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First described inDrosophila melanogaster, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse,Xenopusand zebrafish embryos. The regulated and poorly understood traffic of Vangl2 to the plasma membrane is a key event for its function in development. Here we report on the molecular and functional characterization of a novel 569-amino acid N-terminally extended Vangl2 isoform, Vangl2-Long, that arises from an alternative non-AUG translation initiation site, lying 144 base pair upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, includingDrosophila melanogaster, this N-terminal extension is conserved in all vertebrate Vangl2 sequences and confers a subcellular localization in the Golgi apparatus, probably as a result of an extended retention time in this organelle. Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2 and we show that its down-regulation leads to severe PCP-related phenotypes inXenopusembryos, including shorter body axis and neural tube closure defects. Altogether, our study unveils a novel level of complexity in Vangl2 expression, trafficking and function.

This computational work draws some analogies between control of light in diffusive media and control of drug release in some biochemical cloaks, the latter being a concept first proposed by Tania Puvirajesinghe (Institut Paoli-Calmettes) and Sebastien Guenneau (Institut Fresnel) five years ago.; International audience; We make use of transformation optics technique to realize cloaking operation in the light diffusive regime, for spherical objects. The cloak requires spatially heterogeneous anisotropic diffusivity, as well as spatially varying speed of light and absorption. Analytic calculations of Photon’s fluence confirm minor role of absorption in reduction of far-field scattering, and a monopole fluence field converging to a constant in the static regime in the invisibility region. The latter is in contrast to acoustic and electromagnetic cloaks, for which the field vanishes inside the core. These results are finally discussed in the context of mass diffusion, where cloaking can be achieved with a heterogeneous anisotropic diffusivity.

International audience; Cell polarity is a vital biological process involved in the building, maintenance and normal functioning of tissues in invertebrates and vertebrates. Unsurprisingly, molecular defects affecting polarity organization and functions have a strong impact on tissue homeostasis, embryonic development and adult life, and may directly or indirectly lead to diseases. Genetic studies have demonstrated the causative effect of several polarity genes in diseases; however, much remains to be clarified before a comprehensive view of the molecular organization and regulation of the protein networks associated with polarity proteins is obtained. This challenge can be approached head-on using proteomics to identify protein complexes involved in cell polarity and their modifications in a spatio-temporal manner. We review the fundamental basics of mass spectrometry techniques and provide an in-depth analysis of how mass spectrometry has been instrumental in understanding the complex and dynamic nature of some cell polarity networks at the tissue (apico-basal and planar cell polarities) and cellular (cell migration, ciliogenesis) levels, with the fine dissection of the interconnections between prototypic cell polarity proteins and signal transduction cascades in normal and pathological situations. This review primarily focuses on epithelial structures which are the fundamental building blocks for most metazoan tissues, used as the archetypal model to study cellular polarity. This field offers broad perspectives thanks to the ever-increasing sensitivity of mass spectrometry and its use in combination with recently developed molecular strategies able to probe in situ proteomic networks.

Neural tube defects (NTDs) are congenital birth defects classified according to their resulting morphological characteristics in newborn patients. Current diagnosis of NTDs relies largely on the structural evaluation of fetuses using ultrasound imaging, with biochemical characterization used as secondary screening tools. The multigene etiology of NTDs has been aided by genetic studies, which have discovered panels of genes mutated in these diseases that encode receptors and cytoplasmic signaling molecules with poorly defined functions. Animal models ranging from flies to mice have been used to determine the function of these genes and identify their associated molecular cascades. More emphasis is now being placed on the identification of biochemical markers from clinical samples and model systems based on mass spectrometry, which open novel avenues in the understanding of NTDs at protein, metabolic and molecular levels. This article reviews how the use of proteomics can push forward the identification of novel biomarkers and molecular networks implicated in NTDs, an indispensable step in the improvement of patient management.

