Objective The construction of the reconstructed acellular extracellular matrix(REAECM) of bone and preliminary investigation of biocompatibility of REAECM was performed to supply the experimental basis for the extracellular scaffold of bone tissue engineering. Methods Applying the technique of culture in vitro, biocompatibility of the REAECM cultured with osteoblasts was observed using phase-contrast microscope and scanning eletron microscope at 1-4 weeks. Results The osteoblasts could attach and proliferate, even grow well into the foam of REAECM.Conclusion REAECM is an excellent scaffold material for bony tissue engineering construction, which pocesses a morphological structure of three-dimensional foam. The REAECM showed a good biocompatibility with osteoblasts co-cultured in vitro. There was prospective signs of REAECM to the attachment, proliferation of the osteoblasts

Thermal proteome profiling (TPP) is a combination of cellular thermal shift assay (CETSA) and quantitative mass spectrometry (MS), also termed as MS-CETSA. TPP determines the stability of the entire proteome by measuring the content of soluble proteins in cells or cell lysates at different heating temperatures. Proteins can change their thermostability when interacting with small molecules (e.g., drugs or metabolites), nucleic acids, or other proteins or posttranslational modification, while TPP can identify target proteins based on the difference in thermostability with or without ligand-binding. At present, TPP has been applied to identify the targets and off-targets of drugs and interrogate protein-metabolite and protein-protein interactions. Due to limited understanding of this technology, this review introduced the principles, methods, applications, advantages and limitations of TPP.
Proteome ; Mass Spectrometry

Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.
Humans ; Kinetics ; Mutation ; Phospholipases A2, Secretory ; Phospholipids ; Saccharomycetales ; Substrate Specificity