Atypical EPO profiles in anti-doping analyses

Résumé : Erythropoietin (EPO) is a glycoprotein hormone endogenously produced by the kidney, whose main physiological role is the stimulation of erythropoiesis. Since the beginning of the nineties, recombinant human EPO (rhEPO), a potent anti-anaemia treatment drug, has been manufactured by pharmaceutical industries. However, the erythropoiesis stimulating power of rhEPO was rapidly misused by unscrupulous athletes in order to improve their performances in endurance sports. Endogenous EPO has the same amino-acid backbone as most of recombinant forms; the molecules however differ through their respective glycosylation patterns. This difference constitutes the basis of the usual EPO screening test (IEF) developed in 2000 and still currently used in all anti-doping laboratories of the world. Nowadays, 3 EPO generations have been commercialized. The fight against EPO abuse is a continuous challenge for anti-doping laboratories. The diversity of recombinant EPO forms and the continuous development of new ones considerably confuse the identification of EPO doping. Several facets of this fight were investigated in this work. One of the limiting aspects of doping agents screening is the availability of positive samples. Therefore, 2nd and 3rd generation EPOS, namely NESP and C.E.R.A., were injected to healthy subjects in the frame of pilot clinical studies. These latter allowed to review the current EPO identification criteria defined by the World Anti-Doping Agency (WADA) in the case of NESP and to validate and implement a new assay targeting C.E.R.A. in human serum. Both studies resulted in the determination of the respective detection windows of NESP and C.E.R.A. in biological fluids. Following that, Dynepo, a 1st generation EPO presenting similarities with the endogenous form, was also in the centre of a similar clinical study. Our work aimed to overcome the actual identification criteria, which are not adapted to Dynpeo, and to propose an alternative pattern classification method based on the discriminant analysis of IEF EPO profiles. This method might be validated for other EPO forms in the future. The detection window of this molecule was also determined. Under particular conditions, confounding effects can complicate the identification of EPO in biological matrices. For example, athletes having performed a strenuous physical effort can excrete modified isoforms of endogenous EPO, making it very similar to some recombinant forms. Such phenomena, called effort urines, were reproduced under controlled conditions and, after characterization of effort EPO, an urinary biochemical marker was proposed to unequivocally identify effort urines. It also happens that EPO analyses fail to detect endogenous levels of EPO. Such profiles were thoroughly investigated and potential causes identified. Natural reasons relying on urine properties and test specificity were underlined, but the possible addition of adulterant agents in urine samples was also considered. Therefore, a simple biochemical assay targeting the suspected substances was set up. Our work was based on the characterization of atypical EPO profiles from different origins. Therefore, 3 EPO molecules representing the 3 generations of the drug and 2 confounding effects confusing the results interpretation were studied. These studies resulted in tangible applications for the laboratory, the best example of which being the C.E.R.A. assay, but also in scientific findings allowing to improve our comprehension of EPO doping in sport.

Genetic analyses reveal a role for vitamin D insufficiency in HCV-associated hepatocellular carcinoma development

BACKGROUND: Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99-1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12-2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13-1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. CONCLUSIONS/SIGNIFICANCE: Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Trophic dynamics in the northern Humboldt Current system: insights from stable isotopes and stomach content analyses

