br Isolation of extracellular vesicles and vesicle

Isolation of extracellular vesicles and vesicle-free secretome Three biological replicates of P. aeruginosa PAO001 and FCP001 were obtained from mid-exponential growth phase cultures (OD600 of 0.6). Extracellular vesicles were concentrated using a Centricon Plus-70 filter (>100kDa) and pelleted by ultracentrifuged at 100,000×g for 1h at 4°C. The supernatants were collected, mixed with the flowthrough <100kDa and concentrated using a 10kDa cut-off filter to obtain the vesicle-free secretome. Proteins were solubilised in 0.5M Triethylammonium bicarbonate (TEAB) buffer supplemented with a protease inhibitor cocktail.
Quantitative proteomic analysis
Experimental design, materials and methods
Acknowledgements This work was supported by the National Health and Medical Research Council, Australia, Programme grant (496601) (http://www.nhmrc.gov.au/); Access to proteomic infrastructure in the QIMR Berghofer Protein Discovery Centre was made possible by Bioplatforms Australia and the Queensland State Government provided through the Australian Government National Collaborative Infrastructure Scheme (NCRIS) and Education Investment Fund (EIF). We acknowledge Griffith Medical Research Council, Griffith University Postgraduate International Scholarship and QIMR Berghofer Medical Research Institute for awarding MTS a scholarship to undertake this work. Peter A Ryan of Monash University participated in the initial development of the study design and Buddhika Jayakody of the proteomics Discovery centre, QIMR Berghofer provided assistance with mass spectrometry.
Specifications table Value of the data Data, experimental design, materials and methods
Acknowledgments We thank the patients and their families for their cooperation. We acknowledge the RIKEN BRC for providing us iPS AZD1208 Supplier through the National Bio-Resource Project of the MEXT, Japan. This work was mainly supported by a Grant from the Precursory Research for Embryonic Science and Technology (PRESTO) program in Japan Science and Technology Agency (JST), Kawaguchi, Japan. It was partially supported by a Grant-in-Aid for Young Scientists (B) (24791090), Japan Society for the Promotion of Science (JSPS), a Grant from the Japan Epilepsy Research Foundation (JERF), and a Grant from Kanae Foundation for the promotion of Medical Science in Japan (K.S.). In addition, it was partially supported by a Grant-in-Aid for Scientific Research on Innovated Areas Foundation of Synapse and Neurocircuit Pathology (23110534); a Grant-in-Aid of Health Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare, Japan; a Grant from the Mother and Child Health Foundation in Japan; and a Grant from Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (T.Y.).
Value of the data Introduction Several lines of evidence indicate that Cytomegalovirus (CMV), a ubiquitous β-herpesvirus that infects 60–90% of the population, is a driving force in age-related T cell immunosenescence. Briefly, in CMV-seropositive older adults, aging has been associated with large expansion of CMV-specific CD8+ T cell clones and shrinkage of the T cell repertoire available for other antigens [3–7]. In individuals sharing the widespread HLA-A*0201 allele (referred to as A2), CMV-specific CD8+ T cells recognize the same epitope pp65495–503 (NLVPMVATV), hereafter referred to as NLV [8,9]. Despite the great effort made to date, the founding mechanism of NLV immunodominance has not been resolved. However, studies thus far have indicated that antigen-driven selection is most likely the main parameter contributing to the predominant usage of public TCR by NLV-A2-specific CD8 T cells [10–12]. Motivated by our previously reported finding that pMHC-TCR binding avidity of the transcriptional regulatory protein of the human T-cell leukemia virus type 1 (HTLV-1), Tax, along with that of a number of its variants with A6 TCR was highly correlated to atomic coordination of peptide tertiary structure [1,2], here we investigate the potential correlation between atomic coordination of a number of NLV variant-MHC complexes in respect to that of the NLV-MHC and we additionally inquire whether this correlation may also be indicative of the end effect of pMHC-TCR binding avidity of the same complexes as recognized by the RA14 TCR.