CDC3, EF3 and HIS3) were amplified by a fluorescence labeled PCR. Fluorescent signals were read with an automatic se- quencer, and the data were collected with GeneScan software followed by genotyping with Genotyper soft- ware to analyze polymorphic microsatellite loci. Results Combined analysis of the 3 microsatellite markers showed 18 gene allele associations in C. albicans from genital sites of all men and women, including 10 in women, 11 in men and 3 in both. The allele associations of dominant pathogenetic strains for both sexes were 116:124, 122:131,160:200, which covered 50% of pathogenetic infection. Three common allele associations for both sexes covered 71% of all infections. Genitals and anal canals shared strains of same allele associations in 80% of women and in only 3.8% of men. The strains of same allele associations were identified in both genitals and mouth in 2.7% of women but in none of men. In their genital sites 71% of couples shared the same allele strains, of which 80% were the dominant pathogenetic strains identified in both sexes. Conclusions The improved microsatellite genotyping assay is useful for rapid differentiation, identification of infective source, and contact tracing of C. albicans infection. There are pathogenetic C. albi- cans strains with predominant allele associations in genital infections.

To investigate the effect of microRNA?26a?5p on osteogenic differentiation of human periodontal ligament stem cells (hPDLSC) and its related mechanisms. Methods hPDLSC in periodontal tissues from healthy adults and hPDLSC from periodontitis patients (PPDLSC) were isolated and cultured in vitro , respectively. The PPDLSC were divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups. GroupⅠis control group, and the other four groups were transiently transfected with miR?NC, miR?26a?5p, antimiR?NC and antimiR?26a?5p lentiviral vectors, respectively. The osteogenic differentiation abilities of the cells in vitro were determined by alizarin red staining, alkaline phosphatase (ALP) activity assay and real?time quantitative PCR (qPCR). Totally 40 male mice (6?weeks) were equally divided into five groups with 8 mice in each group. The PPDLSCs cells (1×107/ml) inⅠ,Ⅱ,Ⅲ,ⅣandⅤgroups, which adhered to hydroxyapatine?tricalcium phosphate (HA?TCP), were implanted into the nude mice subcutaneously and the animal models were constructed to analyze the effect of miR?26a?5p on the osteogenic differentiation of PPDLSCs in vivo. PPDLSCs were divided into A, B, C, D groups, and transfected with miR?26a?5p+Wnt5a?Wt, miR?NC+Wnt5a?Wt, miR?26a?5p+Wnt5a?Mut and miR?NC+Wnt5a?Mut in each of the above mentioned 5 groups, respectively. The luciferase activity assay was used to detect the relative luciferase in A, B, C and D groups to analyze the targeting relationship between miR?26a?5p and Wnt5a. Osteogenic differentiation related proteins expression were analyzed by western blotting. Results hPDLSC and PPDLSC were observed consistent with the characteristics of mesenchymal stem cells and had osteogenic differentiation ability in vitro. Compared with hPDLSC [(89.87±8.12) %], the osteogenic capacity of PPDLSC [(31.46±6.56) %] was significantly lower (P<0.05). The ALP activity (1.88±0.59), calcified nodules (79.88± 5.92), the expression of the osteogenic differentiation markers Runt?related transcription factor 2 (Runx2) (2.40 ± 0.70), ALP (2.10 ± 0.60) and osteocalcin (3.00 ± 0.90) mRNA in the PPDLSC from Group Ⅲ were significantly higher in comparison with the control group [(0.88±0.34), (29.69±2.65), (1.30±0.30), (0.09± 0.25), (1.71 ± 0.50)], while those from Group Ⅴ[(0.44 ± 0.07), (14.83 ± 3.05), (0.50 ± 0.11), (0.30 ± 0.08) and (0.80±0.17)] were significantly lower (P<0.05). In vivo studies in nude mice showed that the proportion of the osteogenic region [(34.96±5.65) %] in the miR?26a?5p group was significantly increased in comparison with the control group [(23.28±3.03) %], while in the antimiR?26a?5p group [(8.02±2.27) %] was significantly lower (P<0.05). The luciferase activity of the Group A (0.46 ± 0.06) was significantly lower than Group B (3.46±0.45) (P<0.05). Compared with the control group, the expression levels of Wnt5a protein, calmodulin kinase Ⅱ and protein kinase C proteins in the Group Ⅲwere significantly decreased, while those in the GroupⅤwere significantly increased (P<0.05). Conclusions MicroRNA?26a?5p could promote osteogenic differentiation of PPDLSC in vivo and in vitr o, and its mechanism might be inhibiting the activation of Wnt/Ca2+signaling pathway by targeting Wnt5a.