The northern Humboldt Current system (NHCS) off Peru is one of the most productive world marine regions. It represents less than 0.1% of the world ocean surface but presently sustains about 10% of the world fish catch, with the Peruvian anchovy or anchoveta Engraulis ringens as emblematic fish resource. Compared with other eastern boundary upwelling systems, the higher fish productivity of the NHCS cannot be explained by a corresponding higher primary productivity. On another hand, the NHCS is the region where El Niño, and climate variability in general, is most notable. Also, surface oxygenated waters overlie an intense and extremely shallow Oxygen Minimum Zone (OMZ). In this context, the main objective of this study is to better understand the trophic flows in the NHCS using both stomach content and stable isotope analyses. The study focuses on a variety of organisms from low trophic levels such as zooplankton to top predators (seabirds and fur seals). The approach combines both long-term and specific studies on emblematic species such as anchoveta, and sardine Sardinops sagax and a more inclusive analysis considering the 'global' food web in the recent years (2008 – 2012) using stable isotope analysis. Revisiting anchovy and sardine we show that whereas phytoplankton largely dominated anchoveta and sardine diets in terms of numerical abundance, the carbon content of prey items indicated that zooplankton was by far the most important dietary component. Indeed for anchovy euphausiids contributed 67.5% of dietary carbon, followed by copepods (26.3%). Selecting the largest prey, the euphausiids, provide an energetic advantage for anchoveta in its ecosystem where oxygen depletion imposes strong metabolic constrain to pelagic fish. Sardine feed on smaller zooplankton than do anchoveta, with sardine diet consisting of smaller copepods and fewer euphausiids than anchoveta diet. Hence, trophic competition between sardine and anchovy in the northern Humboldt Current system is minimized by their partitioning of the zooplankton food resource based on prey size, as has been reported in other systems. These results suggest an ecological role for pelagic fish that challenges previous understanding of their position in the foodweb (zooplanktophagous instead of phytophagous), the functioning and the trophic models of the NHCS. Finally to obtain a more comprehensive vision of the relative trophic position of NHCS main components we used stable isotope analyses. For that purpose we analyzed the δ13C and δ15N stable isotope values of thirteen taxonomic categories collected off Peru from 2008 - 2011, i.e., zooplankton, fish, squids and air-breathing top predators. The δ15N isotope signature was strongly impacted by the species, the body length and the latitude. Along the Peruvian coast, the OMZ get more intense and shallow south of ~7.5ºS impacting the baseline nitrogen stable isotopes. Employing a linear mixed-effects modelling approach taking into account the latitudinal and body length effects, we provide a new vision of the relative trophic position of key ecosystem components. Also we confirm stomach content-based results on anchoveta Engraulis ringens and highlight the potential remarkable importance of an often neglected ecosystem component, the squat lobster Pleuroncodes monodon. Indeed, our results support the hypothesis according to which this species forage to some extent on fish eggs and larvae and can thus predate on the first life stages of exploited species. However, the δ13C values of these two species suggest that anchoveta and squat lobster do not exactly share the same habitat. This would potentially reduce some direct competition and/or predation.

Solving ambiguities in contig assembly of Idiomarina loihiensis L2TR chromosome by in silico analyses.

Nucleotide composition analyses of bacterial genomes such as cumulative GC skew highlight the atypical, strongly asymmetric architecture of the recently published chromosome of Idiomarina loihiensis L2TR, suggesting that an inversion of a 600-kb chromosomal segment occurred. The presence of 3.4-kb inverted repeated sequences at the borders of the putative rearrangement supports this hypothesis. Reverting in silico this segment restores (1) a symmetric chromosome architecture; (2) the co-orientation of transcription of all rRNA operons with DNA replication; and (3) a better conservation of gene order between this chromosome and other gamma-proteobacterial ones. Finally, long-range PCRs encompassing the ends of the 600-kb segment reveal the existence of the reverted configuration but not of the published one. This demonstrates how cumulative nucleotide-skew analyses can validate genome assemblies.

A phylogenetic study of the phosphoenolpyruvate carboxylase multigene family in Poaceae: understanding the molecular changes linked to C4 photosynthesis evolution

Phylogenetic reconstructions have supported several independent appearances of C₄ photosynthesis within grasses (Poaceae). These recurrent appearances appear to contradict the large number of biochemical and morphological changes required to change from C₃ to C₄, a paradox that leads to questions about the genetic changes underlying C₄ evolution. In this study, we analysed sequences encoding phosphoenolpyruvate carboxylases (PEPCs) in grasses in order to gain insights into the origin of the ppc-C₄ gene, which encodes a key enzyme in the C₄ pathway. We screened databanks for PEPC genes or cDNAs in grasses. A coding sequence of 1130 base pairs was used to build phylogenetic trees that supported the existence of four distinct PEPC gene lineages. Ppc-C₄ present in all C₄ grasses was also found in two C₃ species. The ppc-C₄ clade was congruent with the species tree, suggesting orthologous evolution. This result would imply that ppc-C₄ appeared without any duplication event. Nevertheless, caution is needed since the sampling of our study is still far from comprehensive. Further investigation with an increased sampling is recommended to elucidate the evolutionary changes underlying ppc-C₄ gene evolution in grasses.

