Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping.

Aim: Transcriptomic biomarkers originating from reticulocytes measured in dried blood spots (DBSs) may be reliable indicators of blood doping. Methods/results: Here, we examined changes in the expression levels of the erythropoiesis-related ALAS2, CA1 and SLC4A1 genes in DBS samples from elite athletes and volunteers of clinical study with recombinant erythropoietin dose. Conclusion: By comparing the mean intraday coefficients of variation for ALAS2L, ALASLC, CA1 and SLC4A1 between manual and automated RNA extractions, an average improvement was observed, whereas the assessment of interday variability provided comparable results for both manual and automated approaches. Our results confirmed that RNA biomarkers on DBS support are efficient to detect blood doping

Genomic Instability Is Associated with Natural Life Span Variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for ∼50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span

Rules Governing Selective Protein Carbonylation

BACKGROUND:Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only some proteins are prone to carbonylation. METHODOLOGY/PRINCIPAL FINDINGS:we used mass spectrometry analysis to identify carbonylated sites in: BSA that had undergone in vitro MCO, and 23 carbonylated proteins in Escherichia coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. CONCLUSIONS/SIGNIFICANCE:We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms

A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA “bar-code” sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Δ mutant and exacerbated by a short-lived atg16Δ autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process

Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting, Autophagy, Biosynthetic, and tRNA Methylation Genes Involved in Life Span Regulation

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved

Effects of calorie restriction on life span of microorganisms

Calorie restriction (CR) in microorganisms such as budding and fission yeasts has a robust and well-documented impact on longevity. In order to efficiently utilize the limited energy during CR, these organisms shift from primarily fermentative metabolism to mitochondrial respiration. Respiration activates certain conserved longevity factors such as sirtuins and is associated with widespread physiological changes that contribute to increased survival. However, the importance of respiration during CR-mediated longevity has remained controversial. The emergence of several novel metabolically distinct microbial models for longevity has enabled CR to be studied from new perspectives. The majority of CR and life span studies have been conducted in the primarily fermentative Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, but studies in primarily respiratory Crabtree-negative yeast and obligate aerobes can offer complementary insight into the more complex mammalian response to CR. Not only are microorganisms helping characterize a conserved cellular mechanism for CR-mediated longevity, but they can also directly impact mammalian metabolism as part of the natural gut flora. Here, we discuss the contributions of microorganisms to our knowledge of CR and longevity at the level of both the cell and the organism

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry.

Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides. We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays. Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS. The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease

Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7–97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research. [Figure not available: see fulltext.]. © 2020, The Author(s)

Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry

Background: Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides. Methods: We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays. Results: Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS. Conclusions: The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease. © 2022 American Association for Clinical Chemistry 2022