Structural characteristics of Saccharomyces cerevisiae mannoproteins: Impact of their polysaccharide part.

While they have many properties of interest in enology, the structure-function relationships of mannoproteins and the part played by their polysaccharide moiety are not yet well understood. Mannoproteins (MP) extracted with ß-glucanase from a laboratory yeast strain (WT), two of its mutants (Mnn2 with unbranched N-glycosylated chains and Mnn4 without mannosyl-phosphorylation), and an enological strain (Com) were purified and thoroughly characterized. The protein moiety of the four MPs had the same amino acid composition. Glycosyl-linkage and net charge analyses confirmed the expected differences in mutant strain MPs. MP-Com had the highest mannose/glucose ratio followed by MP-WT/MP-Mnn4, and MP-Mnn2 (13.5 > 5.6 ≈ 5.2 > 2.2). The molar mass dependencies of Rg, Rh, and [η], determined through HPSEC-MALLS-QELS-Viscosimetry, revealed specific conformational properties of mannoproteins related to their nature of highly branched copolymers with two branching levels. It also clearly showed structural differences between MP-Com, MP-WT/Mnn4, and MP Mnn2, and differences between two populations within the four mannoproteins.

Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography.

Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate, and organize into branched tubular structures with walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space, and show markers of epithelial maturation such as primary cilia and continuous tight junctions. The resulting structures have dimensions typically found in the intralobular and intrahepatic biliary tree and are stable for weeks, without any requirement of bulk supporting material, thereby offering total access to the external side of these biliary epithelial constructs.

Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees.

BACKGROUND AND AIMS: Globally, liver diseases account for 2 million deaths per year. For those with advanced liver disease the only curative approach is liver transplantation. However, less than 10% of those in need get a liver transplant due to limited organ availability. To circumvent this challenge, there has been a great focus in generating a bioengineered liver. Despite its essential role in liver functions, a functional biliary system has not yet been developed. In this framework, exploration of epithelial cell self-organogenesis and microengineering-driven geometrical cell confinement allow to envision the bioengineering of a functional biomimetic intrahepatic biliary tract. APPROACH: three-dimensional (3D) bile ducts were built in vitro by restricting cell adhesion to two-dimensional (2D) patterns to guide cell self-organization. Tree shapes mimicking the configuration of the human biliary system were micropatterned on glass slides, restricting cell attachment to these areas. Different tree geometries and culture conditions were explored to stimulate self-organogenesis of normal rat cholangiocytes (NRCs) used as a biliary cell model, either alone or in co-culture with human umbilical endothelial cells (HUVECs). RESULTS: Pre-seeding the micropatterns with HUVECs promoted luminogenesis with higher efficiency to yield functional branched biliary tubes. Lumen formation, apico-basal polarity, and preservation of the cholangiocyte phenotype were confirmed. Moreover, intact and functional biliary structures were detached from the micropatterns for further manipulation. CONCLUSION: This study presents physiologically relevant 3D biliary duct networks built in vitro from 2D micropatterns. This opens opportunities for investigating bile duct organogenesis, physiopathology, and drug testing.

Acacia gums new fractions and sparkling base wines: How their biochemical and structural properties impact foamability?

Foam is the first attribute observed when sparkling wine is served. Bentonite is essentially used to flocculate particles in sparkling base wines but can impair their foamability. Gums from Acacia senegal and Acacia seyal improved the foamability of different bentonite-treated base wines. Our main goal was to see how the supplementation with new fractions separated from Acacia gums by Ion Exchange Chromatography affected foamability of sparkling base wines, deepening the relation between foam behavior and characteristics of wine and gums. High molar mass fractions increased the maximal foam height and the foam height during the stability period in, respectively, 11 out and 8 out of 16 cases (69% and 50%, respectively). The properties of the supplementing gums fractions obtained by IEC and, although to a minor extent, the wine characteristics, affected positively and/or negatively the foam behavior. Wine foamability also depended on the relationship between wine and gums fractions properties.

Recent advances in the knowledge of wine oligosaccharides.

Oligosaccharides are carbohydrates with a low polymerization degree containing between three and fifteen monosaccharide residues covalently linked through glycosidic bonds. Oligosaccharides are related to plant defense responses and possess beneficial attributes for human health. Research has focused in wine oligosaccharides only in the last decade. In this paper, a summary of these works is provided. They include: (i) wine oligosaccharides origins, (ii) techniques for isolating oligosaccharide fraction and determining their content, composition and structure, (iii) their dependence on the grape origin and cultivar and winemaking process, and (iv) the connection between oligosaccharides and wine sensorial attributes. Further research is required regarding the impact of agricultural aspects and winemaking techniques on wine oligosaccharides. The knowledge concerning their influence on sensorial and physicochemical properties of wines and on human health should also be improved. The implementation of laboratory methods will provide better understanding of these compounds and their performance within wine's matrix.

Fractionation and characterization of polyphenolic compounds and macromolecules in red wine by asymmetrical flow field-flow fractionation.

