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International audience; Incellulosite-specific unnatural amino acid incorporation based on amber stop codon reassignment is a powerful tool to modify protein at defined positions. This technique is herein applied to the selective functionalization of the Pneumococcal surface adhesin A protein at three distinct positions. N-propargyloxycarbonyl-L-lysine residues were incorporated and their alkyne groups reacted using click-chemistry with a synthetic azido-functionalized tetrasaccharide representative of one repeat unit of the Streptococcus pneumoniaeserotype 14 capsular polysaccharide. Anti-PsaA antibody response induced in mice by the trivalent glycoconjugate was determined in comparison with corresponding monovalent and randomly functionalized conjugates. Our results suggest that controlled was superior to random conjugation for preserving antigenicity. In definitive, the reported strategy offers a unique opportunity to study the impact of carbohydrate antigen-carrier protein connectivity on immunogenicity.

User-led approaches are increasingly common in the field of social services and call for significant change in social service culture and practices. This research1 aims to study the implementation ...

L’article porte sur une experience de theâtre-forum realisee dans le cadre d’un projet de recherche visant l’augmentation de la capacite d’agir de membres d’un organisme communautaire, le Reseau d’Appui aux familles Monoparentales et recomposees de l’Estrie (RAME). Il expose le positionnement de l’Ecole de travail social quant a l’implication des personnes au sein de ses formations et montre comment le theâtre-forum renforce les capacites d’agir personnelles et collectives par la transformation du vecu en savoirs partages. Particulierement, cet article decrit l’experience vecue par huit participantes ayant coconstruit et presente un theâtre-forum devant public. Les resultats s’appuient sur la perspective des participantes recueillies au cours de cinq entrevues individuelles et d’une rencontre preparatoire a la redaction de cet article ou trois participantes etaient presentes. Les resultats portent sur les defis qu’elles ont du relever au cours des differentes activites et des retombees individuelles et collectives qu’elles observent au terme du projet. L’article se conclut en exposant les apprentissages utiles pour l’actualisation d’un exercice de coconstruction comme celui du projet de recherche avec le RAME.

Here, we report on the first combined one-pot use of the two so-called "click reactions": the thiol-ene coupling and the copper-catalyzed alkyne-azide cycloaddition. These reactions were employed in an alternating and one-pot fashion to combine appropriately functionalized monomeric carbohydrate building blocks to create mimics of trisaccharides and tetrasaccharides as single anomers, with only minimal purification necessary. The deprotected oligosaccharide mimics were found to bind both plant lectins and human galectin-3.

International audience; Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually peptides incorporated within the protein sequences which compose the VLPs or conjugated to them. We herein described the conjugation of a synthetic tetrasaccharide mimicking the Streptococcus pneumoniae serotype 14 capsular polysaccharide to recombinant adenoviral type 3 dodecahedron, formed by the self-assembling of twelve penton bases and investigated the induced immune response when administered subcutaneously (s.c.). Whether formulated in the form of a dodecahedron or disassembled, the glycoconjugate induced an anti-protein response after two and three immunizations equivalent to that observed when the native dodecahedron was administered. On the other hand, the glycoconjugate induced a weak anti-IgM response which diminishes after two doses but no IgM-to-IgG switch was observed in mice against the serotype 14 capsular polysaccharide. In definitive, the whole conjugation process preserved both particulate nature and immunogenicity of the adenoviral dodecahedron. Further studies are needed to fully exploit adenoviral dodecahedron potential in terms of plasticity towards sequence engineering and of its capacity to stimulate the immune system via the intranasal route of administration as well as to shift the response to the carbohydrate antigen by playing both with the carbohydrate to protein ratio and the length of the synthetic carbohydrate antigen.

International audience; Chemo-enzymatic strategies are useful to provide both regio-and stereoselective access to bioactive oligosaccharides. We show herein that a glycosynthase mutant of a Thermus thermophilus -glycosidase can react with unnatural glycosides such as 6-azido-6-deoxy-D-glucose/glucosamine to lead to -D-galactopyranosyl-D-glucopyranoside or -D-galactopyranosyl-2-acetamido-2-deoxy-D-glu-copyranoside derivatives bearing a unique azide function. Taking advantage of the orthogonality between the azide and the hydroxyl functional groups, the former was next selectively reacted to give rise to a library of galectin-3 inhibitors. Combining enzyme substrate promiscuity and bioorthogonality thus appears as a powerful strategy to rapidly access to sugar-based ligands.

