Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens.

Noroviruses (NoVs) are one of the leading causes of gastroenteritis in children and adults. For the last 2 decades, genogroup II genotype 4 (GII.4) NoVs have been circulating worldwide. GII.4 NoVs can be divided into variants, and since 2002 they have circulated in the population before being replaced every 2 or 3 years, which raises questions about the role of their histo-blood group antigen (HBGA) ligands in their evolution. To shed light on these questions, we performed an analysis of the interaction between representative GII.4 variants and HBGAs, and we determined the role of selected amino acids in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the amino acids, directly implicated in interactions with HBGAs. However, the ablation of the threonine residue at position 395 (ΔT395), an epidemiological feature of the post-2002 variants, was not deleterious to the binding of the virus-like particle (VLP) to the H antigen, while binding to A and B antigens was severely hampered. Nevertheless, the ΔT395 VLPs gained the capacity to bind to the Lewis x and sialyl-Lewis x antigens in comparison with the wild-type VLP, demonstrating that amino acid residues outside the HBGA binding site can modify the binding properties of NoVs. We also analyzed the attachment of baculovirus-expressed VLPs from six variants (Bristol, US95/96, Hunter, Yerseke, Den Haag, and Osaka) that were isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs. We showed that the six variants could all attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, Den Haag and Osaka variants additionally bound to carbohydrates present in the saliva of Lewis-positive nonsecretors. The carbohydrate binding profile and the genetic and mutagenesis analysis suggested that GII.4 binding to Lewis x and sialyl-Lewis x antigens might be a by-product of the genetic variation of the amino acids located in the vicinity of the binding site. Analysis of the binding properties for the six variants by surface plasmon resonance showed that only post-2002 variants (i.e., Hunter, Yerseke, Den Haag, and Osaka) presented strong binding to A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post-2002 variants. The combination of increased affinity for ABH antigens and of a newly acquired ability to recognize glycans from Lewis-positive nonsecretors could have contributed to the epidemiological importance of strains such as the Den Haag GII.4 subtype.

Surface plasmon resonance imaging in arrays coupled with mass spectrometry (SUPRA-MS): proof of concept of on-chip characterization of a potential breast cancer marker in human plasma.

Protein biomarker discovery and validation are crucial for diagnosis, prognosis, and theranostics of human pathologies; "omics" approaches bring new insights in this field. In particular, the combination of immuno-sensors in array format with mass spectrometry efficiently extends the classical immunoassay format and includes molecular characterization. Here, we coupled surface plasmon resonance imaging (SPRi) with MALDI-TOF mass spectrometry in a hyphenated technique which enables multiplexed quantification of binding by SPRi and molecular characterization of interacting partners by subsequent MS analysis. This adds specificity, because MS enables differentiation of molecules that are difficult to distinguish by use of antibodies, for example truncation variants or protein isoforms. Proof of concept was established for detection, identification, and characterization of a potential breast cancer marker, the LAG3 protein, at ~1 µg mL(-1), added to human plasma. The analytical performance of this new method, dubbed "SUPRA-MS", was established, particularly its specificity (S/N > 10) and reliability (100 % LAG3 identification with high significant mascot score >87.9). The adjusted format for rapid, collective, and automated on-chip MALDI-MS analysis is robust at the femtomole level and has numerous potential applications in proteomics.

Label-free sensing and atomic force spectroscopy for the characterization of protein-DNA and protein-protein interactions: application to estrogen receptors.

In this paper we describe a new surface plasmon resonance (SPR) biosensor dedicated to potential estrogenic compounds prescreening, by developing an estrogen receptor (ER) specific DNA chip. Through the covalent binding of a DNA strain wearing the estrogen response element (ERE) to an activated 6-mercapto-1-hexadecanoic acid and 11-mercapto-1-undecanol self-assembled monolayer on gold surface, the SPR biosensor allows to detect specifically, quickly, and without any labeling the binding of ER in the presence of estrogen. In parallel, we investigated the ER interaction with itself, in order to study the formation of ER dimer apparently needed to activate the gene expression through ERE interaction. For that, we engaged force spectroscopy experiments that allowed us to prove that ER needs estrogen for its dimerization. Moreover, these ER/ER intermolecular measurements enabled to propose an innovative screening tool for anti-estrogenic compounds, molecules of interest for hormono-dependent cancer therapy.

