Objective To establish a rapid and convenient method for determination of genomic DNA methylation in cells.Methods Five standard substances (dC, mdC, dA, dT and dG) were separated by high-performance capillary electrophoresis.Bare fused-silica capillary was used and eletrophoresis buffer was 48 mmol/L NaHCO3 with 60 mmol/L SDS, pH 9.6.The temperature of separation was controlled at 25 ℃ and a voltage of 20 kV was applied.The separation of the mixture was performed at a wavelength of 256 nm with UV-Vis detection and injection time was 5 seconds at 0.7 psi.Under optimal condition,genomic DNA methylation in methotrexate drug-resistant A549 cells was detected.Results The optimal condition was made by adjusting SDS concentration(40, 60, 80 mmol/L), pH value of running buffer(9.4,9.6, 9.8), voltage(15, 17, 19, 20, 22 kV), injection time(5, 10, 15, 20, 30 s) and capillary temperature(15, 20, 25, 30 ℃).The method for determination of genomic DNA methylation in cells was established.Five substances were completely separated by high-performance capillary electrophoresis in 10 mins.Intra-day coefficient of variation was less than 0.2% and inter-day coefficient of variation was less than 2%.The minimal detection limit was 2 μmol/L.Percentage of mdC in A549 parent cells was (4.80 ±0.52) %.Percentage of mdC in 15, 30, 40 μmol/L methotrexate drug-resistant A549 cells were (4.20±0.44) %, ( 3.70 ± 0.36 ) %, (3.10±0.35 ) %, respectively.Conclusions Genomic DNA methylation can be quantificated by high-performance capillary electrophoresis efficiently, rapidly, conveniently and sensitively.Genomic DNA methylation in methotrexate drug resistance cells decreases significantly.

Objective To summarize the experience of cannulation for extracorporeal membrane oxygenation (ECMO) in infants less than 5 kg. Methods Eleven infants with ECMO support who weighed less than 5 kg were admitted to critical care medicine of Guangzhou Women and Children's Medical Center from June 2016 to June 2018 were enrolled. Retrospective analysis of support type, configuration, site of cannula and complication of ECMO was performed. Results The 11 infants consisted of 9 males and 2 females. The weight on ECMO of 1.96-4.60 kg, with an average of (3.14±0.65) kg; age 0.1-30.0 days, with a median of 5.6 (1.5, 8.3) days. Four cases were given ECMO because of congenital diaphragmatic hernia with severe pulmonary hypertension and other 7 cases were cannulated due to complication of congenital cardiac surgery. All infants were received veno-arterial (V-A) ECMO. In 4 cases, the cannulas were placed in the right internal jugular vein for drainage (8-10 French catheter with 6.0-7.5 cm depth) and the right carotid artery for infusion (6-8 French catheter with 2.5-3.5 cm depth); the average time of cannulation in right carotid and jugular vessels was (73±20) minutes (range 55-100 minutes). In other 7 cases, the cannulas were inserted into the right atrium (12-14 French catheter with 2.8-3.0 cm depth) for draining blood and returning it to the ascending aorta (6-8 French catheter with 1.0-2.0 cm depth); the average time of cannulation in central vessels was (64±31) minutes (range 35-110 minutes) with exclusion of 2 cases intraoperative cannulation. There were three infants with complications. One episode was shown in vascular rupture, one in catheter site hemorrhage and one in cannula malposition with later repositioning. There was no case of insertion site infection, cannula-related bloodstream infection and accident detached cannula. Conclusion Cannulation for ECMO can be performed in infants less than 5 kg with a high rate of success and a low rate of complication owing to appropriate catheter and skillful cannulation.