Galectins play key roles in numerous biological processes. Their mode of action depends on their localization which can be extracellular, cytoplasmic, or nuclear and is partly mediated through interactions with β-galactose containing glycans. Galectins have emerged as novel therapeutic targets notably for the treatment of inflammatory disorders and cancers. This has stimulated the design of carbohydrate-based inhibitors targeting the carbohydrate recognition domains (CRDs) of the galectins. Pursuing this approach, we reasoned that linear oligogalactosides obtained by straightforward iterative click chemistry could mimic poly-lactosamine motifs expressed at eukaryote cell surfaces which the extracellular form of galectin-3, a prominent member of the galectin family, specifically recognizes. Affinities toward galectin-3 consistently increased with the length of the representative oligogalactosides but without reaching that of oligo-lactosamines. Elucidation of the X-ray crystal structures of the galectin-3 CRD in complex with a synthesized di- and tri-galactoside confirmed that the compounds bind within the carbohydrate-binding site. The atomic structures revealed that binding interactions mainly occur with the galactose moiety at the non-reducing end, primarily with subsites C and D of the CRD, differing from oligo-lactosamine which bind more consistently across the whole groove formed by the five subsites (A-E) of the galectin-3 CRD.

Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.

International audience

Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.

Under nitrogen-limiting conditions, legumes are able to form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce root nodules. These new root organs satisfy plant nitrogen needs by reducing atmospheric nitrogen to ammonium. However, the senescence of these organs disturbs the assimilation of nitrogen. In this study, we present different histological, biochemical, and genetic markers of the natural nodule senescence in Medicago truncatula over a 10-week period following bacterial inoculation. During aging the length and the weight of nodules increased, whereas the nitrogen-fixing capacity of the nodules decreased. The development of the nodule senescence zone correlated with a reduction in leghemoglobin levels without significant reduction in the total protein concentration of the nodule. In contrast, no difference in glutathione and homoglutathione concentration was detected at the onset of senescence at 6 and 8 weeks after bacterial infection. Furthermore, we observed a significant decrease in the relative transcription levels of Nodule Cysteine Rich 001, MtLb1, sucrose synthase 1, thioredoxin S1 and thioredoxin S2 genes, which are involved in nodule development and functioning, thus demonstrating that natural senescence impacts the transcription of genes involved in the expansion and the metabolism of the nitrogen-fixing zone. Finally, the induction of amine oxidase and cysteine protease CP6 transcription was unstable, suggesting that these two genes are related to senescence but are not robust gene markers of the natural senescence process. Considered together, our results define novel biochemical and genetic markers for natural nodule senescence and show that leghemoglobin gene transcription and protein concentration are robust markers that closely correlate with nitrogen fixation efficiency.

Cet article est un exercice réflexif sur la combinaison de la réflexivité et de la délibération éthique comme choix épistémologiques et méthodologiques dans le cadre d’une recherche qualitative avec des professionnels en exercice dans le domaine de la protection de l’enfance. Il a pour but de présenter et d’analyser les rouages de cette expérience de recherche. L’appréhension d’une pratique complexe ainsi que les choix épistémologiques et méthodologiques sont d’abord présentés puis les principaux constats de la démarche sont mis en lumière, ce qui permet de voir que les résultats sont profitables tant pour l’amélioration des pratiques professionnelles de protection de l’enfance que pour les réflexions autour de la recherche universitaire appliquée.

Pneumococcal infections remain a major public health concern worldwide. The currently available vaccines in the market are based on pneumococcal capsular polysaccharides but they still need to be improved to secure an optimal coverage notably in population at risk. To circumvent this, association of virulence pneumococcal proteins to the polysaccharide valencies has been proposed with the hope to observe an additive - if not synergistic - protective effect. Along this line, the use of the highly conserved and ubiquitous pneumococcal surface adhesin A (PsaA) as a protein carrier for a synthetic pneumococcal oligosaccharide is demonstrated herein for the first time. A tetrasaccharide mimicking functional antigenic determinants from the S. pneumoniae serotype 14 capsular polysaccharide (Pn14TS) was chemically synthesised. The mature PsaA (mPsaA) was expressed in E. coli and purified using affinity chromatography. The Pn14PS was conjugated to mPsaA using maleimide-thiol coupling chemistry to obtain mPsaA-Pn14PS conjugate (protein/sugar molar ratio: 1/5.4). The mPsaA retained the structural conformation after the conjugation and lyophilisation. The prepared glycoconjugate adjuvanted with α-galactosylceramide, a potent activator of invariant Natural Killer T cells, was tested in mice for its immunological response upon subcutaneous injection in comparison with mPsaA alone and a model BSA conjugate (BSA-Pn14PS, used here as a control). Mice immunised with the mPsaA-Pn14TS produced a robust IgG response against mPsaA and against the capsular polysaccharide from pneumococcal serotype 14. These data provide the basis for novel pneumococcal vaccine development.