Analyte capture in an array of functionalized droplets for a regenerable biosensor.

We describe in this work an advanced microfluidic chip for the capture of bioanalyte on the surface of droplets arranged in a dense array. We show the procedure for generating, functionalizing, and arranging the droplets inside the device for capturing a specific bioanalyte. Then, we demonstrate the capacity of the array to capture analyte from a cross-flowing liquid, using a biotin/streptavidin model. The paper also proposes to use the droplets array, after integration with acoustic detection, as a regenerable detection interface for bioanalyte sensing. We model the arrangement of droplet in dense array and show that they present a larger effective capture surface and shorter capture distance than standard flat surface biosensor of the same footprint. As the droplets can be easily evacuated and replaced inside the device analysis chamber, the proposed biosensor would allow biointerface regeneration and chain measurement without dismounting the device.

Strategy of macromolecular grafting onto a gold substrate dedicated to protein-protein interaction measurements.

Many biotechnology applications use proteins immobilized on surface. For biosensor, the sensing layer is a key component interfacing the transducer and the sample. Strategies employed to activate the bidimensional surface act directly on the performance of the biosensor. In this paper we propose a novel strategy for engineered proteins self-assembly. Our original supramolecular structure allows a direct and fast covalent attachment of proteins onto bare gold substrate through a homobifunctional cross-linker, 1,4-di-([2'-pyridyldithio]propionamido)butane (DPDPB). In this work, engineered proteins and linker-protein complexes were synthesized and characterized by gel electrophoresis, chromatography and spectroscopy experiments. Macromolecular construction "DPDPB-GST tag-GEC1 protein" was conceived in order to guarantee a 2D architecture enhancing the capabilities of the target (tubulin) to recognize its partner (GEC1). Surface plasmon resonance measurements clearly showed potential of this particular self-assembled protein layer compared to a commercial immunosensor interface. At the concentrations tested, the recognition process occurs between tubulin and the immobilized GEC1; moreover enhanced binding was obtained with the home-made 2D sensing layer more than with 3D carboxymethyl dextran matrix.

Unique supramolecular assembly of a redox protein with nucleic acids onto hybrid bilayer: towards a dynamic DNA chip.

Highly controlled supramolecular assemblies combining a genetically engineered redox protein, cytochrome b5, and modified oligonucleotides are presented. Modified b5 and DNA are covalently assembled through a hetero bifunctional cross-linker to give a unique hybrid molecular species. Moreover, the assembly includes a histidine tag head able to bind to modified phospholipids which lead to a new generation of self-assembled dynamic DNA chips. The interaction of the construction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using Biacore technology. The biochip, presented herein, features unique properties including tunable surface density of probes, very low non-specific interactions and optimization of hybridization efficiency. In addition, we demonstrated that the phase transition of the lipidic layer can modulate the dynamic of the association of the complex to the supported membrane. Potential applications of this new device are multiple including high sensitivity and high selectivity biochips, especially for studies of the DNA-ligands interactions in a biomimetic environment.

cIAP1 regulates TNF-mediated cdc42 activation and filopodia formation.

Tumour necrosis factor-α (TNF) is a cytokine endowed with multiple functions, depending on the cellular and environmental context. TNF receptor engagement induces the formation of a multimolecular complex including the TNFR-associated factor TRAF2, the receptor-interaction protein kinase RIP1 and the cellular inhibitor of apoptosis cIAP1, the latter being essential for NF-κB activation. Here, we show that cIAP1 also regulates TNF-induced actin cytoskeleton reorganization through a cdc42-dependent, NF-κB-independent pathway. Deletion of cIAP1 prevents TNF-induced filopodia and cdc42 activation. The expression of cIAP1 or its E3-ubiquitin ligase-defective mutant restores the ability of cIAP1(-/-) MEFs to produce filopodia, whereas a cIAP1 mutant unable to bind TRAF2 does not. Accordingly, the silencing of TRAF2 inhibits TNF-mediated filopodia formation, whereas silencing of RIP1 does not. cIAP1 directly binds cdc42 and promotes its RhoGDIα-mediated stabilization. TNF decreases cIAP1-cdc42 interaction, suggesting that TNF-induced recruitment of cIAP1/TRAF2 to the receptor releases cdc42, which in turn triggers actin remodeling. cIAP1 also regulates cdc42 activation in response to EGF and HRas-V12 expression. A downregulation of cIAP1 altered the cell polarization, the cell adhesion to endothelial cells and cell intercalation, which are cdc42-dependent processes. Finally, we demonstrated that the deletion of cIAP1 regulated the HRas-V12-mediated transformation process, including anchorage-dependent cell growth, tumour growth in a xenograft model and the development of experimental metastasis in the lung.

