Wetting layer thickness and early evolution of epitaxially strained thin films

We propose a physical model which explains the existence of finite thickness wetting layers in epitaxially strained films. The finite wetting layer is shown to be stable due to the variation of the non-linear elastic free energy with film thickness. We show that anisotropic surface tension gives rise to a metastable enlarged wetting layer. The perturbation amplitude needed to destabilize this wetting layer decreases with increasing lattice mismatch. We observe the development of faceted islands in unstable films.Comment: 4 pages, 3 eps figure

Analysis of three epoetin alpha products by LC and LC-MS indicates differences in glycosylation critical quality attributes, including sialic acid content

Erythropoietin (EPO) is one of the main therapeutics used to treat anaemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions and O-acetylation pattern. Reverse-phase ultra high pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labelled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labelling was applied for the first time to the erythropoiesis stimulating agent (ESA) products, which facilitated novel, in-depth characterisation, and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moie-ties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatisation, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy

L1cam as an e-selectin ligand in colon cancer

POCI-01-0145-FEDER-007728 ref. 140_596817822Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.publishersversionpublishe

Plant N-glycan breakdown by human gut Bacteroides

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source

A 20-year multicenter analysis of dialysis-dependent patients who had aortic or mitral valve replacement: Implications for valve selection

Objective Valve selection in dialysis-dependent patients can be difficult because long-term survival is diminished and bleeding risks during anticoagulation treatment are greater in patients with renal failure. In this study we analyzed long-term outcomes of dialysis-dependent patients who underwent valve replacement to help guide optimal prosthetic valve type selection. Methods Dialysis-dependent patients who underwent aortic and/or mitral valve replacement at 3 institutions over 20 years were examined. The primary outcome was long-term survival. A Cox regression model was used to estimate survival according to 5 ages, presence of diabetes, and/or heart failure symptoms. Results Four hundred twenty-three available patients were analyzed; 341 patients had biological and 82 had mechanical valves. Overall complication and 30-day mortality rates were similar between the groups. Thirty-day readmission rates for biological and mechanical groups were 15% (50/341) and 28% (23/82; P = .005). Five-year survival was 23% and 33% for the biological and mechanical groups, respectively. After adjusting for age, New York Heart Association (NYHA) class, and diabetes using a multivariable Cox regression model, survival was similar between groups (hazard ratio, 0.93; 95% confidence interval, 0.66-1.29; P = .8). A Cox regression model on the basis of age, diabetes, and heart failure, estimated that patients only 30 or 40 years old, with NYHA class I-II failure without diabetes had a >50% estimated 5-year survival (P < .001). Conclusions Dialysis-dependent patients who underwent valve replacement surgery had poor long-term survival. Young patients without diabetes or NYHA III or IV symptoms might survive long enough to justify placement of a mechanical valve; however, a biological valve is suitable for most patients

Exploring the sequence-function space of microbial fucosidases

Microbial α-l-fucosidases catalyse the hydrolysis of terminal α-l-fucosidic linkages with diverse substrate/linkage specificities and can be used in transglycosylation reactions to synthesise oligosaccharides. Based on sequence identity, α-l-fucosidases have been classified in distinct glycoside hydrolases (GHs) families in the carbohydrate-active enzymes (CAZy) database. Here, we explored the sequence-function space of fucosidases from GH29 family. Based on sequence similarity network (SSN) analyses, 16 GH29 α-l-fucosidases were selected for functional characterisation. Using activity assays combined with HPAEC-PAD and LC-FD-MS/MS analyses, we determined the substrate and linkage specificities of these enzymes against a range of defined oligosaccharides and glycoconjugates, revealing a range of specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated ligands. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1-6 linkages and FA2G2 N-glycan was further determined by X-ray crystallography and saturation transfer difference NMR. TLC combined with electrospray ionization – MS and NMR confirmed the capacity of this enzyme to carry out transfucosylation reactions with GlcNAc and Fuc1,3GlcNAc as acceptors. Taken together, these experimental data validate the use of SSN as a reliable bioinformatics approach to predict the substrate specificity and transfucosylation activity of GH29 fucosidases.<br/

Exploring the sequence-function space of microbial fucosidases

Microbial α-l-fucosidases catalyse the hydrolysis of terminal α-l-fucosidic linkages with diverse substrate/linkage specificities and can be used in transglycosylation reactions to synthesise oligosaccharides. Based on sequence identity, α-l-fucosidases have been classified in distinct glycoside hydrolases (GHs) families in the carbohydrate-active enzymes (CAZy) database. Here, we explored the sequence-function space of fucosidases from GH29 family. Based on sequence similarity network (SSN) analyses, 16 GH29 α-l-fucosidases were selected for functional characterisation. Using activity assays combined with HPAEC-PAD and LC-FD-MS/MS analyses, we determined the substrate and linkage specificities of these enzymes against a range of defined oligosaccharides and glycoconjugates, revealing a range of specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated ligands. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1-6 linkages and FA2G2 N-glycan was further determined by X-ray crystallography and saturation transfer difference NMR. TLC combined with electrospray ionization – MS and NMR confirmed the capacity of this enzyme to carry out transfucosylation reactions with GlcNAc and Fuc1,3GlcNAc as acceptors. Taken together, these experimental data validate the use of SSN as a reliable bioinformatics approach to predict the substrate specificity and transfucosylation activity of GH29 fucosidases.<br/

Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074–like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a “liganded” PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain

Fucosidases from the human gut symbiont Ruminococcus gnavus

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers

Hall noise and transverse freezing in driven vortex lattices

We study driven vortices lattices in superconducting thin films. Above the critical force $F_c$ we find two dynamical phase transitions at $F_p$ and $F_t$, which could be observed in simultaneous noise measurements of the longitudinal and the Hall voltage. At $F_p$ there is a transition from plastic flow to smectic flow where the voltage noise is isotropic (Hall noise = longitudinal noise) and there is a peak in the differential resistance. At $F_t$ there is a sharp transition to a frozen transverse solid where the Hall noise falls down abruptly and vortex motion is localized in the transverse direction.Comment: 4 pages, 3 figure