Searching for carbonylome biomarkers of aging – development and validation of the proteomic method for quantification of carbonylated protein in human plasma

Aim To develop a method for measuring protein carbonylation in human plasma and serum samples, which was previously implied in numerous age-related phenotypes. Methods Protein expression and carbonylation were analyzed in plasma samples obtained from 12 healthy human individuals by using a novel method that combines affinity-based albumin and immunoglobulin G removal, and aminooxy dyeing in one- or two-dimensional gels. In addition, carbonylome profile of plasma and serum was compared. Coefficients of variation and intra-class correlation coefficients were used in statistical analysis. Results Following a step-wise laboratory development and optimization process, we measured the protein expression and carbonylation for 813 proteins from the plasma. The analysis of repeated measurements suggested excellent coefficients of variation, which rarely exceeded 10%. The average value of intra-class correlation based on absolute agreement (ICC) for protein expression was 0.97 ± 0.02, while for carbonylation it was 0.73 ± 0.24. The removal of the most extreme protein outlier in carbonylation assessment increased the average ICC to 0.87 ± 0.04. Low protein spot volume substantially reduced repeatability. Serum carbonylation estimates were similar to those from plasma, with the ICC in the range of 0.86-0.89. Conclusion We developed a reliable method for the measurement of human plasma protein carbonylation, which can be used for the assessment of carbonylome biomarkers of aging

Identification of amino acids in mitochondrially encoded proteins that correlate with lifespan.

International audienceAnimals show a huge diversity in their lifespan that can vary from a few weeks to over a hundred years in vertebrates. Size is a key element in this variation and the positive correlation between size and maximum lifespan can be observed in each class of vertebrate. Some groups and species clearly stand out in this size-lifespan relationship and the ones with exceptionally long lifespan have been studied to understand the biological causes of their low aging rate. Amongst the potential explanations of animals' lifespan variations, mitochondria and mitochondrially encoded genes have drawn attention because of their importance in the aging process. To understand both the extent of lifespan variations and their dependence to genes and amino acids variations in mitochondrial genes and DNA (mtDNA), we analyze in a systematic way all 13 proteins encoded by mitochondria in all vertebrates for which we had information on weight, maximum lifespan and mtDNA sequence. This comparison allows us to visualize positions, and even specific amino acids, in these sequences that correlate with lifespan. With this approach, we draw a map of 356 amino acid residues, at 296 positions within the sequence, that correlate with longer or shorter lifespan. We also compared this map with the human mitochondrial polymorphism to determine its potential as a predictive tool

Volatile Oil Chemical Composition of Wild, Edible <i>Centaurea scabiosa</i> L. and Its Cytotoxic Activity

Centaurea species are well known as a source of phytopharmaceuticals having both beneficial and harmful influences on human health. Centaurea scabiosa L. is a wild edible plant used in Mediterranean cuisine in the Dalmatian region of Croatia. We have assessed the volatile oil’s chemical composition using GC/MS chromatography and its cytotoxic activity on human fibroblasts using the MTT test. Data on chromosome number, obtained by classical karyological methods, and genome size, assessed by flow cytometry, of the same plant material of C. scabiosa, were also given. The major chemical compounds found in C. scabiosa volatile oil were heptacosane, caryophyllene oxide, alloaromadendrene epoxide, α-cyperone, and α-bisabolol. This volatile oil showed no cytotoxicity on human fibroblasts in a dose range of 0.01–1 g/L. The chromosome number of a C. scabiosa sample from Croatia showed 2n = 20 + 2B chromosomes. The total genome DNA amount of 2C = 3.3 ± 0.01 pg or 1 Cx = 1628 Mbp presents the first report on the genome size of this species from Croatia. The presented results support the idea of using this plant in the human diet. To our knowledge, this is the first report on edible C. scabiosa species in general and in particular from Croatia

Protection against Streptococcus pneumoniae serotype 1 acute infection shows a signature of Th17- and IFN-γ-mediated immunity.

International audienceAcute pneumonia caused by Streptococcus pneumoniae is a major cause of child mortality. Antibodies are considered the main effectors of protection in this clinical presentation of pneumococcal invasive disease. To get new insights into the mechanisms involved in the protective immunity, we established a murine experimental model of protection against acute pneumococcal pneumonia and then evaluated the transcriptional, humoral and cellular responses in protected and non-protected animals. We found that intranasal inoculation of a sublethal dose of S. pneumoniae serotype 1 conferred complete protection against a subsequent challenge with a lethal dose of the same strain. Sublethal infection elicited a strong IgM and IgG antibody response against the capsular polysaccharide, as assessed one week later, and an exacerbated influx of neutrophils into the lungs immediately after the lethal challenge. Genome-wide microarray-based transcriptional analysis of whole lungs showed 149 differentially expressed genes among which we found upregulation of Il17a, Ifng and several IL-17A- and IFN-γ-related genes in protected versus non-protected mice. Kinetics analysis showed higher expression levels of Il17a in protected animals at all time points whereas Ifng was upregulated early in the protected mice and later in the non-protected animals. Intracelluar cytokine staining demonstrated that CD4(+) T cells account for a great proportion of the IL-17A produced in the lungs of protected animals. Overall, these results showed that an upregulation of IL-17A- and a timely regulation of IFN-γ-related gene expression, together with development of a Th17 response, are relevant characteristics of the protective immunity against S. pneumoniae acute pneumonia