We begin with a brief historical survey of discoveries of quasi-crystals and graphene, and then introduce the concept of transformation crystallography, which consists of the application of geometric transforms to periodic structures. We consider motifs with three-fold, four-fold and six-fold symmetries according to the crystallographic restriction theorem. Furthermore, we define motifs with five-fold symmetry such as quasi-crystals generated by a cut-and-projection method from periodic structures in higher-dimensional space. We analyze elastic wave propagation in the transformed crystals and (Penrose-type) quasi-crystals with the finite difference time domain freeware SimSonic. We consider geometric transforms underpinning the design of seismic cloaks with square, circular, elliptical and peanut shapes in the context of honeycomb crystals that can be viewed as scaled-up versions of graphene. Interestingly, the use of morphing techniques leads to the design of cloaks with interpolated geometries reminiscent of Victor Vasarely's artwork. Employing the case of transformed graphene-like (honeycomb) structures allows one to draw useful analogies between large-scale seismic metamaterials such as soils structured with columns of concrete or grout with soil and nanoscale biochemical metamaterials. We further identify similarities in designs of cloaks for elastodynamic and hydrodynamic waves and cloaks for diffusion (heat or mass) processes, as these are underpinned by geometric transforms. Experimental data extracted from field test analysis of soil structured with boreholes demonstrates the application of crystallography to large scale phononic crystals, coined as seismic metamaterials, as they might exhibit low frequency stop bands. This brings us to the outlook of mechanical metamaterials, with control of phonon emission in graphene through extreme anisotropy, attenuation of vibrations of suspension bridges via low frequency stop bands and the concept of transformed meta-cities. We conclude that these novel materials hold strong applications spanning different disciplines or across different scales from biophysics to geophysics.

Microarray technologies inspired the development of carbohydrate arrays. Initially, carbohydrate array technology was hindered by the complex structures of glycans and their structural variability. The first designs of glycoarrays focused on the HTP (high throughput) study of protein–glycan binding events, and subsequently more in-depth kinetic analysis of carbohydrate–protein interactions. However, the applications have rapidly expanded and now achieve successful discrimination of selective interactions between carbohydrates and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Combining array technology with other HTP technologies such as mass spectrometry is expected to allow even more accurate and sensitive analysis. This review provides a broad overview of established glycoarray technologies (with a special focus on glycosaminoglycan applications) and their emerging applications to the study of complex interactions between glycans and whole living cells.

The non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway plays a crucial role in embryonic development. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed Wnt/PCP signalling as a therapeutic target. Here we show that the archetypal Wnt/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumour growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2–p62/SQSTM1–JNK pathway. This proliferative signalling cascade is upregulated in breast cancer patients with shorter survival and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2–p62/SQSTM1 interaction. VANGL2–JNK signalling is thus a potential target for breast cancer therapy., Defects in non-canonical Wnt/planar cell polarity signalling have recently been linked to breast cancer aggressiveness. Puvirajesinghe et al. identify VANGL2, p62/SQSTM1 and JNK as important players in this pathway which may be amenable to therapeutic intervention in breast cancer.

International audience; Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not requirea functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development.

Contains fulltext : 152758.PDF (Publisher’s version ) (Open Access) INTRODUCTION: Sulf1 and Sulf2 are cell surface sulfatases, which remove specific 6-O-sulfate groups from heparan sulfate (HS) proteoglycans, resulting in modulation of various HS-dependent signaling pathways. Both Sulf1 and Sulf2 knockout mice show impairments in brain development and neurite outgrowth deficits in neurons. METHODOLOGY AND MAIN FINDINGS: To analyze the molecular mechanisms behind these impairments we focused on the postnatal cerebellum, whose development is mainly characterized by proliferation, migration, and neurite outgrowth processes of precursor neurons. Primary cerebellar granule cells isolated from Sulf1 or Sulf2 deficient newborns are characterized by a reduction in neurite length and cell survival. Furthermore, Sulf1 deficiency leads to a reduced migration capacity. The observed impairments in cell survival and neurite outgrowth could be correlated to Sulf-specific interference with signaling pathways, as shown for FGF2, GDNF and NGF. In contrast, signaling of Shh, which determines the laminar organization of the cerebellar cortex, was not influenced in either Sulf1 or Sulf2 knockouts. Biochemical analysis of cerebellar HS demonstrated, for the first time in vivo, Sulf-specific changes of 6-O-, 2-O- and N-sulfation in the knockouts. Changes of a particular HS epitope were found on the surface of Sulf2-deficient cerebellar neurons. This epitope showed a restricted localization to the inner half of the external granular layer of the postnatal cerebellum, where precursor cells undergo final maturation to form synaptic contacts. CONCLUSION: Sulfs introduce dynamic changes in HS proteoglycan sulfation patterns of the postnatal cerebellum, thereby orchestrating fundamental mechanisms underlying brain development.