Liver X receptor ß activation induces pyroptosis of human and murine colon cancer cells.

Liver X receptors (LXRs) have been proposed to have some anticancer properties, through molecular mechanisms that remain elusive. Here we report for the first time that LXR ligands induce caspase-1-dependent cell death of colon cancer cells. Caspase-1 activation requires Nod-like-receptor pyrin domain containing 3 (NLRP3) inflammasome and ATP-mediated P2 × 7 receptor activation. Surprisingly, LXRß is mainly located in the cytoplasm and has a non-genomic role by interacting with pannexin 1 leading to ATP secretion. Finally, LXR ligands have an antitumoral effect in a mouse colon cancer model, dependent on the presence of LXRß, pannexin 1, NLRP3 and caspase-1 within the tumor cells. Our results demonstrate that LXRß, through pannexin 1 interaction, can specifically induce caspase-1-dependent colon cancer cell death by pyroptosis.

Improving immunosensor performances using an acoustic mixer on droplet microarray.

A major drawback of protein microarrays is the lack of control of ligand immobilization at the surface of the chip which limits their performances and thus their impacts in in vitro diagnosis. To improve antibody (Ab) grafting during the spotting process on commercialized gold SPRi chips, we propose to produce a chaotic flow in every spotted droplet, by using an acoustic field, in order to disrupt the steady state of the reaction of Ab grafting. Our results show that acoustic mixing during Ab binding at the biochips surface increases their biorecognition performances of a mean factor of 2.7 in comparison with Ab layer grafted in a passive mode. Moreover, it increases statistically the homogeneity of the response over all the surface of the chips.

Revisited BIA-MS combination: entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels.

We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.

An electrochemical approach of the redox behavior of water insoluble ubiquinones or plastoquinones incorporated in supported phospholipid layers.

Physiological mole fractions of long isoprenic chain ubiquinone (UQ[10]) and plastoquinone (PQ9) were incorporated inside a supported bilayer by vesicle fusion. The template of the bilayer was an especially designed microporous electrode that allows the direct electrochemistry of water insoluble molecules in a water environment. The artificial structure, made by self-assembly procedures, consisted of a bilayer laterally in contact with a built-in gold electrode at which direct electron transfers between the redox heads of the quinones molecules and the electrode can proceed. The mass balances of quinone and lipid in the structure were determined by radiolabeling and spectrophotometry. A dimyristoyl phosphatdylcholine stable surface concentration of 250 +/- 50 pmol x cm(-2), unaffected by the presence of the quinone, was measured in the fluid monolayer. The mole fraction of quinone was between 1 and 3 mol%, remaining unchanged when going from the vesicles to the supported layers. The lipid molecules and the quinone pool were both laterally mobile, and cyclic voltammetry was used to investigate the redox properties of UQ10 and PQ9 over a wide pH range. Below pH 12, the two electrons-two protons electrochemical process at the gold electrode appeared under kinetic control. Thus all thermodynamic deductions must be anchored in the observed reversibility of the quinone/hydroquinol anion transformation at pH > 13. Within the experimental uncertainty, the standard potentials and the pK(a)'s of the pertinent redox forms of UQ10 and PQ9 were found to be essentially identical. This differs slightly from the literature in which the constants were deduced from the studies of model quinones in mixed solvents or of isoprenic quinones without a lipidic environment.

Electrochemical measurement of lateral diffusion coefficients of ubiquinones and plastoquinones of various isoprenoid chain lengths incorporated in model bilayers.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.