High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma

A simple, rapid and sensitive reversed-phase high-performance liquid chromatographic method with UV detection was developed for the quantification of di(2-ethylhexyl) phthalate (DEHP) in parenteral nutrition admixtures containing fat emulsion and in plasma samples of children daily treated by total parenteral nutrition. The analyte and the internal standard, di-n-heptyl phthalate, were extracted twice using hexane and the organic layer separated and dried under nitrogen. The residues were reconstituted with acetonitrile and 20 μl was injected into a Waters Spherisorb C₁₈ column, the UV detector was set at 202 nm. The mobile phase was acetonitrile–aqueous buffer (triethylamine 0.08% adjusted to pH 2.8 with 1 M phosphoric acid) mixture (88:12, v/v) and it was pumped at 1 ml/min. Average recoveries were 97% or greater. This method was successfully used to investigate the amounts of DEHP which can leach from bags and tubing into fat emulsion and which could contaminate children under long-term parenteral nutrition. On the other hand, the circulating DEHP concentrations were estimated in four children under regular long-term parenteral nutrition.     

Schistosoma mansoni: IL-4 is necessary for concomitant immunity in mice.

To ask whether type-2 immune responses serve an essential role in concomitant immunity, that is the prevention of superinfection with Schistosoma mansoni, we compared resistance to a challenge infection in infected wild-type (WT) mice and in infected IL-4-/- mice, which are unable to mount Th2 responses during schistosomiasis. Although WT mice are protected from superinfection, resistance is abrogated in the absence of interleukin (IL)-4. We conclude that IL-4 or IL-4-dependent responses, or both, are necessary for resistance to S. mansoni superinfection in mice.     

Free-living amoebae promote Candida auris survival and proliferation in water

Candida auris is an emerging species responsible for life-threatening infections. Its ability to be resistant to most systemic antifungal classes and its capacity to persist in a hospital environment have led to health concerns. Currently, data about environmental reservoirs are limited but remain essential in control of C. auris spread. The aim of our study was to explore the interactions between C. auris and two free-living amoeba (FLA) species, Vermamoeba vermiformis and Acanthamoeba castellanii, potentially found in the same water environment. Candida auris was incubated with FLA trophozoites or their culture supernatants. The number of FLA and yeasts was determined at different times and transmission electron microscopy (TEM) was performed. Supernatants of FLAs promoted yeast survival and proliferation. Internalization of viable C. auris within both FLA species was also evidenced by TEM. A water environmental reservoir of C. auris can therefore be considered through FLAs and contamination of the hospital water networks would consequently be possible.     

Genus delimitation,biogeography and diversification of Choristoneura Lederer (Lepidoptera: Tortricidae) based on molecular evidence

Widely known for pest species that include major modulators of temperate forests, the genus Choristoneura is part of the species‐rich tribe Archipini of leafroller moths (Tortricidae). Delimitation of the genus has remained unresolved because no phylogeny has included species endemic to Africa and studies have often omitted the type species of the genus. Further taxonomic confusion has been generated by the transfer of Archips occidentalis (Walsingham) to Choristoneura, creating a homonym with Choristoneura occidentalis Freeman, an important defoliator of North American forests. To define the limits of the genus, we reconstructed a phylogeny using DNA sequences for mitochondrial cytochrome oxidase subunit I and nuclear ribosomal 28S genes. Our ingroup included 23 Choristoneura species‐level taxa, complemented by a large sample of outgroups comprising 82 species of Archipini and other Tortricidae. We generated a time‐calibrated tree using fossil and secondary calibrations and we inferred biogeographic and diversification processes in Choristoneura. Our analysis recovered the genus as polyphyletic, with Archips occidentalis, Choristoneura simonyi and Choristoneura evanidana excluded from the main clade. Based on the recovered phylogenies and a redefinition, we restrict Choristoneura primarily to species with a northern hemisphere distribution. Our analysis supports A. occidentalis as the sister group of Cacoecimorpha pronubana, C. simonyi as the sister of ‘Xenotemna’ pallorana, and C. evanidana as the sister of Archips purpurana. A new combination is proposed: Archips evanidana comb.n. ; the availability of ‘Xenotemna’ as a valid name is discussed and A. occidentalis is considered as an orphaned name within the Archipini. We found support for a Holarctic origin of Choristoneura about 23 Ma, followed by early divergence in the Palearctic region. The main divergence occurred at 16 Ma, with one clade in the Nearctic and another in the Palearctic. Subsequent cladogenetic events were synchronous and related to herbivorous specialization, with each clade divided into coniferophagous and polyphagous lineages. Their specialization as conifer feeders temporally matched the expansion of boreal forest during the Miocene.     

Origin of abnormality in a human simelian foetus as elucidated by our knowledge of vertebrate development

In this paper we attempt to explain the abnormality of a simelian foetus with reference to our present knowledge of vertebrate development. The various developmental defects seem to have a single common origin: the speeding-up of the progression of cell differentiation in the notochord anlage--which is the organization centre of the embryo--during the regression of the Hensen's node. Cell activity involved in the morphogenetic movements in the chordamesoderm probably stopped before it should have. The elongation of the notochord anlage was not completed, resulting in the defective development of the posterior part of the foetus. A number of pairs of posterior trunk somites were not induced. Consequently (1) the pelvic limb buds, whose posterior parts were missing, fused, bringing in further developmental deviations in the limb skeleton and abdominal muscles; (2) there are no vertebrae between the first sacral vertebra and the misshaped coccyx formed by the tail bud. The derivatives of the posterior endoderm (hindgut, bladder and ureters) were not induced either. The cauda equina is deficient. The absence of functional kidneys and the presence of embryonic urinary tubules in the pelvic cysts which are wrapped up by gut epithelium suggest the induction of the metanephric mesenchyme by ectopic endoderm. The speeding-up of differentiation in the notochord anlage also probably resulted in the excessive extension of its anterior region which is the organizer of brain structures. This explains the overdevelopment of the nose and of the neurocranium, and the low position of the ear. A gene mutation as well as a mechanical stress are the possible causes of the abnormal behaviour of the notochord anlage.     

Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors

The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.     

Autophosphorylation-independent and -dependent Functions of Focal Adhesion Kinase during Development

Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fakΔ) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAKΔ is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK^Δ/Δ embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK^Δ/Δ embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5–14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK^Δ/Δ fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAKΔ. FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.     

Premiere decouverte d'une faune de petits mammiferes dans le Paleogene du bassin d'Aurillac (Cantal): Implications stratigraphiques

The so-called “série carbonatée” of Aurillac-Arpajon basin is generally ascribed to the upper Oligocene; a newlydiscovered micrommalian fauna shows that the basis of this sequence is really older and at least of a middle oligocene age.     

Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N-hydroxyguanidines by hydrogen peroxide.

Nitric oxide (NO) is a potent intra- and intercellular messenger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of l-arginine catalysed by hemeproteins called NO-synthases with intermediate formation of Nomega-hydroxy-l-arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model compound, N-(4-chlorophenyl)-N'-hydroxyguanidine, were tested for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydrogen peroxide. Nitrite and nitrate production along with selective formation of 4-chlorophenylcyanamide was observed from incubations of N-(4-chlorophenyl)-N'-hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyanamide, Ndelta-cyano-L-ornithine, was too unstable under the conditions used and l-citrulline was the only product identified. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl and formation of MP8-Fe-NO complexes were observed by EPR spectroscopy and low-temperature UV/visible spectroscopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxidation of exogenous hydroxyguanidines by hydrogen peroxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N-hydroxyguanidines by peroxidases of physiological interest remains to be established in vivo.