International audience; We review recent advances in the control of diffusion processes in thermodynamicsand life sciences through geometric transforms in the Fourier and Fick equations,which govern heat and mass diffusion, respectively. We propose to further encom-pass transport properties in the transformed equations, whereby the temperatureis governed by a three-dimensional, time-dependent, anisotropic heterogeneousconvection-diffusion equation, which is a parabolic partial differential equationcombining the diffusion equation and the advection equation. We perform twodimensional finite element computations for cloaks, concentrators and rotators ofa complex shape in the transient regime. We precise that in contrast to invisibilitycloaks for waves, the temperature (or mass concentration) inside a diffusion cloakcrucially depends upon time, its distance from the source, and the diffusivity ofthe invisibility region. However, heat (or mass) diffusion outside cloaks, concen-trators and rotators is unaffected by their presence, whatever their shape or posi-tion. Finally, we propose simplified designs of layered cylindrical and sphericaldiffusion cloaks that might foster experimental efforts in thermal and biochem-ical metamaterials.

Neural tube defects (NTDs) are congenital birth defects classified according to their resulting morphological characteristics in newborn patients. Current diagnosis of NTDs relies largely on the structural evaluation of fetuses using ultrasound imaging, with biochemical characterization used as secondary screening tools. The multigene etiology of NTDs has been aided by genetic studies, which have discovered panels of genes mutated in these diseases that encode receptors and cytoplasmic signaling molecules with poorly defined functions. Animal models ranging from flies to mice have been used to determine the function of these genes and identify their associated molecular cascades. More emphasis is now being placed on the identification of biochemical markers from clinical samples and model systems based on mass spectrometry, which open novel avenues in the understanding of NTDs at protein, metabolic and molecular levels. This article reviews how the use of proteomics can push forward the identification of novel biomarkers and molecular networks implicated in NTDs, an indispensable step in the improvement of patient management.

Here, we adapt the concept of transformational thermodynamics, whereby the flux of temperature is controlled via anisotropic heterogeneous diffusivity, for the diffusion and transport of mass concentration. The n-dimensional, time-dependent, anisotropic heterogeneous Fick's equation is considered, which is a parabolic partial differential equation also applicable to heat diffusion, when convection occurs, for example in fluids. This theory is illustrated with finite element computations for a liposome particle surrounded by a cylindrical multilayered cloak in a water-based environment, and for a spherical multilayered cloak consisting of layers of fluid with an isotropic homogeneous diffusivity, deduced from an effective medium approach. Initial potential applications could be sought in bio-engineering., Comment: 7 pages, 3 figures

International audience; The glycosaminoglycan heparan sulphate (HS) has a heterogeneous structure; evidence shows that specific structures may be responsible for specific functions in biological processes such as blood coagulation and regulation of growth factor signalling. This review summarises the different experimental tools and methods developed to provide more rapid methods for studying the structure and functions of HS. Rapid and sensitive methods for the facile purification of HS, from tissue and cell sources are reviewed. Data sets for the structural analysis are often complex and include multiple sample sets, therefore different software and tools have been developed for the analysis of different HS data sets. These can be readily applied to chromatographic data sets for the simplification of data (e.g., charge separation using strong anion exchange chromatography and from size separation using gel filtration techniques. Finally, following the sequencing of the human genome, research has rapidly advanced with the introduction of high throughput technologies to carry out simultaneous analyses of many samples. Microarrays to study macromolecular interactions (including glycan arrays) have paved the way for bioassay technologies which utilize cell arrays to study the effects of multiple macromolecules on cells. Glycan bioassay technologies are described in which immobilisation techniques for saccharides are exploited to develop a platform to probe cell responses such as signalling pathway activation. This review aims at reviewing available techniques and tools for the OPEN ACCESS Metabolites 2012, 2 1061 purification, analysis and bioassay of HS saccharides in biological systems using " glycomics " approaches.

International audience; Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of -helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.

International audience; Array methodologies have become powerful tools for interrogation of glycan-protein interactions but have critically lacked the ability to generate cell response data. Here, we report the development of a slide-based array method exemplified by measurement of activation of fibroblast growth factor signaling by heparin saccharides. Heparan sulfate-deficient Swiss 3T3 cells were overlaid onto an aminosilane-coated slide surface onto which heparin saccharides had been spotted and immobilized. The cells were transiently stimulated with FGF2 and immunofluorescence measured to assess downstream ERK1/2 phosphorylation. Activation of this signaling pathway response was restricted to cells exposed to heparin saccharides competent to activate FGF2 signaling. Differential activation of the overlaid cells by different-sized heparin saccharides was demonstrated by quantitative measurement of fluorescence intensity. This "glycobioarray" platform has significant potential as a generic tool for functional glycomics screening.

