An in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry / 生物工程学报

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.

Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1 / 中华医学遗传学杂志

OBJECTIVE@#To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1).@*METHODS@#In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene.@*RESULTS@#Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly.@*CONCLUSION@#A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.

Comparative Study of Total Laparoscopic Hysterectomy for Complicated Hysteromyoma / 中国微创外科杂志

Objective-To-investigate-the-feasibility-of-total-laparoscopic-hysterectomy-for-complicated-hysteromyoma-,-such-as-broad-ligament-myoma,-lower-uterine-fibroids,-or-cervical-myoma.-Methods-From-December-2009-to-December-2012,-we-performed-laparoscopic-hysterectomy-on-166-patients-with-complicated-hysteromyoma-and-on-170-patients-with-common-hysteromyoma-.-The-operation-time-,-intraoperative-blood-loss-,-recovery-time-of-gastrointestinal-function-,-postoperative-morbidity-,-and-the-rate-of-conversion-to-laparotomy-were-compared-between-the-two-groups-respectively-.-Results-No-significant-differences-were-detected-in-operation-time-[(112.2-±15.3)-min-vs.(110.3-±16.1)-min,-t=1.088,-P=0.277],-intraoperative-blood-loss-[(117.0-±35.3)-ml-vs.(110.8-±37.8)-ml,-t=-1.525,-P=0.128],-recovery-time-of-gastrointestinal-function-[(20.8-±2.7)-h-vs.(21.3-±2.3)-h,-t=1.797,-P=0.073],-postoperative-morbidity-[6.3%(10/159)-vs.7.3%(12/165),χ2-=0.124,-P=0.725],-and-the-rate-of-conversion-to-laparotomy-(4.2%vs.2.9%,χ2-=0.397,-P=0.529)-between-the-two-groups-.Both-the-groups-were-followed-up-at-1,-2,-and-6-months-after-procedure-,-and-none-of-the-patients-showed-severe-postoperative-complications-.-Conclusions-Total-laparoscopic-hysterectomy-for-complicated-hysteromyoma-is-feasible-.To-prevent-complications-,-it-should-be-converted-to-laparotomy-timely-when-the-myoma-has-a-wide-base-or-is-located-at-lower-position-.

Effects of ibrolipim against atherosclerosis in minipigs / 中国病理生理杂志

AIM: To explore the application mechanism of NO-1886 (ibrolipim), a synthetic compound, improving dyslipidemia and inhibiting atherosclerosis in Guizhou minipigs fed with high fat/high sucrose diet. METHODS: Fifteen Chinese Guizhou minipigs were randomized into three groups with similar body weight [(n=5 in normal control group (CD); n=5 in high fat/high sucrose group (HFSD); n=5 in high fat/high sucrose supplemented ibrolipim group (HFSD+ibrolipim)]. Blood samples were withdrawn from the eyehole sinus venosus of the animals at the end of each month after fasting overnight. The animals were sacrificed at the end of 8 months. The concentrations of cholesterol ester in plasma HDL were analyzed by HPLC. The aortic fatty streak-lesions were quantified following lipid staining with Sudan IV. Lipid droplets in liver were observed by Oil red O staining. RESULTS: Compared with CD, fasting plasma TC, TG and FFA levels of HFSD were elevated significantly. The aortic fatty streak-lesions were clearly presented in the animals' aortas. The intima became rougher and thicker. A lot of lipoid foam cells migrated to regions of intima and smooth muscle cells, which associated with the injuries of internal elastic lamina. Extensive fat deposited in the liver were observed. Supplementing of 1.0% ibrolipim into high fat/high sucrose diet induced the decrease in plasma TG and FFA concentrations and an increase in plasma HDL-C concentration compared with HFSD. A little fat deposited in the liver were observed. CONCLUSION: ibrolipim prevents AS in high fat/high sucrose diet feeding minipigs through decreasing the plasma TG and elevating the plasma HDL-C.