Translocation of the cytoplasmic domain of ADAM13 to the nucleus is essential for Calpain8-a expression and cranial neural crest cell migration

ADAMs are transmembrane metalloproteases that control cell behavior by cleaving both cell adhesion and signaling molecules. The cytoplasmic domain of ADAMs can regulate the proteolytic activity by controlling the subcellular localization and/or the activation of the protease domain. Here, we show that the cytoplasmic domain of ADAM13 is cleaved and translocates into the nucleus. Preventing this translocation renders the protein incapable of promoting cranial neural crest (CNC) cell migration in?vivo, without affecting its proteolytic activity. In addition, the cytoplasmic domain of ADAM13 regulates the expression of multiple genes in CNC, including the protease Calpain8-a. Restoring the expression of Calpain8-a is sufficient to rescue CNC migration in the absence of the ADAM13 cytoplasmic domain. This study shows that the cytoplasmic domain of ADAM metalloproteases can perform essential functions in the nucleus of cells and may contribute substantially to the overall function of the protein.     

ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection

Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85α protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes.     

Metagenomic exploration of antibiotic resistance in soil

The ongoing development of metagenomic approaches is providing the means to explore antibiotic resistance in nature and address questions that could not be answered previously with conventional culture-based strategies. The number of available environmental metagenomic sequence datasets is rapidly expanding and henceforth offer the ability to gain a more comprehensive understanding of antibiotic resistance at the global scale. Although there is now evidence that the environment constitutes a vast reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin, a better understanding of their diversity, prevalence and ecological significance may help predict the emergence and spreading of newly acquired resistances. Recent applications of metagenomic approaches to the study of ARGDs in natural environments such as soil should help overcome challenges concerning expanding antibiotic resistances.     

Global patterns of bacterial beta-diversity in seafloor and seawater ecosystems

Background

Marine microbial communities have been essential contributors to global biomass, nutrient cycling, and biodiversity since the early history of Earth, but so far their community distribution patterns remain unknown in most marine ecosystems.

Methodology/Principal Findings

The synthesis of 9.6 million bacterial V6-rRNA amplicons for 509 samples that span the global ocean''s surface to the deep-sea floor shows that pelagic and benthic communities greatly differ, at all taxonomic levels, and share <10% bacterial types defined at 3% sequence similarity level. Surface and deep water, coastal and open ocean, and anoxic and oxic ecosystems host distinct communities that reflect productivity, land influences and other environmental constraints such as oxygen availability. The high variability of bacterial community composition specific to vent and coastal ecosystems reflects the heterogeneity and dynamic nature of these habitats. Both pelagic and benthic bacterial community distributions correlate with surface water productivity, reflecting the coupling between both realms by particle export. Also, differences in physical mixing may play a fundamental role in the distribution patterns of marine bacteria, as benthic communities showed a higher dissimilarity with increasing distance than pelagic communities.

Conclusions/Significance

This first synthesis of global bacterial distribution across different ecosystems of the World''s oceans shows remarkable horizontal and vertical large-scale patterns in bacterial communities. This opens interesting perspectives for the definition of biogeographical biomes for bacteria of ocean waters and the seabed.     

Biotin synthesis in plants. The first committed step of the pathway is catalyzed by a cytosolic 7-keto-8-aminopelargonic acid synthase

Biochemical and molecular characterization of the biotin biosynthetic pathway in plants has dealt primarily with biotin synthase. This enzyme catalyzing the last step of the pathway is localized in mitochondria. Other enzymes of the pathway are however largely unknown. In this study, a genomic-based approach allowed us to clone an Arabidopsis (Arabidopsis thaliana) cDNA coding 7-keto-8-aminopelargonic acid (KAPA) synthase, the first committed enzyme of the biotin synthesis pathway, which we named AtbioF. The function of the enzyme was demonstrated by functional complementation of an Escherichia coli mutant deficient in KAPA synthase reaction, and by measuring in vitro activity. Overproduction and purification of recombinant AtbioF protein enabled a thorough characterization of the kinetic properties of the enzyme and a spectroscopic study of the enzyme interaction with its substrates and product. This is the first characterization of a KAPA synthase reaction in eukaryotes. Finally, both green fluorescent protein-targeting experiments and western-blot analyses showed that the Arabidopsis KAPA synthase is present in cytosol, thus revealing a unique compartmentation of the plant biotin synthesis, split between cytosol and mitochondria. The significance of the complex compartmentation of biotin synthesis and utilization in the plant cell and its potential importance in the regulation of biotin metabolism are also discussed.     

3-Thio-1,2,4-triazoles, novel somatostatin sst2/sst5 agonists

Novel 3-thio-1,2,4-triazoles have been obtained via a solution-phase parallel synthesis strategy, affording potent non-peptidic human somatostatin receptor subtypes 2 and 5 agonists.     

Non-linear mapping for exploratory data analysis in functional genomics

Background  

Several supervised and unsupervised learning tools are available to classify functional genomics data. However, relatively less attention has been given to exploratory, visualisation-driven approaches. Such approaches should satisfy the following factors: Support for intuitive cluster visualisation, user-friendly and robust application, computational efficiency and generation of biologically meaningful outcomes. This research assesses a relaxation method for non-linear mapping that addresses these concerns. Its applications to gene expression and protein-protein interaction data analyses are investigated