IbeA is an important invasion determinant contributing to Escherichia coli K1 entry into brain microvascular endothelial cells (BMEC) that is a key step in the pathogenesis of E. coli meningitis. Our previous studies ...IbeA is an important invasion determinant contributing to Escherichia coli K1 entry into brain microvascular endothelial cells (BMEC) that is a key step in the pathogenesis of E. coli meningitis. Our previous studies have shown that IbeA-induced signaling and E. coli K1 invasion is mediated by two IbeA-binding proteins, vimentin, which is constitutively present in the surface of human BMECs (HBMECs), and PSF, which is inducibly expressed in both mesenchymal (endothelium) and non-mesenchymal (epithelium) cells. However, it is unknown whether p54nrb, a PSF partner protein, could contribute to the pathogenesis of E. coli K1 meningitis. Here, we reported that a 54-kDa protein was identified by copurification with PSF through IbeA-affinity chromatography as an IbeA-binding protein, which is identical to p54nrb. Both p54nrb and PSF are RNA-binding proteins and share significant sequence homology. The specific interaction between IbeA and p54nrb was confirmed by Western blot and ligand overlay assays. Recombinant p54nrb blocked E. coli K1 invasion of human BMEC very effectively. Overexpressed p54nrb as a GFP fusion protein in the transfected 293T cells significantly enhanced E. coli K1 invasion. Furthermore, higher levels of surface p54nrb in the transfected 293T cells were detected by flow cytometry. These results suggest that the IbeA invasion protein of E. coli K1 interacts with p54nrb for bacterial invasion of human BMEC.展开更多
液相色谱-串联质谱技术(liquid chromatography-tandem mass spectrometry, LC-MS/MS)结合了液相色谱的良好分离性能和串联质谱的高灵敏度、高特异性、高通量等优势,已经成为临床检验领域重要的新技术之一。目前LC-MS/MS在临床检验方面...液相色谱-串联质谱技术(liquid chromatography-tandem mass spectrometry, LC-MS/MS)结合了液相色谱的良好分离性能和串联质谱的高灵敏度、高特异性、高通量等优势,已经成为临床检验领域重要的新技术之一。目前LC-MS/MS在临床检验方面,已经成为临床小分子化合物的主要检测方法,此外在激素检测、新生儿筛查、治疗药物监测、维生素检测、蛋白质与多肽定量等项目应用也日趋成熟。虽然LC-MS/MS技术凭借其独特的优势,已经在临床检验领域占得一席之地,但是LC-MS/MS临床检验应用也存在诸多的问题,在质谱检验中,其自动化程度较低、前处理操作复杂、仪器操作复杂、信息化管理程度低、对检验人员能力要求较高,因此阻碍了LC-MS/MS优秀的检验技术在临床的应用,也限制了LC-MS/MS在临床检验中的发展,本文将探讨首都医科大学附属北京妇产医院临床质谱检验中心在LC-MS/MS检测技术工作效率提升方面所做的探索和尝试。展开更多
文摘IbeA is an important invasion determinant contributing to Escherichia coli K1 entry into brain microvascular endothelial cells (BMEC) that is a key step in the pathogenesis of E. coli meningitis. Our previous studies have shown that IbeA-induced signaling and E. coli K1 invasion is mediated by two IbeA-binding proteins, vimentin, which is constitutively present in the surface of human BMECs (HBMECs), and PSF, which is inducibly expressed in both mesenchymal (endothelium) and non-mesenchymal (epithelium) cells. However, it is unknown whether p54nrb, a PSF partner protein, could contribute to the pathogenesis of E. coli K1 meningitis. Here, we reported that a 54-kDa protein was identified by copurification with PSF through IbeA-affinity chromatography as an IbeA-binding protein, which is identical to p54nrb. Both p54nrb and PSF are RNA-binding proteins and share significant sequence homology. The specific interaction between IbeA and p54nrb was confirmed by Western blot and ligand overlay assays. Recombinant p54nrb blocked E. coli K1 invasion of human BMEC very effectively. Overexpressed p54nrb as a GFP fusion protein in the transfected 293T cells significantly enhanced E. coli K1 invasion. Furthermore, higher levels of surface p54nrb in the transfected 293T cells were detected by flow cytometry. These results suggest that the IbeA invasion protein of E. coli K1 interacts with p54nrb for bacterial invasion of human BMEC.
文摘液相色谱-串联质谱技术(liquid chromatography-tandem mass spectrometry, LC-MS/MS)结合了液相色谱的良好分离性能和串联质谱的高灵敏度、高特异性、高通量等优势,已经成为临床检验领域重要的新技术之一。目前LC-MS/MS在临床检验方面,已经成为临床小分子化合物的主要检测方法,此外在激素检测、新生儿筛查、治疗药物监测、维生素检测、蛋白质与多肽定量等项目应用也日趋成熟。虽然LC-MS/MS技术凭借其独特的优势,已经在临床检验领域占得一席之地,但是LC-MS/MS临床检验应用也存在诸多的问题,在质谱检验中,其自动化程度较低、前处理操作复杂、仪器操作复杂、信息化管理程度低、对检验人员能力要求较高,因此阻碍了LC-MS/MS优秀的检验技术在临床的应用,也限制了LC-MS/MS在临床检验中的发展,本文将探讨首都医科大学附属北京妇产医院临床质谱检验中心在LC-MS/MS检测技术工作效率提升方面所做的探索和尝试。