International audience; Background: Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results.Methodology and Main Findings: A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2Lp protein in mutant mice compared to the wild type mice.Conclusion: Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.

The heparan sulfate (HS) family of glycosaminoglycans are highly complex and structurally diverse polysaccharides with information encoded within the chains that imparts the ability to bind selectively to a wide range of proteins-the "HS interactome"-and to regulate their biological activities. However, there are two key questions which need to be addressed; first, the extent of structural variation of expressed HS structures-the "heparanome"-in specific biological contexts and second, the degree of functional selectivity exerted by these structures in regulating biological processes. There is a clear need to develop more systematic and high throughput approaches in order to address these questions. Here, we describe a cohort of protocols for profiling different aspects of HS structure and activity, focusing particularly on disaccharide building blocks and larger oligosaccharide domains, the latter representing the functional units of HS chains. A range of other complementary methods in the literature are also discussed. Together these provide a new and more comprehensive toolkit to investigate HS structure and activity in a higher throughput manner in selected biological systems. The implementation of such a glycomics strategy will enable development of a systems biology view of HS structure-function relationships and help to resolve the significant puzzle of the extensive interactome of HS, which remains a key question in the glycobiology field. We anticipate that the next decade will see major advances in our understanding of the complex biology of HS.

The heparan sulfate (HS) family of glycosaminoglycans are highly complex and structurally diverse polysaccharides with information encoded within the chains that imparts the ability to bind selectively to a wide range of proteins-the "HS interactome"-and to regulate their biological activities. However, there are two key questions which need to be addressed; first, the extent of structural variation of expressed HS structures-the "heparanome"-in specific biological contexts and second, the degree of functional selectivity exerted by these structures in regulating biological processes. There is a clear need to develop more systematic and high throughput approaches in order to address these questions. Here, we describe a cohort of protocols for profiling different aspects of HS structure and activity, focusing particularly on disaccharide building blocks and larger oligosaccharide domains, the latter representing the functional units of HS chains. A range of other complementary methods in the literature are also discussed. Together these provide a new and more comprehensive toolkit to investigate HS structure and activity in a higher throughput manner in selected biological systems. The implementation of such a glycomics strategy will enable development of a systems biology view of HS structure-function relationships and help to resolve the significant puzzle of the extensive interactome of HS, which remains a key question in the glycobiology field. We anticipate that the next decade will see major advances in our understanding of the complex biology of HS.

International audience; Heparan sulphate (HS) is a glycosaminoglycan present in all metazoan organisms. It is an unbranched chain made up of repeating disaccharide units of uronic acid and glucosamine sugars, and is present in both cells and the extracellular matrix. It is one of the most structurally diverse biological molecules and its biosynthesis involves a variety of enzymic modification steps. Unlike the genome and the transcriptome, HS synthesis is not template driven. Nevertheless, the HS structure and function are highly regulated with modification steps occurring in discrete regions of the polysaccharide chain to give rise to diverse structures interacting with, and regulating, many different proteins. The resulting variation leads to diverse biological roles of HS. To study this structural diversity, rapid isolation and characterization of HS from small amounts of tissues, followed by digestion with bacterially derived enzymes (heparitinases) and chromatography techniques can be used to separate HS oligosaccharides of different size and charge. However, this leads to complex datasets where comparison of just a few samples leads to difficulties in data analysis. Using automatically integrated peak data obtained from chromatographic software, one can apply the effective disc technique to the data points to obtain the centre of mass in each dataset, for example from different murine tissues. This allows facile comparative analysis of different datasets. When the cloud of points displays some preferential direction (anisotropy), it is preferable to compute its effective ellipse. Analysis of the dynamics of the cloud of points for repeated experiments allows the quantification of their reproducibility through evaluation of an average Lyapunov exponent characterizing the area-preserving nature of a sequence of effective ellipses. These basic mathematical approaches allow a more systematic comparison of datasets derived from structural analysis using basic spreadsheet software calculations and contribute to the development of system biology strategies for tackling biocomplexity of HS polysaccharides.