Staphylococcus aureus clfB and spa alleles of the repeat regions are segregated into major phylogenetic lineages.

To reliably differentiate among Staphylococcus aureus isolates we recently developed the Double Locus Sequence Typing (DLST) based on the analysis of partial sequences of clfB and spa genes. This method is highly discriminatory and gives unambiguous definition of types. The highly clonal population structure of S. aureus suggests that isolates with identical clfB or spa alleles belong to the same clonal complex (CC) defined by Multi-Locus Sequence Typing (MLST). To test this hypothesis as well as to investigate putative intra-CC genetic structure, we analyzed a total of 289 isolates (186 MSSA and 103 MRSA) with DLST-, spa- and MLST-typing. Among the 289 strains, 242 were clustered into 7 major MLST CCs, 40 into minor CCs and 7 were not grouped into CCs. A total of 205 DLST- and 129 spa-types were observed. With one exception, all DLST-clfB, DLST-spa and spa-type alleles were segregated into CCs. DLST-types sharing an identical allele (clfB or spa) were clustered using eBURST. Except for one strain, all isolates from each DLST cluster belonged to the same CC. However, using both DLST- and spa-typing we were not able to disclose a clear intra-CC structure. Nevertheless, the high diversity of these loci confirmed that they are good markers for local epidemiological investigations.

Détermination des vents extrêmes à l'aide d'analyses statistiques et de modélisations numériques dans une topographie accidentée en Suisse.

Les constructions en Suisse et en Europe doivent être dimensionnées pour subir sans dommages des vents extrêmes (rafales) avec une période de retour d'au moins 50 ans. Des analyses de Gumbel ont donc été effectuées sur des séries de mesures d'une vingtaine d'années à 67 endroits de la Suisse, afin de déterminer les vitesses extrêmes des vents pour différents courants régionaux. Elles ont abouti à de bons résultats pour la majorité des endroits. Mais ces vitesses extrêmes ne peuvent être extrapolées linéairement aux autres régions dans une topographie aussi accidentée que la Suisse. Un modèle numérique tridimensionnel des écoulements non hydrostatique a été expérimenté pour calculer les champs de vent extrêmes en Suisse avec une résolution horizontale de 2 km et une période de retour de 50 ans. Ces modélisations ont permis de reproduire de manière satisfaisante ces vents extrêmes pour l'ensemble du pays. Toutefois, elles sous-estiment leurs intensités sur les reliefs jurassiens et alpins, comparativement aux vallées alpines et au Plateau Suisse.

The abrupt climate change at the Eocene-Oligocene boundary and the emergence of South-East Asia triggered the spread of sapindaceous lineages.

Background and Aims Paleoclimatic data indicate that an abrupt climate change occurred at the Eocene-Oligocene (E-O) boundary affecting the distribution of tropical forests on Earth. The same period has seen the emergence of South-East (SE) Asia, caused by the collision of the Eurasian and Australian plates. How the combination of these climatic and geomorphological factors affected the spatio-temporal history of angiosperms is little known. This topic is investigated by using the worldwide sapindaceous clade as a case study. Methods Analyses of divergence time inference, diversification and biogeography (constrained by paleogeography) are applied to a combined plastid and nuclear DNA sequence data set. Biogeographical and diversification analyses are performed over a set of trees to take phylogenetic and dating uncertainty into account. Results are analysed in the context of past climatic fluctuations. Key Results An increase in the number of dispersal events at the E-O boundary is recorded, which intensified during the Miocene. This pattern is associated with a higher rate in the emergence of new genera. These results are discussed in light of the geomorphological importance of SE Asia, which acted as a tropical bridge allowing multiple contacts between areas and additional speciation across landmasses derived from Laurasia and Gondwana. Conclusions This study demonstrates the importance of the combined effect of geomorphological (the emergence of most islands in SE Asia approx. 30 million years ago) and climatic (the dramatic E-O climate change that shifted the tropical belt and reduced sea levels) factors in shaping species distribution within the sapindaceous clade.