BACKGROUNDStent thrombosis is one of severe complications after sirolimus-eluting stent implantation. Rapamycin (sirolimus) promotes arterial thrombosis in in vivo studies. However, the underlying molecular and transcriptional mechanisms of this adverse effect have not been thoroughly investigated. This study was designed to examine the effects of rapamycin on the expression of the gene, Kruppel-like factor 2 (KLF2), and its transcriptional targets in mice.

METHODSMice were randomly divided into four groups: the control group (intraperitoneal injection with 2.5% of dimethyl sulfoxide (DMSO) only), rapamycin group (intraperitoneal injection with 2 mg/kg of rapamycin only), Ad-LacZ + rapamycin group (carotid arterial incubation with Ad-LacZ plus intraperitoneal injection with 2 mg/kg of rapamycin 10 days later), and Ad-KLF2 + rapamycin group (carotid arterial incubation with Ad-KLF2 plus intraperitoneal injection with 2 mg/kg rapamycin 10 days later). The carotid arterial thrombosis formation was induced by FeCl3 and the time of arterial thrombosis was determined. Finally, the RNA and protein of carotid arteries were extracted for KLF2, tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), endothelial nitric oxide synthase (eNOS), thrombomodulin (TM) mRNA and protein analysis.

RESULTSCompared with controls, treatment with rapamycin inhibited KLF2, eNOS and TM mRNA and protein expression, and enhanced TF and PAI-1 mRNA and protein expression, and shortened time to thrombotic occlusion from (1282 ± 347) seconds to (715 ± 120) seconds (P < 0.01) in vivo. Overexpression of KLF2 strongly reversed rapamycin-induced effects on KLF2, eNOS, TM, TF and PAI-1 expression. KLF2 overexpression increased the time to thrombotic occlusion to control levels in vivo.

CONCLUSIONSRapamycin induced an inhibition of KLF2 expression and an imbalance of anti- and pro-thrombotic gene expression, which promoted arterial thrombosis in vivo. Overexpression of KLF2 increased KLF2 expression and reversed time to thrombosis in vivo.

Animals ; Carotid Arteries ; metabolism ; Drug-Eluting Stents ; adverse effects ; Kruppel-Like Transcription Factors ; analysis ; genetics ; physiology ; Mice ; Mice, Inbred C57BL ; Nitric Oxide Synthase Type III ; physiology ; Plasminogen Activator Inhibitor 1 ; physiology ; Sirolimus ; pharmacology ; Thrombomodulin ; physiology ; Thrombosis ; chemically induced

Altitude ; Heart Rate ; Humans ; Oxygen ; Oxygen Consumption

Objective To investigate the role of overexpression of Smad7,the inhibitory factor of TGF-?/Smads signaling,in epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells.Methods Peritoneal fibrosis rat model was built by daily intraperitoneal injection with 4.25% Dineal (100 ml/kg) and lipopolysaccharide(LPS) (0.6 mg/kg) at day 8,10,12,22,24,26. Smad7 or control empty vectors was transferred at day 0,14 and was induced by doxycline in the daily drinking water (200 mg/L).Rats were sacrificed on day 28 and the expression of TGF-beta/ Smads,?-SMA and E-cadherin was examined.Results Compared with normal rats,empty vector rats showed higher expression of phosphorylated Smad2/3.?-SMA expression was elevated but E-cadherin was reduced.Under electron microscope,the mesothelial cells removed to submesothelial zone and showed large bundles of actin microfilaments and dense bodies within the cytoplasm. Basement membrane was broken.After induction of Smad7 in peritoneal fibrosis rats,the morphology of mesothelial ceils normalized partly,phosphorylated Smad2/3 was reduced.Moreover,expression of E-cadherin was increased,expression of?-SMA was dramatically reduced.Conclusion Inhibition of TGF-?/Smad signaling by Smad7 overexpression may inhibit the epithelial-mesenchymal transition of mesothelial cell,which may provide a new therapeutic method for peritoneal fibrosis by overexpression of Smad7.

