Objective To estimate the application value of colony PCR in the detection of pathogenic filamentous fungi. Methods Colony PCR was established and performed to amplify the internal transcribed spacer (ITS) region of 19 species (strains) of filamentous fungus followed by sequencing analysis. At the same time, DNA extracts from 8 of the 19 species of filamentous fungus were subjected to conventional PCR. Hha I and Hinf I endonucleases were used for restriction fragment length polymorphism (RFLP) analysis of the conventional and colony PCR products. Comparison analysis was carried out between the colony and conventional PCR. Results Of the 19 strains, 16(84.2%) yielded positive results by colony PCR; sequence analysis of the PCR products of ITS region revealed a 96% - 100% similarity with the reference sequence (NCBI database)of corresponding fungi. The amplification product length and RFLP profile of these products from the 8 species of filamentous fungus, except for those from Aspergillus nidulans, were consistent between the colony and conventional PCR. Conclusions Compared with conventional PCR, colony PCR-based detection of filamentous fungi is easy to operate, time and labor-saving, with high accuracy and reliability, and can be applied to the rapid identification of filamentous fungi.

Objective To develop a PCR-RFLP method to rapidly identify filamentous fungi causing deep infection. Methods Universal fungal primers were used to amplify the internal transcribed spacer (ITS) region of Aspergillus fumigatus, Aspergillus Bavus, Aspergillus terreus, Aspergillus niger, Aspergillus versicolor, Aspergillus nidulans, Scedosporium apiospermum and Fusarium moniliforme followed by restriction fragment length polymorphism (RFLP) analysis with restrictive endonucleases Hha I, Hae III, Hinf I, Taq I and Msp I. Then, 22 clinical and 2 environmental fungal isolates were identified with the developed PCR-RFLP method. Results The RFLP analysis of PCR products with restrictive endonucleases Hha I and Hinf I allowed discrimination of 8 filamentous fungi causing invasive infection, and it took only 1 day to carry out the whole procedure from DNA extraction to PCR and restriction digestion. The identification results of 22 clinical strains and 2 environmental isolates with this PCR-RFLP method were completely consistent with those with conventional morphological method. Conclusion PCR-RFLP analysis is an efficient method for rapid identification of cultured filamentous fungi.

Humans ; Occupational Exposure ; Occupational Health ; Risk Factors ; Sanitation

Objective To genotype Trichosporon spp. with rDNA-ITSAGSl-RFLP analysis followed by cluster analysis, and attempt to apply this method to rapid species identification of human pathogenic Trichosporon spp.. Methods Fourteen strains of Trichosporon, which belonged to 8 species, were collected. The rDNA-ITS/IGSl regions were amplified by PCR and sequenced. Simultaneously, the amplicons were digested separately with restriction enzymes, including Hae III, Hha I , Hae IH and Hha I , Hinf I , Msp I and Taq I . Results The 8 species of Trichosporon could be classified into 4 subgroups with rDNA-ITS-RFLP, while inter-species identification of all the 14 strains from 8 species of Trichosporon could be realized with rDNA-IGSl-RFLP. Also, those genotypes of T. asahii which had relative long phylogenic distance could even be discriminated with rDNA-IGSl-RFLP. Conclusion The rDNA-ITS/IGSl-RFLP analysis is expected to be used in rapid interspecific identification of genus Trichosporon.

ObjectiveTo evaluate the biological characteristics of rat bone marrow mesenchymal stem cells (BMSCs) transfected with hypoxia-inducible factor-1α(HIF-1 α) gene.MethodsThe rat BMSCs of 3rd generation and the vector expressing HIF-1α gene (pcDNA3.1-HIF-1α) were provided by department of anesthesia,Tangdu Hospital,the 4th Military Medical University.BMSCs expressing HIF-1α gene (BMSCs-HIF-1α cells) were constructed by transfection of vector pcDNA3.1-HIF-1α into BMSCs by means of electroporation.Successful transfection of HIF-1α gene was confirmed by immuno-cytochemistry.Simple BMSCs and BMSCs-pcDNA3.1 cells were used as control cells.After being cultured in hypoxic condition HIF-1α expression was detected by Western blot analysis.Flow cytometry was used to determine the proportion of cells in G1,G2 and S phase and detect apoptosis.The proliferation index (PI) was calculated.The cell growth curve was described by MTT assay and the number of the 3 types of cells was recorded.ResultsA large number of deep blue granules were observed in the nuclei of BMSCs-HIF-1α cells using immuno-cytochemistry but no such granule was found in the two types of control cells.HIF-1α expression was significantly up-regulated and apoptosis rate (the number of apoptotic cella/the total number of cells examined) decreased in BMSC-HIF-1α cells compared with the control cells.The proportion of cells in S and G2 phase was significantly higher and the proportion of cells in G1 phase was significantly lower and PI higher in BMSCs-HIF-1α cells than in the control cells.The number of BMSCs-HIF-lα cells was significantly higher than the number of the two types of control cells at day 3-8 of culture.There was no significant difference in the above variables between BMSCs and BMSCs-pcDNA3.1 cells.ConclusionBMSCs-HIF-1α is successfully constructed by transfection of vector pcDNA3.1-HIF-1α gene into BMSCs by means of electroporation.

Objective:To establish the normal reference value of lumbar bone mineral density (BMD) under quantitative CT (QCT) in Chinese healthy adult females and to explore the regional differences.Methods:Total of 35 431 healthy women who met the inclusion criteria of Chinese health quantitative CT big data program were selected in this study. The BMD of the central plane of L 1 and L 2 vertebrae was measured by Mindways′s QCT system, and the mean value was taken. One-way analysis of variance was used to compare the BMD differences of lumbar vertebrae in women of different ages and regions. The subjects were grouped by an age interval of 10 years, and the level of BMD in different regions of the same age group were compaired. Results:The peak BMD of Chinese healthy adult women appeared in the age group of 20-29 years (Northeast China(183.01±24.58) mg/cm 3, North China (188.93±24.80) mg/cm 3, East China (187.54±27.71) mg/cm 3, South China (186.22±33.72) mg/cm 3, Central China (176.33±24.91) mg/cm 3, Southwest China(182.25±28.00) mg/cm 3), and then it decreased with age. The level of BMD in different regions decreased with the age. Before the age of 70 years, BMD in Central and Southwest China was always at a low level((176.23±24.91) to (90.38±28.12) mg/cm 3, 182.25±28.00 to (88.55±25.68) mg/cm 3), lower than those in Northeast China ((183.01±24.58) to (99.69±27.85) mg/cm 3), North China ((188.93±24.80) to (95.89±26.12) mg/cm 3), East China ((187.54±27.71) to (95.65±27.86) mg/cm 3). After 70 years of age, BMD tended to be the same in different regions ( P>0.05). The BMD values in Central China and Southwest China were similar in the age group of 40-60 years ( P>0.05). The BMD values in the health adult femles in the age group of 60 years in different regions of Chinawere all lower than those of bone mass abnormality (all P<0.05). The detection rate of osteoporosis in females over 50 years was the highest in Southwest China (25.65%) and it was the lowest in North China (17.30%). Conclusions:This study establishes reference values of BMD under QCT in healthy Chinese women, which can be used as a reference basis for identifying women with low BMD who are at risk of osteoporosis. The BMD value is the lowest in Southwest China and the highest in South China.

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*