A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than ,20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to ,60:1 in a screen for proteins that bind to phosphotyrosine residues. Methods for acquiring quantitative proteomics data are continually developing with very accurate stable isotope labeling (SIL) and label-free approaches. SIL provides chemically equivalent but isotopically different internal standards for each peptide/protein for direct comparison of mass spectral signal intensities that represent relative abundance. Common SIL strategies include protein level labeling strategies such as stable isotope labeling of amino acids in cell culture (SILAC) [1], a global method whereby all translated proteins have isotope labels metabolically incorporated at selected amino acid residues, and isotope-coded affinity tags (ICAT) [2], a technique that labels cysteine residues at the protein level. Peptide level labeling strategies include multiplexed isobaric tags for relative and absolute quantification (iTRAQ

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Tyrannosaurus rex

We sequenced collagen protein fragments derived from fossilized bones of two extinct taxa: a 160,000- to 600,000-year-old mastodon [specimen number Museum of the Rockies (MOR) 605] (9) and a 68-million-year-old dinosaur (Tyrannosaurus rex, MOR 1125) (10), results that are supported by immunological and molecular analyses published in this issue by Schweitzer et al. (11). We first looked for tryptic peptide fragments from extracts of fossilized bone that matched identically with sequences from an orthologous protein or proteins from extant taxa, thereby identifying the protein(s) of interest. This is a common procedure for conserved proteins from taxa that share genomic information. Next, we generated a protein sequence database of likely drifts in amino acids in other tryptic peptides by comparing amino acid sequences ofthe orthologs from multiple related extant taxa. This approach produced a manageable number of theoretical protein sequences. The predicted peptide fragmentation pattern from these theoretical protein sequences were then compared with the fragmentation patterns of additional peptides derived from extracts of fossilized bone that did not match peptides in public sequence databases (fig. S1).Published as part of John M. Asara, Mary H. Schweitzer, Lisa M. Freimark, Matthew Phillips & Lewis C. Cantley, 2007, Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry, pp. 280-284 in Science 316 (5822) on page 281, DOI: 10.1126/science.1137614, http://zenodo.org/record/374443

Les équipages sans nom / Claude Baille

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Rapid Detection of Powassan Virus in a Patient With Encephalitis by Metagenomic Sequencing

Abstract We describe a patient with severe and progressive encephalitis of unknown etiology. We performed rapid metagenomic sequencing from cerebrospinal fluid and identified Powassan virus, an emerging tick-borne flavivirus that has been increasingly detected in the United States

Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection

Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection. Author summary Different genotypes of B. burgdorferi have been associated with different rates of dissemination, but the genetic basis of these differences is not known. We report the genomes of 299 B. burgdorferi isolates from patients with Lyme disease. We find that whole genome sequence (WGS) type A isolates are a genetically divergent group of isolates characterized by an enlarged pan-genome, an expanded surface lipoproteome encoded on a unique set of plasmids, including lp28-1 and lp56, and increased rates of dissemination. Using genome-wide association methods applied to the B. burgdorferi pan-genome, we identify loci associated with dissemination. The near-clonal nature of B. burgdorferi populations means that relationships of individual loci to dissemination are relatively weak after adjusting for the lineage structure among the isolates, implying that experimental studies and larger cohorts are needed to identify the causal alleles within a lineage mediating these effects. Across the isolates studied, an increasing number of surface-expressed lipoproteins was associated with an increased probability of dissemination in humans. The results underscore how strain-specific genetic variation—particularly among surface lipoproteins located on plasmids—is linked to the phenotype of human dissemination. More broadly, this approach provides a foundation for future studies linking spirochete genotype to the diverse clinical phenotypes of Lyme disease in humans