Environmental metagenomics: An innovative resource for industrial biocatalysis

The current existing enzymes have been identified from cultivable micro-organisms, most frequently from bacteria. These bacterial biocatalytic capabilities have been widely used for biotransformations, resulting in the development of profitable industrial bioprocesses in the fields of feed and food processing, textiles, agro-chemistry, cosmetics, pharmaceuticals and fine chemistry. However, the originality of this bioresource is progressively drying up, while requests from industry for novel biocatalytic activities are increasing in the face of economic and environmental pressure. Metagenomics, through access to the huge reservoir of uncultivated bacteria which represents the majority of the present biodiversity, opens the door to new industrial sources of enzymes. Surmounting hurdles encountered with this technology (e.g. DNA extraction to obtain high quality DNA libraries with proper statistical representativity, setting up of relevant high throughput screenings assays, combining functional and genome-based identifications), gives unique opportunities to access novel biocatalysts that better fit with the required industrial specifications, thus providing new biocatalysis tool boxes.     

3D Evaluation of the acetabular coverage assessed by biplanar X-rays or single anteroposterior X-ray compared with CT-scan

Considering the increasing development of three dimensional (3D) imaging, the 3D assessment of the acetabular coverage is to become the most interesting tool for the detection of acetabular pathologies. Biplanar X-rays based methods allow a 3D reconstruction of the hip with a reduced radiation dose. This study proposes a 3D assessment method of the acetabular coverage from biplanar X-rays or from an anteroposterior X-ray (conventional clinical imaging). An in vitro evaluation of the method was performed on six hip joints in comparison with computed tomography. The global coverage, the local coverage and the acetabular rim orientation were estimated in 3D. The mean global acetabular coverage was 40% with an estimated mean accuracy of 1.3% for the biplanar X-rays based method. This study evaluated a 3D assessment method of the acetabular coverage from biplanar X-rays or anteroposterior X-ray and open the way for clinical in vivo applications.     

Egg box conformation of oligogalacturonides: the time-dependent stabilization of the elicitor-active conformation increases its biological activity

Circular dichroism spectrometry was used on oligogalacturonides (OGAs) and showed the existence of a calcium/sodium-induced conformational state that is intermediate between single-isolated chains and calcium-associated multimer chains. This conformation is interpreted as being egg box dimers. Using the 2F4 monoclonal antibody that specifically binds such an egg box dimer conformation of pectin, the stability of OGA dimers was investigated over a period of 24 hours. The extent to which egg box dimers were recognized by the antibody was dependent on the temperature and duration of preincubation of the OGA. This suggests a "maturation" process of the egg-box structure that consists in a progressive increase in the length of the junction sequences between two chains that slide along each other in order to form a maximum number of calcium bridges and dimer ends. The maturation of egg boxes induced both a significant increase in their binding to wall-associated kinase 1 (WAK1) and an increased extracellular alkalinization when applied to Arabidopsis thaliana cell suspensions. The chemical modification of the reducing end of the OGAs largely diminished their elicitating activity but did not hinder either dimerization or binding of these end-reduced egg boxes to WAK1. We conclude that there are at least two different perception systems for egg box dimers. One binds egg box junctions and the other binds egg box ends. The relevance of these results is discussed in terms of pectic signal perception and plant-pathogen interaction.     

Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ

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3D Pharmacophore,hierarchical methods,and 5-HT4 receptor binding data

5-Hydroxytryptamine subtype-4 (5-HT₄) receptors have stimulated considerable interest amongst scientists and clinicians owing to their importance in neurophysiology and potential as therapeutic targets. A comparative analysis of hierarchical methods applied to data from one thousand 5-HT₄ receptor–ligand binding interactions was carried out. The chemical structures were described as chemical and pharmacophore fingerprints. The definitions of indices, related to the quality of the hierarchies in being able to distinguish between active and inactive compounds, revealed two interesting hierarchies with the Unity (1 active cluster) and pharmacophore fingerprints (4 active clusters). The results of this study also showed the importance of correct choice of metrics as well as the effectiveness of a new alternative of the Ward clustering algorithm named Energy (Minimum E-Distance method). In parallel, the relationship between these classifications and a previously defined 3D 5-HT₄ antagonist pharmacophore was established.     

Mitochondrial Phylogenomics of Modern and Ancient Equids

The genus Equus is richly represented in the fossil record, yet our understanding of taxonomic relationships within this genus remains limited. To estimate the phylogenetic relationships among modern horses, zebras, asses and donkeys, we generated the first data set including complete mitochondrial sequences from all seven extant lineages within the genus Equus. Bayesian and Maximum Likelihood phylogenetic inference confirms that zebras are monophyletic within the genus, and the Plains and Grevy’s zebras form a well-supported monophyletic group. Using ancient DNA techniques, we further characterize the complete mitochondrial genomes of three extinct equid lineages (the New World stilt-legged horses, NWSLH; the subgenus Sussemionus; and the Quagga, Equus quagga quagga). Comparisons with extant taxa confirm the NWSLH as being part of the caballines, and the Quagga and Plains zebras as being conspecific. However, the evolutionary relationships among the non-caballine lineages, including the now-extinct subgenus Sussemionus, remain unresolved, most likely due to extremely rapid radiation within this group. The closest living outgroups (rhinos and tapirs) were found to be too phylogenetically distant to calibrate reliable molecular clocks. Additional mitochondrial genome sequence data, including radiocarbon dated ancient equids, will be required before revisiting the exact timing of the lineage radiation leading up to modern equids, which for now were found to have possibly shared a common ancestor as far as up to 4 Million years ago (Mya).     

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

Background

Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.

Methodology/Principal Findings

We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.

Conclusions/Significance

These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.     

Focal adhesion kinase plays a dual role in TRAIL resistance and metastatic outgrowth of malignant melanoma

Despite remarkable advances in therapeutic interventions, malignant melanoma (MM) remains a life-threating disease. Following high initial response rates to targeted kinase-inhibition metastases quickly acquire resistance and present with enhanced tumor progression and invasion, demanding alternative treatment options. We show 2^nd generation hexameric TRAIL-receptor-agonist IZI1551 (IZI) to effectively induce apoptosis in MM cells irrespective of the intrinsic BRAF/NRAS mutation status. Conditioning to the EC₅₀ dose of IZI converted the phenotype of IZI-sensitive parental MM cells into a fast proliferating and invasive, IZI-resistant metastasis. Mechanistically, we identified focal adhesion kinase (FAK) to play a dual role in phenotype-switching. In the cytosol, activated FAK triggers survival pathways in a PI3K- and MAPK-dependent manner. In the nucleus, the FERM domain of FAK prevents activation of wtp53, as being expressed in the majority of MM, and consequently intrinsic apoptosis. Caspase-8-mediated cleavage of FAK as well as FAK knockdown, and pharmacological inhibition, respectively, reverted the metastatic phenotype-switch and restored IZI responsiveness. FAK inhibition also re-sensitized MM cells isolated from patient metastasis that had relapsed from targeted kinase inhibition to cell death, irrespective of the intrinsic BRAF/NRAS mutation status. Hence, FAK-inhibition alone or in combination with 2nd generation TRAIL-receptor agonists may be recommended for treatment of initially resistant and relapsed MM, respectively.Subject terms: Metastasis, Target identification