Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.     

Le genre Cratopus aux îles Seychelles (Coleoptera : Curculionidae)

Les espèces seychelloises du genre Cratopus Schoenherr 1823 sont révisées, et quatre nouveaux taxons sont décrits. Espèce de grande taille (16–19 mm), C. roberti n. sp. possède dix stries élytrales, des fémurs antérieurs portant de petits denticules, y compris sur la dent, et un revêtement squamuleux vert métallique, avec une bande blanche le long de la base et des côtés des élytres, ainsi que sur les côtés du prothorax. Long de 8–10 mm, C. venustus n. sp. se reconnait à ses téguments noirs et brillants, sa vestiture réduite bleu-vert métallique, ses douze stries élytrales de forts points, ainsi qu’à ses interstries 8 et 9 caréniformes. C. griseovestitus northislandensis n. ssp. diffère de la forme nominative C. griseovestitus griseovestitus Linell 1887 par ses téguments coriacés et sa vestiture métallique du dessus dense, à reflets cuivre doré ou verts, et non gris blanchâtre ou bleuâtre. C. griseovestitus fregata n. ssp. diffère de son côté de la forme nominative par ses téguments bruns plus ou moins foncés, saupoudrés de poils et de squamules très petits. Les synonymies de C. parcesquamosus Fairmaire 1893 et de C. abbotti Linell 1907 avec C. aurostriatus Fairmaire 1892 sont confirmées, et enfin un tableau de détermination des espèces seychelloises du genre est donné. Les Cratopus des Seychelles se divisent en un ensemble de trois espèces du groupe de griseovestitus, à large répartition, habitant les côtes et les îles coralliennes basses et probablement d’arrivée récente, et un ensemble de six espèces limitées aux îles granitiques, où cinq d’entre elles ont une distribution limitée à l’intérieur d’une île ou deux. Elles sont certainement de souche beaucoup plus ancienne. Les Cratopus des Seychelles sont folivores à l’état adulte, et souvent polyphages. Une liste de leurs plantes-hôtes connues est donnée.     

Dating the “red beds” of the Eastern Moroccan High Plateaus: Evidence from late Late Cretaceous charophytes and dinosaur eggshells

A new locality in the poorly known “red beds” of Tendrara (High Plateaus, Morocco) has yielded four charophytes species (Feistiella anluensis, Lamprothamnium stipitatum, Peckisphaera portezueloensis, Platychara caudata) and dinosaur eggshells (Pseudomegaloolithus atlasi). These red beds, which overly the Cenomanian-Turonian marine deposits, generally assigned to “Senonian” based on geometric position, are directly dated by these fossils: the charophytes species and dinosaur oospecies association indicates a Campano-Maastrichtian or Maastrichtian age for these calm floodplain deposits.     

Phenoscope: an automated large‐scale phenotyping platform offering high spatial homogeneity

Increased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non‐destructive, real‐time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high‐throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro‐environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro‐environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large‐scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.     

Structural Basis for Highly Effective HIV-1 Neutralization by CD4-Mimetic Miniproteins Revealed by 1.5 Å Cocrystal Structure of gp120 and M48U1

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Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.     

Life cycle assessment of a biobased chainsaw oil made on the farm in Wallonia

Purpose

The environmental issue is a particular concern for chainsaw oils because these fluids represent a total loss system. The aim of this study is to quantify the environmental impacts of a biobased chainsaw oil made on the farm in Wallonia (a region of Belgium) and to compare it with a model mineral chainsaw oil. With this study, the aim is also to participate in the development of the life cycle assessment (LCA) methodology applied to the biolubricant sector since LCAs on these products are quite limited and rarely sufficiently detailed.

Method

In this LCA, the attributional approach is applied. Seven impact categories are studied. The methods for life cycle impact assessment are IPCC, ReCiPe, CML and USEtox. The functional unit is 1 kg of base oil. Seven sensitivity analyses are performed.

Results and discussion

Results indicate that the biobased chainsaw oil made on the farm has a lower impact for the global warming potential, the abiotic depletion potential, the ozone depletion potential and the photochemical oxidation potential. On the contrary, it has larger acidification, aquatic eutrophication and aquatic ecotoxicity potential impacts. Regarding the contribution of the life cycle stages of the biobased chainsaw oil, the agricultural stage causes the highest contribution in all impact categories. For the mineral chainsaw oil, the refining stage is preponderant for all impact categories except for the global warming potential for which the end-of-life stage contributes the most. When taking additives into account, conclusions regarding the comparison between the oils are not reversed. Even if it was necessary to consume more biobased than mineral chainsaw oil, conclusions regarding the comparison of the oils would not be reversed. In the same way, a different allocation procedure for rapeseed oil and rape meal, a different rape seeds yield or different extraction yields in the refining stage of the mineral base oil do not change the results of the comparison. For the biobased chainsaw oil, the substitution of only one active substance in the agricultural stage could result in an important decrease of the freshwater ecotoxicity impact.

Conclusions

The biobased chainsaw oil has a lower impact in four out of the seven impact categories and a higher impact in three impact categories. By providing a detailed LCA on a biobased chainsaw oil, this study contributes to the development of LCA applied to biobased lubricants.     

Évaluation fonctionnelle par TEP 18F-FDopa des paragangliomes et phéochromocytomes non métastatiques : impact de la localisation lésionnelle et du statut génétique

ObjectiveParaganglioma (PGL) and pheochromocytoma (PCC) are neuroendocrine tumors most often benign associated with hereditary syndromes in about 30% of cases. This study aims to define the impact of tumor location and patient genotype on the clinical value of ¹⁸F-FDopa PET by assessing in detail the false negative occurrences.Patients and methodsA retrospective study was conducted on a cohort of 53 cases with non-metastatic sporadic or inherited PGL/PCC (SDHx or VHL related syndromes), investigated with ¹⁸F-FDopa PET.ResultsOverall detection sensitivity of ¹⁸F-FDopa PET was 88%. Seventy-three lesions were found using this technique, including 49 head-and-neck PGL (HNP), two thoracic PGL (1 sympathetic and 1 parasympathetic), eight extra-adrenal retroperitoneal PGL and 15 PCC. The 10 missed lesions were seven extra-adrenal abdominal PGL (2 SDHB, 2 SDHD), two HNP (1 sporadic, 1 SDHD) and one PCC (1 SDHD).Conclusion¹⁸F-FDopa PET is a sensitive technique for the evaluation of non-metastatic head and neck and adrenal PGLs. Exploration of extra-adrenal retroperitoneal PGL associated with SDHB or SDHD syndrome is the main limitation of this technique, encouraging the use of alternative functional imaging modalities like FDG-PET. Negativity of ¹⁸F-FDopa PET in the initial assessment of a PGL should prompt to search for a SDHx mutation.     

Intérêt de l’imagerie hybride TEMP/TDM dans la localisation des glandes parathyroïdes ectopiques : à propos de 3 cas

The presence of a parathyroid gland in ectopic position constitutes one of the leading causes of surgery failure. For this purpose, scintigraphy takes a major place in the anatomical localization of pathological parathyroid and so facilitates its treatment. The advent of hybrid cameras combining single photon emission tomography to CT (SPECT/CT) has further strengthened the contribution of this imaging modality in the precise localization of ectopic parathyroid and its anatomical relationships. The objective of our work is to illustrate the interest of hybrid imaging SPECT/CT, compared to planar scintigraphy and ultrasounds within the framework of primary hyperparathyroidism, particularly ectopic situation through three cases.