Automated glycan assembly of a S. pneumoniae serotype 3 CPS antigen

Vaccines against S. pneumoniae, one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS) that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA) of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides

Metal-free photoanodes for C-H functionalization

Organic semiconductors, such as carbon nitride, when employed as powders, show attractive photocatalytic properties, but their photoelectrochemical performance suffers from low charge transport capability, charge carrier recombination, and self-oxidation. High film-substrate affinity and well-designed heterojunction structures may address these issues, achieved through advanced film generation techniques. Here, we introduce a spin coating pretreatment of a conductive substrate with a multipurpose polymer and a supramolecular precursor, followed by chemical vapor deposition for the synthesis of dual-layer carbon nitride photoelectrodes. These photoelectrodes are composed of a porous microtubular top layer and an interlayer between the porous film and the conductive substrate. The polymer improves the polymerization degree of carbon nitride and introduces C-C bonds to increase its electrical conductivity. These carbon nitride photoelectrodes exhibit state-of-the-art photoelectrochemical performance and achieve high yield in C-H functionalization. This carbon nitride photoelectrode synthesis strategy may be readily adapted to other reported processes to optimize their performance

Reshaping Silica Particles: Mesoporous Nanodiscs for Bimodal Delivery and Improved Cellular Uptake

The role played by the shape of mesoporous silica nanoparticles has been investigated for intra- and extracellular delivery. Specifically, we have developed the bottom-up synthesis of flat disc-shaped mesoporous silica nanoparticles, the Nanodiscs (NDs). Due to their peculiar shape and large porous system, NDs present a higher cellular uptake than commonly investigated spherical mesoporous nanoparticles. Moreover, NDs are able to efficiently perform exhaustive delivery of their therapeutic cargo when loaded with the anticancer drug Doxorubicin and administered in vitro to cancerous HeLa cells. Thanks to their aspect ratio, NDs can also be readily assembled into well-organized monolayers to be employed in HeLa cells adhesion experiments upon preliminary functionalization with a specific targeting ligand. In these conditions NDs are able to deliver a hydrophobic dye to adhered cells via the highly accessible vertically aligned pores and their flat surface that ensures optimal cell contact. This initial investigation on the performance of NDs in both intra- and extracellular delivery activities suggests the great potential of these particles.This work was financially supported by the European Research Council (ERC) Advanced Grant “MAGIC” (grant N° 247365), the Marie Skłodowska-Curie fellowship (MSCA-IEF) “POP-SILICA” (grant N° 627788) and the SACS Project (grant N° 310651), the Foundation ARC through the project “Thera-HCC” (grant N° IHU201301187), the Région Alsace, and the Département du Bas-Rhin. LDC especially acknowledges AXA Research funds. PHS and SVA acknowledge the Max Planck Society for generous funding

Computer Controlled Automated Assay for Comprehensive Studies of Enzyme Kinetic Parameters

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework (“robot lab assistant”) that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions

Novel interactions of transglutaminase-2 with heparan sulphate proteoglycans: reflection on physiological implications

This mini-review brings together information from publications and recent conference proceedings that have shed light on the biological interaction between transglutaminase-2 and heparan sulphate proteoglycans. We subsequently draw hypothesis of possible implications in the wound healing process. There is a substantial overlap in the action of transglutaminase-2 and the heparan sulphate proteoglycan syndecan-4 in normal and abnormal wound repair. Our latest findings have identified syndecan-4 as a possible binding and signalling partner of fibronectinbound TG2 and support the idea that transglutaminase-2 and syndecan-4 acts in synergy

Glycans as receptors for influenza pathogenesis

Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.National Institute of General Medical Sciences (U.S.) (Glue Grant U54 GM62116)National Institutes of Health (U.S.) (Grant GM57073)Singapore-MIT Alliance for Research and Technolog

Bioinformatics and molecular modeling in glycobiology

The field of glycobiology is concerned with the study of the structure, properties, and biological functions of the family of biomolecules called carbohydrates. Bioinformatics for glycobiology is a particularly challenging field, because carbohydrates exhibit a high structural diversity and their chains are often branched. Significant improvements in experimental analytical methods over recent years have led to a tremendous increase in the amount of carbohydrate structure data generated. Consequently, the availability of databases and tools to store, retrieve and analyze these data in an efficient way is of fundamental importance to progress in glycobiology. In this review, the various graphical representations and sequence formats of carbohydrates are introduced, and an overview of newly developed databases, the latest developments in sequence alignment and data mining, and tools to support experimental glycan analysis are presented. Finally, the field of structural glycoinformatics and molecular modeling of carbohydrates, glycoproteins, and protein–carbohydrate interaction are reviewed