Establishment of a recombined mannose-binding lectin protein-magnetic beads-enriched binding recombinant enzyme-assisted polymerase chain reaction assay for Candida in blood samples / 中华流行病学杂志

Objective: To establish a nested recombinant enzyme-assisted polymerase chain reaction (RAP) technique combined with recombined mannose-binding lectin protein (M1 protein)-magnetic beads enrichment for the detection of Candida albicans (C. albicans) and Candida tropicalis (C. tropicalis) in blood samples for the early diagnosis of candidemia albicans and candidiemia tropicalis. Methods: The primer probes for highly conserved regions of the internal transcribed spacerregions of C. albicans and C. tropicalis were deigned to establish RAP assays for the detections of C. albicans and C. tropicalis; The sensitivity and reproducibility of nucleic acid tests with gradient dilutions of standard strains and specificity of nucleic acid tests with common clinical pathogens causing bloodstream infection were condcuted. M1 protein-magnetic bead enriched plasma C. albicans and C. tropicalis were used for RAP and PCR in with simulated samples and the results were compared. Results: The sensitivity of the established dual RAP assay was 2.4-2.8 copies/reaction, with higher reproducibility and specificity. M1 protein-magnetic bead enrichment of pathogen combined with the dual RAP assay could complete the detections of C. albicans and C. tropicalis in plasma within 4 hours. Fie the pathogen samples at concentration <10 CFU/ml, the number of the samples tested by RAP was higher than that tested by PCR after enrichment. Conclusion: In this study, a dual RAP assay for the detections of C. albicans and C. tropicalis in blood sample was developed, which has the advantages of accuracy, rapidity, and less contaminants and has great potential for rapid detection of Candidemia.

Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells / 北京大学学报(医学版)

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

Etiology and clinical features of fever of unknown origin.

OBJECTIVE: To investigate the etiology and clinical features of fever of unknown origin (FUO). METHODS: The clinical data including etiology, diagnostic approaches, and clinical features were retrospectively analyzed in 816 patients with FUO who were presented in our department from January 2000 to January 2009. RESULTS: Of these 816 FUO cases, 766 (93.9%) were confirmed to be with infective diseases(40.4%, n=330), connective tissue diseases (34.4%, n=281), malignant tumors (10.9%, n=89), other known diseases (8.1%, n=66), and unknown diseases (6.1%, n=50). The most common infective disease was tuberculosis (49.7%, 164/330), the most common connective tissue disease was adult-onset Stills disease (AOSD)(55.2%, 155/281), the most common malignant tumor was lymphoma(56.2%, 50/89), and the most common "other known disease" was Crohns disease(22.7%, 15/66). All lung cancer cases had obstructive pneumonia. Significantly more elderly patients suffered from infective diseases (49.4% vs.32.0%) and malignant tumor (15.6% vs. 6.4%) compared with the non-elderly (both P=0.0000), while the proportion of connective tissue diseases was significantly less than that of the non-elderly (17.9% vs. 50.1%, P=0.0000). CONCLUSIONS: Most FUO can be confirmed after careful examinations and analysis. The main cause of FUO is infective diseases, especially tuberculosis in the elderly. The connective tissue diseases and malignant tumors are also important causes.

Effects of different Helicobacter pylori culture filtrates on growth of gastric epithelial cells.

AIM: To study the effects of different Helicobacter pylori (H pylori) culture filtrates on growth of gastric epithelial cells. METHODS: Broth culture filtrates of H pylori were prepared. Gastric epithelial cells were treated with the filtrates, and cell growth was determined by growth curve and flow cytometry. DNA damage of gastric epithelial cells was measured by single-cell microgel electrophoresis. RESULTS: Gastric epithelial cells proliferated actively when treated by CagA-gene-positive broth culture filtrates, and colony formation reached 40%. The number of cells in S phase increased compared to controls. Comet assay showed 41.2% comet cells in GES-1 cells treated with CagA-positive filtrates (P < 0.05). CONCLUSION: CagA-positive filtrates enhance the changes in morphology and growth characteristics of human gastric epithelial tumor cells. DNA damage maybe one of the mechanisms involved in the growth changes.

[Risk factors of elderly patients with total gastrectomy for gastric cancer].

OBJECTIVE: To analyze the relative risk factors of elderly patients with total gastrectomy for gastric cancer. METHODS: The risk factors for hospital death and postoperative complications in 131 elderly patients undergoing total gastrectomy for gastric cancer from Aug. 1994 to Aug. 2004 were analyzed retrospectively. RESULTS: The risk factors for hospital death and postoperative complications included coexistent diseases, hemoglobin level less than 80 g/L, albumin level less than 35 g/L, body mass index(BMI) less than 18.5 kg/m (2), intraoperative blood loss more than 1000 ml, operating time longer than 5 h, combined resection of the spleen or pancreas. The relative risks were 1.57, 1.74, 2.97, 4.23, 2.21, 2.28, 3.80 respectively for hospital death, and 1.50, 1.90, 2.38, 2.12, 2.45, 1.66, 3.41 for postoperative complications. CONCLUSION: The risk factors of the elderly patients with total gastrectomy for gastric cancer should be considered carefully during the perioperative period. It can increase the security of the procedure to control these risk factors.