Objective: To investigate the effect of microRNA-26a-5p on osteogenic differentiation of human periodontal ligament stem cells (hPDLSC) and its related mechanisms. Methods: hPDLSC in periodontal tissues from healthy adults and hPDLSC from periodontitis patients (PPDLSC) were isolated and cultured in vitro, respectively. The PPDLSC were divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups. Group Ⅰ is control group, and the other four groups were transiently transfected with miR-NC, miR-26a-5p, antimiR-NC and antimiR-26a-5p lentiviral vectors, respectively. The osteogenic differentiation abilities of the cells in vitro were determined by alizarin red staining, alkaline phosphatase (ALP) activity assay and real-time quantitative PCR (qPCR). Totally 40 male mice (6-weeks) were equally divided into five groups with 8 mice in each group. The PPDLSCs cells (1×10⁷/ml) in Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups, which adhered to hydroxyapatine-tricalcium phosphate (HA-TCP), were implanted into the nude mice subcutaneously and the animal models were constructed to analyze the effect of miR-26a-5p on the osteogenic differentiation of PPDLSCs in vivo. PPDLSCs were divided into A, B, C, D groups, and transfected with miR-26a-5p+Wnt5a-Wt, miR-NC+Wnt5a-Wt, miR-26a-5p+Wnt5a-Mut and miR-NC+Wnt5a-Mut in each of the above mentioned 5 groups, respectively. The luciferase activity assay was used to detect the relative luciferase in A, B, C and D groups to analyze the targeting relationship between miR-26a-5p and Wnt5a. Osteogenic differentiation related proteins expression were analyzed by western blotting. Results: hPDLSC and PPDLSC were observed consistent with the characteristics of mesenchymal stem cells and had osteogenic differentiation ability in vitro. Compared with hPDLSC [(89.87±8.12)%], the osteogenic capacity of PPDLSC [(31.46±6.56)%] was significantly lower (P<0.05). The ALP activity (1.88±0.59), calcified nodules (79.88±5.92), the expression of the osteogenic differentiation markers Runt-related transcription factor 2 (Runx2) (2.40±0.70), ALP (2.10±0.60) and osteocalcin (3.00±0.90) mRNA in the PPDLSC from Group Ⅲ were significantly higher in comparison with the control group [(0.88±0.34), (29.69±2.65), (1.30±0.30), (0.09±0.25), (1.71±0.50)], while those from Group Ⅴ[(0.44±0.07), (14.83±3.05), (0.50±0.11), (0.30±0.08) and (0.80±0.17)] were significantly lower (P<0.05). In vivo studies in nude mice showed that the proportion of the osteogenic region [(34.96±5.65)%] in the miR-26a-5p group was significantly increased in comparison with the control group [(23.28±3.03)%], while in the antimiR-26a-5p group [(8.02±2.27)%] was significantly lower (P<0.05). The luciferase activity of the Group A (0.46±0.06) was significantly lower than Group B (3.46±0.45) (P<0.05). Compared with the control group, the expression levels of Wnt5a protein, calmodulin kinase Ⅱ and protein kinase C proteins in the Group Ⅲ were significantly decreased, while those in the GroupⅤ were significantly increased (P<0.05). Conclusions MicroRNA-26a-5p could promote osteogenic differentiation of PPDLSC in vivo and in vitro, and its mechanism might be inhibiting the activation of Wnt/Ca²⁺ signaling pathway by targeting Wnt5a.