Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.     

Structural identification of a bacterial quorum-sensing signal containing boron

Cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development. Unlike other autoinducers, which are specific to a particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species. AI-2 has been proposed to serve as a 'universal' signal for inter-species communication. The chemical identity of AI-2 has, however, proved elusive. Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with autoinducer. The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized autoinducers. Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2. Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.     

Multi-membrane hydrogels

Polysaccharide-based hydrogels are useful for numerous applications, from food and cosmetic processing to drug delivery and tissue engineering. The formation of hydrogels from polyelectrolyte solutions is complex, involving a variety of molecular interactions. The physical gelation of polysaccharides can be achieved by balancing solvophobic and solvophilic interactions. Polymer chain reorganization can be obtained by solvent exchange, one of the processing routes forming a simple hydrogel assembly. Nevertheless, many studies on hydrogel formation are empirical with a limited understanding of the mechanisms involved, delaying the processing of more complex structures. Here we use a multi-step interrupted gelation process in controlled physico-chemical conditions to generate complex hydrogels with multi-membrane 'onion-like' architectures. Our approach greatly simplifies the processing of gels with complex shapes and a multi-membrane organization. In contrast with existing assemblies described in the literature, our method allows the formation of free 'inter-membrane' spaces well suited for cell or drug introduction. These architectures, potentially useful in biomedical applications, open interesting perspectives by taking advantage of tailor-made three-dimensional multi-membrane tubular or spherical structures.     

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.     

A warm layer in Venus' cryosphere and high-altitude measurements of HF, HCl, H2O and HDO

Venus has thick clouds of H2SO4 aerosol particles extending from altitudes of 40 to 60 km. The 60-100 km region (the mesosphere) is a transition region between the 4 day retrograde superrotation at the top of the thick clouds and the solar-antisolar circulation in the thermosphere (above 100 km), which has upwelling over the subsolar point and transport to the nightside. The mesosphere has a light haze of variable optical thickness, with CO, SO2, HCl, HF, H2O and HDO as the most important minor gaseous constituents, but the vertical distribution of the haze and molecules is poorly known because previous descent probes began their measurements at or below 60 km. Here we report the detection of an extensive layer of warm air at altitudes 90-120 km on the night side that we interpret as the result of adiabatic heating during air subsidence. Such a strong temperature inversion was not expected, because the night side of Venus was otherwise so cold that it was named the 'cryosphere' above 100 km. We also measured the mesospheric distributions of HF, HCl, H2O and HDO. HCl is less abundant than reported 40 years ago. HDO/H2O is enhanced by a factor of approximately 2.5 with respect to the lower atmosphere, and there is a general depletion of H2O around 80-90 km for which we have no explanation.     

BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous ciliopathy. Although nine BBS genes have been cloned, they explain only 40-50% of the total mutational load. Here we report a major new BBS locus, BBS10, that encodes a previously unknown, rapidly evolving vertebrate-specific chaperonin-like protein. We found BBS10 to be mutated in about 20% of an unselected cohort of families of various ethnic origins, including some families with mutations in other BBS genes, consistent with oligogenic inheritance. In zebrafish, mild suppression of bbs10 exacerbated the phenotypes of other bbs morphants.     

Forecasting Mixed‐Frequency Time Series with ECM‐MIDAS Models

This paper proposes a mixed‐frequency error correction model for possibly cointegrated non‐stationary time series sampled at different frequencies. We highlight the impact, in terms of model specification, of the choice of the particular high‐frequency explanatory variable to be included in the cointegrating relationship, which we call a dynamic mixed‐frequency cointegrating relationship. The forecasting performance of aggregated models and several mixed‐frequency regressions are compared in a set of Monte Carlo experiments. In particular, we look at both the unrestricted mixed‐frequency model and at a more parsimonious MIDAS regression. Whereas the existing literature has only investigated the potential improvements of the MIDAS framework for stationary time series, our study emphasizes the need to include the relevant cointegrating vectors in the non‐stationary case. Furthermore, it is illustrated that the choice of dynamic mixed‐frequency cointegrating relationship does not matter as long as the short‐run dynamics are adapted accordingly. Finally, the unrestricted model is shown to suffer from parameter proliferation for samples of relatively small size, whereas MIDAS forecasts are robust to over‐parameterization. We illustrate our results for the US inflation rate. Copyright © 2014 John Wiley & Sons, Ltd.     

Estimating and Predicting the General Random Effects Model

This paper extends the ‘remarkable property’ of Breusch (Journal of Econometrics 1987; 36 : 383–389) and Baltagi and Li (Journal of Econometrics 1992; 53 : 45–51) to the three‐way random components framework. Indeed, like its one‐way and two‐way counterparts, the three‐way random effects model maximum likelihood estimation can be obtained as an iterated generalized least squares procedure through an appropriate algorithm of monotonic sequences of some variance components ratios, θ_i (i = 2, 3, 4). More specifically, a search over θ_iwhile iterating on the regression coefficients estimates β and the other θ_jwill guard against the possibility of multiple local maxima of the likelihood function. In addition, the derivations of related prediction functions are obtained based on complete as well as incomplete panels. Finally, an application to international trade issues modeling is presented. Copyright © 2014 John Wiley & Sons, Ltd.     

Maximum Transfer Distance Between Partitions

In this paper, we study a distance defined over the partitions of a finite set. Given two partitions P and Q, this distance is defined as the minimum number of transfers of an element from one class to another, required to transform P into Q. We recall the algorithm to evaluate this distance and we give some formulae for the maximum distance value between two partitions having exactly or at most p and q classes, for given p and q.