Sébastien Fort

Oligosaccharide/Silicon-Containing Block Copolymers with 5 nm Features for Lithographic Applications

Block copolymers demonstrate potential for use in next-generation lithography due to their ability to self-assemble into well-ordered periodic arrays on the 3-100 nm length scale. The successful lithographic application of block copolymers relies on three critical conditions being met: high Flory-Huggins interaction parameters (χ), which enable formation of <10 nm features, etch selectivity between blocks for facile pattern transfer, and thin film self-assembly control. The present paper describes the synthesis and self-assembly of block copolymers composed of naturally derived oligosaccharides coupled to a silicon-containing polystyrene derivative synthesized by activators regenerated by electron transfer atom transfer radical polymerization. The block copolymers have a large χ and a low degree of polymerization (N) enabling formation of 5 nm feature diameters, incorporate silicon in one block for oxygen reactive ion etch contrast, and exhibit bulk and thin film self-assembly of hexagonally packed cylinders facilitated by a combination of spin coating and solvent annealing techniques. As observed by small angle X-ray scattering and atomic force microscopy, these materials exhibit some of the smallest block copolymer features in the bulk and in thin films reported to date.

The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2.

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

A Brief and Informationally Rich Naming System for Oligosaccharide Motifs of Heteroxylans Found in Plant Cell Walls

The one-letter code system proposed here is a simple method to accurately describe structurally diverse oligosaccharides derived from heteroxylans. Substitutions or ‘molecular decoration(s)’ of main-chain d-xylosyl moieties are designated by unique letters. Hence, an oligosaccharide is described by a series of single letters, beginning with the non-reducing d-xylosyl unit. Superscripted numbers are used to indicate the linkage position(s) of main-chain substitution(s) and, where necessary, superscripted lowercase letter(s) indicate the nature of non-glycosidic groups (e.g., methyl, acetyl, or phenolic derivative moieties) that can be present on the substituents. Although relatively simple and practical to use, this abbreviated system lends itself to the naming of a large number of different combinations of structural building blocks and substituents. In its present state, this system is, therefore, adequate to name and differentiate all currently known complex oligosaccharides derived from heteroxylans and is sufficiently flexible to accommodate new structures as they become available.

Activation of Symbiosis Signaling by Arbuscular Mycorrhizal Fungi in Legumes and Rice

Arbuscular mycorrhizal fungi produce a variety of signaling molecules that are shown to promote symbiosis signaling in a range of plant species. Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

Lipo-chitooligosaccharidic Symbiotic Signals Are Recognized by LysM Receptor-Like Kinase LYR3 in the Legume Medicago truncatula

While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.

Development of catalytically active silver colloid nanoparticles stabilized by dextran.

Colloidal silver nanoparticles (Ag-NPs) with a mean diameter of 6.1 nm and a narrow size distribution were prepared by reduction of the correspondent metal salt with injection of NaBH(4), in the presence of dextran, and characterized by UV-vis, TEM, and DLS. The concentration of all reactants involved in the formation of the nanoparticles was optimized with the use of a new multivariate method, which revealed a significant reduction in the number of experiments when compared with the vast majority of univariate methods described in the literature. The Ag-NPs-dextran composite was able to efficiently catalyze the p-nitrophenol reduction in water by NaBH(4) with a rate constant normalized to the surface area of the nanoparticles per unit volume (k(1)) of 1.41 s(-1) m(-2) L, which is higher than values ever reported for Ag-NPs catalytic systems.

Self-Assembly of Amphiphilic Glycoconjugates into Lectin-Adhesive Nanoparticles

This work describes the synthesis and self-assembly of carbohydrate-clicked rod-coil amphiphilic systems. Copper-catalyzed Huisgen cycloaddition was efficiently employed to functionalize the hydrophilic extremity of PEG-b-tetra(p-phenylene) conjugates by lactose and N-acetyl-glucosamine ligands. The resulting amphiphilic systems spontaneously self-assembled into nanoparticles when dissolved in aqueous media, as evidenced by dynamic light scattering (DLS), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The formation of highly monodisperse micelles having a mean diameter of 10 nm was observed for systems containing a PEG 900 core, and a decrease in the hydrophilic moiety (PEG 600) led to the formation of vesicles with a broader size distribution. The presence of carbohydrate residues on the surfaces of the micelles and their ability to establish specific interactions with wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were further highlighted by light-scattering measurements, thus confirming the attractive applications of such sugar micelles in biosensor devices.

Synthesis and evaluation as glycosidase inhibitors of 2,5-imino-d-glucitol and 1,5-imino-d-mannitol related derivatives

Selectively functionalized 2,5-imino-D-glucitol and 1,5-imino-D-mannitol derivatives were synthesized and tested as precursors of hydrolytically resistant pseudo-disaccharides. Among them N-acetyl-6-amino-6-deoxy-2,5-imino-D-glucitol (11) and N-acetyl-6-amino-6-deoxy-1,5-imino-D-mannitol (12) were found potent and specific inhibitors against beta-D-glucosidase and alpha-L-fucosidase, respectively.

Control of 10 nm scale cylinder orientation in self-organized sugar-based block copolymer thin films

The present paper describes the orientational control of 10 nm scale cylinders in sugar-based block copolymer thin films by simply varying the composition of the annealing co-solvent. The affinity of the block copolymer to the solvent vapor could be systematically adjusted in this way.