BACKGROUNDThe incidence of vancomycin-resistant enterococci (VRE) appeared to be increasing in China, but very few nosocomial outbreaks have been reported. Our hospital had experienced an outbreak of VRE since March 2008 to March 2009. The objective of this study was to analyze the molecular features of the isolates and the control measures used to eradicate a VRE outbreak in a tertiary institution in China.

METHODSWe characterized VRE isolates from 21 infected and 11 colonized inpatients from a single hospital by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), the analysis of Tn1546-like elements and virulence genes detection. Infection control measures, including more environmental disinfection, screening for VRE colonization, contact precautions, education and strict antibiotic restriction, were implemented to control the outbreak.

RESULTSDuring the outbreak, a total of 32 VRE strains were obtained. There were 21 strains found in Emergency Intensive Care Unit (EICU), 9 isolates from Geriatric Ward, and two from other units. All the isolates harbored the vanA gene, however, four of them exhibited the VanB phenotype. Meanwhile, MLST analysis revealed that all isolates belonged to clonal complex (CC) 17. With the infection-control measures, the epidemic was constrained in two units (EICU and Geriatric Ward). After March 2009, no further case infected with VRE was detected in the following one-year period.

CONCLUSIONThe outbreak was controlled by continuous implementation of the infection control programme, and more rigorous infection control policy is needed.

China ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium ; drug effects ; genetics ; pathogenicity ; Gram-Positive Bacterial Infections ; microbiology ; transmission ; Hospitals ; Humans ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Polymerase Chain Reaction ; Vancomycin Resistance ; genetics ; physiology

OBJECTIVETo setup a quantitative assay for detection of cyclooxygenase-2 (COX-2) methylation in human gastric mucosa samples.

METHODSA standard analysis system was established by denaturing high performance liquid chromatography (DHPLC) under the condition of 55 degrees C oven temperature and a linear acetonitrile gradient (4.0/min). While, a total of 10 cases of gastric biopsy samples were detected for methylation status of COX-2.

RESULTSThe complete methylated human promyelocytic leukemia cells (HL-60) and unmethylated gastric cancer cell line (MGC803) were used as positive and negative control. The proportion of the methylated copies of COX-2 was calculated according to the peak heights of methylated (M) and unmethylated (U) COX-2 in same PCR amplicon. The formula was Y = 1.0608 x M/(M + U), R(2) = 0.9894. Among 10 biopsy samples, the proportions of methylated copies of COX-2 in 2 cases of dysplasia were higher than superficial gastritis and chronic atrophy gastritis (24.5%, 18.4% vs 7.6%, 9.6%).

CONCLUSIONThe methylation of COX-2 promoter CpG islands can be detected in human gastric mucosa samples by quantitative DHPLC assay, which could be used in the population-based study of precancerous gastric lesions.

Cell Line, Tumor ; Chromatography, High Pressure Liquid ; methods ; CpG Islands ; Cyclooxygenase 2 ; genetics ; DNA Methylation ; Gastric Mucosa ; metabolism ; pathology ; Humans ; Promoter Regions, Genetic ; Stomach Diseases ; genetics ; metabolism ; pathology

OBJECTIVETo evaluate the relationship between cyclooxygenase-2 (COX-2) methylation and expression, and precancerous gastric lesions.

METHODSMethylation status of COX-2 was evaluated by quantitative denaturing high performance liquid chromatography (DHPLC) in 1201 subjects with different gastric lesions. COX-2 expression was assessed by immunohistochemistry and Helicobacter pylori (H pylori) infection status was determined by 13C-urea breath test (13 C-UBT).

