Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （...Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （MHC） molecule, H-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal Protein A （SPA） column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.展开更多
Novel visualization methods and strategies are necessary to cope with the deluge of datasets present in any scientific field to make discoveries and find answers to previously unanswered questions.These methods and st...Novel visualization methods and strategies are necessary to cope with the deluge of datasets present in any scientific field to make discoveries and find answers to previously unanswered questions.These methods and strategies should not only present scientific findings as images in a concise way but also need to be effective and expressive,which often remain untested.Here,we present Versus,a tool to enable easy image quality assessment and image ranking,utilizing a two-alternative forced choice methodology(2AFC)and an efficient ranking algorithm based on a binary search.The tool provides a systematic way of setting up evaluation experiments via the web without the necessity to install any additional software or require any programming skills.Furthermore,Versus can easily interface with crowdsourcing platforms,such as Amazon’s Mechanical Turk,or can be used as a stand-alone system to carry out evaluations with experts.We demonstrate the use of Versus by means of an image evaluation study,aiming to determine if hue,saturation,brightness,and texture are good indicators of uncertainty in three-dimensional protein structures.Drawing from the power of crowdsourcing,we argue that there is demand and also great potential for this tool to become a standard for simple and fast image evaluations,with the aim to test the effectiveness and expressiveness of scientific visualizations.展开更多
基金supported by a grant from the National Natural Science Foundation of China(NO.30271201).
文摘Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （MHC） molecule, H-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal Protein A （SPA） column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.
基金This work was supported by CSIRO’s OCE Science Leader programme and Computational and Simulation Sciences platformpartly by the Australian Research Council under Linkage Project LP140100574。
文摘Novel visualization methods and strategies are necessary to cope with the deluge of datasets present in any scientific field to make discoveries and find answers to previously unanswered questions.These methods and strategies should not only present scientific findings as images in a concise way but also need to be effective and expressive,which often remain untested.Here,we present Versus,a tool to enable easy image quality assessment and image ranking,utilizing a two-alternative forced choice methodology(2AFC)and an efficient ranking algorithm based on a binary search.The tool provides a systematic way of setting up evaluation experiments via the web without the necessity to install any additional software or require any programming skills.Furthermore,Versus can easily interface with crowdsourcing platforms,such as Amazon’s Mechanical Turk,or can be used as a stand-alone system to carry out evaluations with experts.We demonstrate the use of Versus by means of an image evaluation study,aiming to determine if hue,saturation,brightness,and texture are good indicators of uncertainty in three-dimensional protein structures.Drawing from the power of crowdsourcing,we argue that there is demand and also great potential for this tool to become a standard for simple and fast image evaluations,with the aim to test the effectiveness and expressiveness of scientific visualizations.