RESULTSThe percent of COX-2 methylation was increased steadily with the severity of gastric lesions, showing 10.6% of which with superficial gastritis/chronic atrophic gastritis (SG/CAG), 11.8% with intestinal metaplasia (IM) and 13.8% with indefinite dysplasia/dysplasia (Ind DYS/DYS) (chi2 = 8.312, P = 0.016). Stratified analysis indicated that the percents of COX-2 methylation in subjects with H pylori negative still increased with the severity of gastric lesions,of 8.8% in SG/CAG, 10.6% in IM and 14.1% in Ind DYS/DYS (chi2 = 6.629, P= 0.036). Moreover,the methylated proportion of COX-2 was negatively associated with the expression in gastric lesions, from 13.3% with mild expression to 7.6% with strong expression (chi2 = 10.400, P = 0.015).

CONCLUSIONOur findings indicated that COX-2 methylation was significantly associated with precancerous gastric lesions and H pylori infection, suggesting that promoter methylation of COX-2 might play an important role in the progression of gastric lesions.

Adult ; China ; epidemiology ; Cyclooxygenase 2 ; genetics ; metabolism ; DNA Methylation ; Female ; Gastric Mucosa ; metabolism ; pathology ; Helicobacter Infections ; epidemiology ; metabolism ; pathology ; Humans ; Male ; Middle Aged ; Promoter Regions, Genetic ; Stomach Diseases ; epidemiology ; metabolism ; pathology

OBJECTIVETo investigate the associations of serum gastrin-17 (G-17) concentration with helicobacter pylori (Hp) infection.

METHODSA (13)C-urea breath and ELISA test to determine the Helicobacter pylori status and to detect the serum gastrin concentration was conducted in 242 villagers in Linqu of Shandong Province, a high gastric cancer prevalence area in China.

RESULTSOf 242 subjects, 65 of 111 were found Hp-positive in males (58.56%), compared with 65 of 131 in females (49.62%) (chi(2) = 1.932, P = 0.165). The statistical difference was not observed among different age groups (chi(2) = 4.185, P = 0.123). The average level of G-17 among 242 subjects was (24.43 +/- 25.46) pmol/L and it was statistically higher in females (29.87 +/- 28.18) pmol/L than that in males (18.01 +/- 20.11) pmol/L (Z = -3.618, P < 0.001). However, there was no statistical difference found among age groups (chi(2) = 1.948, P = 0.378). The G-17 level in Hp-negative group (35.50 +/- 30.92) pmol/L was observed significantly higher than in Hp-positive group (14.90 +/- 13.79) pmol/L (Z = 5.368, P = 0.0001).

CONCLUSIONThe G-17 concentration was found higher in Hp-negative subjects than in Hp-positive subjects, and higher in female than in male, but no difference was found among age groups.

Adult ; China ; epidemiology ; Female ; Gastrins ; blood ; Helicobacter Infections ; blood ; epidemiology ; Helicobacter pylori ; isolation & purification ; Humans ; Male ; Middle Aged ; Rural Population ; Sampling Studies

Objective:To establish the HPLC fingerprint of raw products,rice-processed products and honey-processed products of Codonopsis Radix from Shanxi,and establish determination of their chemical constituents,which was used to analyze the changes of types and contents of chemical constituents in Codonopsis Radix from Shanxi before and after processing. Method:Agilent ZORBAX SB-C₁₈ column(4.6 mm×250 mm,5 μm) was adopted,the separation process was carried out using a binary gradient elution system composed of 0.5% phosphoric acid aqueous solution and acetonitrile,the column temperature was 30℃ and the detection wavelength was 220 nm. Result:Compared with the corresponding reference fingerprint,the similarities of HPLC fingerprint of 10 batches of raw products and processed products were >0.90.In raw products,rice-processed products and honey-processed products of Codonopsis Radix from Shanxi,the contents of lobetyolin were (0.33±0.049),(0.24±0.034),(0.18±0.047) mg·g^-1,the contents of atractylenolide Ⅲ were (0.20±0.046),(0.40±0.046),(0.31±0.060) mg·g^-1,the contents of 5-hydroxymethylfurfural(5-HMF) were (0.74±0.16),(1.45±0.19),(1.54±0.12) mg·g^-1,respectively. Conclusion:Different processing methods have little effect on types of chemical constituents in Codonopsis Radix from Shanxi,but have great effect on the contents of some chemical constituents.

Objective:To explore the repairing effects of 3D-printed nano-β-tricalcium phosphate (β-TCP) scaffolds loaded with vancomycin and bone morphogenetic protein-2 (BMP-2) for seawater -soaked tibial bone defects in rabbits. Methods:A total of 27 male New Zealand White rabbits were assigned to the normal group using a random number table method, with each group consisting of 9 rabbits. The rabbit tibial bone defect model was created using the osteotomy surgical method. Eight hours after operation, the wounds in the control group and seawater group were immersed in seawater for 2 hours, and those in the normal group were not immersed. After an observation period of 5-7 days, no significant redness or purulent discharge was observed in the wound appearance, then debridement was performed followed by corresponding implantations: the control group with gelatin sponges loaded with vancomycin and BMP-2, and the other two groups with 3D-printed nano-β-TCP scaffolds loaded with vancomycin and BMP-2. After filling the bone defects with the respective materials, all groups underwent layer-by-layer suturing of the wound, followed by disinfection with iodine and injection of gentamicin to prevent infection. The affected limbs were then immobilized using a plaster cast. The affected limbs were imaged using anteroposterior X-ray at 4, 8 and 16 weeks after operation, and the repair effects were evaluated using the Lane-Sandhu X-ray scoring system. At 16 weeks after operation, the bone defect tissues were collected for HE staining to observe bone tissue growth.Results:At 4 weeks after operation, the Lane-Sandhu X-ray score in the control group was significantly lower than that in the normal group [(2.8±1.1)points vs. (1.1±0.9)points] ( P<0.05), and that in the seawater group [(2.2±1.0)points] was not significantly different from those in the other two groups (all P>0.05). At 8 weeks after operation, the seawater group [(6.1±0.9)points] and the control group [(2.8±1.0)points] exhibited lower Lane-Sandhu X-ray score compared to the normal group [(8.2±1.0)points] (all P<0.05), and the seawater group showed a higher score compared to the control group ( P<0.05). At 16 weeks after operation, the control group [(3.8±1.0)points] exhibited a lower Lane-Sandhu X-ray score compared to the normal group [(10.0±1.3)points] and the seawater group [(9.3±1.2)points] (all P<0.05), while no significant difference was noted between the latter two ( P>0.05). At 16 weeks after operation, histological observations revealed varying degrees of bone tissue formation in three groups, with the normal group showing the best bone defect repair effect, followed by the seawater group. Conclusion:The 3D-printed nano-β-TCP scaffolds loaded with vancomycin and BMP-2 are effective for the treatment of seawater -soaked bone defects, which can promote bone tissue repair.

OBJECTIVETo screen the MYBPC3 gene mutations in Han Chinese patients with hypertrophic cardiomyopathy (HCM).

METHODSSixty-six patients with HCM were enrolled for the study. The exons in the functional regions of MYBPC3 were amplified with PCR and the products were sequenced.

RESULTSFour novel mutations and four common polymorphisms were identified in this patient cohort. A Lys301fs mutation in exon10 was evidenced in a H30, and when he was 47 years old, he had the chest tightness, shortness of breath with septal hypertrophy of 18.7mm; a Asp463stop mutation in exon17 was detected in a H48, he was 24 years old 24-year-old when a medical examination showed ventricular septal hypertrophy of 15.4 mm; both Gly523Arg mutation in exon18 and Tyr847His mutation in exon26 were found in a H53 with onset age 36 years old, feeling chest tightness after excise and his ventricular septal hypertrophy was 27 mm that time. MYBPC3 mutations occurred in 4.5% patients in this cohort. These mutations were not found in 100 non-HCM control patients.

CONCLUSIONMYBPC3 mutation is presented in a small portion of Han Chinese patients with HCM.

Adult ; Asian Continental Ancestry Group ; genetics ; Cardiomyopathy, Hypertrophic ; genetics ; Carrier Proteins ; genetics ; DNA Mutational Analysis ; Exons ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Mutation ; Phenotype ; RNA, Messenger ; genetics