Red wine is a complex matrix containing macromolecules such as condensed tannins and polysaccharides. Wine macromolecular components and their interactions have been reported to impact taste properties such as astringency but the colloidal systems formed in wine are not well known. A key prerequisite to characterize these systems is the ability to work under analytical conditions as close as possible to the colloid environment, preserving the sample structure and limiting the denaturation of macromolecular complexes. A method of Asymmetric Flow Field-Flow Fractionation (AF4) coupled with UV detection, multi-angle light scattering (MALS), and differential refractometer index (dRI) (AF4-UV-MALS-dRI) has been developed to analyse macromolecules, including tannins and polysaccharides, and macromolecular complexes, in red wine. This method separates objects according to their hydrodynamic radius and does not require calibration to determine molecular weight (Mw). AF4 can provide native separation of wine colloidal matter while working with simulated wine as mobile phase. The channel was equipped with a 350-µm spacer and the membrane made in regenerated cellulose had a cut-off of 5kDa. Different parameters of crossflow rate were investigated using a generic red wine to optimize separation conditions. Then, purified fractions of polysaccharides and tannins were analysed using the selected AF4 parameters. The comparison of the peaks obtained for these fractions and for the wine sample allowed us to determine the retention time associated with these macromolecules. The AF4 fractogram of wine was divided into four fractions. The first three were assigned to higher Mw tannins coeluted with lower Mw polysaccharides such as rhamnogalacturonan II (F1), to intermediate Mw polysaccharides (F2), and to higher Mw mannoproteins (F3) whereas the last fraction (F4) was not identified. Furthermore, our results have shown that AF4-UV-MALS-dRI could be an efficient technique to separate large size tannins as well as polysaccharides and macromolecular complexes.

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Multimethod Approach for Extensive Characterization of Gallnut Tannin Extracts.

Gallotannins extracted from gallnuts are commonly added to wine to improve its properties. They consist of mixtures of galloylester derivatives of glucose. However, their composition and properties are not well-established. In this study, methods based on liquid chromatography coupled to ultraviolet-visible detection and mass spectrometry, size-exclusion chromatography, and one-dimensional (31P) and two-dimensional (1H diffusion ordered spectroscopy, 31P total correlated spectroscopy, and 1H/13C heteronuclear single-quantum correlation and heteronuclear multiple-bond correlation) nuclear magnetic resonance spectroscopies have been implemented for extensive chemical characterization of three commercial gallnut tannin extracts. Differences in the proportions of the different constituents (gallic, digallic, and trigallic acids and galloylglucose derivatives) and in the structure and molecular weight distributions of gallotannins were demonstrated between the three extracts, with chains containing 8.5, 12.2, and 12.4 galloyl groups on average for TAN A, TAN B1, and TAN B2, respectively. The antioxidant capacities of the extracts, evaluated using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method, were similar and related mostly to their total tannin content, with only a limited impact of the tannin composition.

Improvement of the foamability of sparkling base wines by the addition of Acacia gums.

In sparkling wine, foam characteristics are one of the major attributes. The foam quality depends on wine components. Bentonite is added to the base wine to facilitate the riddling process, but causes a loss of foamability. Acacia gum can be used as additive in wine. We have studied if the addition of Acacia senegal gum (AsenG), Acacia seyal gum (AseyG) and different AsenG fractions could improve the foamability of different base wines treated with bentonite. The foamability differs depending on the gum or the gum fraction treatment but also on the wine, being these differences linked to some aspects of their respective compositions and molecular parameters. AsenG and AseyG increase the foamability (by Mosalux - sparging procedure), respectively, in five and seven out of eight base wines treated with bentonite. Therefore, AsenG and AseyG are potential treatments increasing the foamability of these wines.

Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice.

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

Inhibition Mechanisms of Wine Polysaccharides on Salivary Protein Precipitation.

In this work, high-performance liquid chromatography, fluorescence quenching, nephelometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to study the effect of polysaccharides naturally present in wine [rhamnogalacturonan II (RG II) and arabinogalactan proteins (AGPs)] on the interaction between salivary proteins (SP) together present in saliva and tannins (punicalagin (PNG) and procyanidin B2). In general, the RG II fraction was more efficient to inhibit SP precipitation by tannins, especially for acidic proline-rich proteins (aPRPs) and statherin/P-B peptide, than AGPs. The RG II fraction can act mainly by a competition mechanism in which polysaccharides compete by tannin binding. However, in the presence of Na+ ions in solution, no RG II effect was observed on SP-tannin interactions. On the other hand, dependent upon the saliva sample as well as the tannin studied, AGPs can act by both mechanisms, competition and ternary (formation of a ternary complex with SP-tannin aggregates enhancing their solubility).

The impact of distillation process on the chemical composition and potential prebiotic activity of different oligosaccharidic fractions extracted from grape seeds.

The study was designed to evaluate how the distillation process is able to affect the composition and bioactivity of oligosaccharides contained in the grape seeds. Different oligosaccharidic fractions have been extracted both before and after grape pomace distillation in order to valorize this by-product. A multistep solid-phase extraction approach (C-18 and carbograph cartridges) has been applied to purify and fractionate the oligosaccharidic compounds. Chemical characterization of the fractions was performed using a UPLC-ESI-MSn method. Complex oligosaccharides consist principally of neutral oligosaccharides rich in arabinose and glucose. Then, the oligosaccharides contained in the different fractions as potential functional ingredients with prebiotic activity toward well-known probiotic bacteria, such as Lactobacillus acidophilus and Lactobacillus plantarum, were evaluated. Data showed how, in some combination, oligosaccharidic fractions obtained may be considered a novel "functional ingredient" with potential prebiotic activity mainly towards L. acidophilus.

Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds.

The integration of bile duct epithelial cells (cholangiocytes) in artificial liver culture systems is important in order to generate more physiologically relevant liver models. Understanding the role of the cellular microenvironment on differentiation, physiology, and organogenesis of cholangiocytes into functional biliary tubes is essential for the development of new liver therapies, notably in the field of cholangiophaties. In this study, we investigated the role of natural or synthetic scaffolds on cholangiocytes cyst growth, lumen formation and polarization. We demonstrated that cholangiocyte cyst formation efficiency can be similar between natural and synthetic matrices provided that the mechanical properties of the hydrogels are matched. When using synthetic matrices, we also tried to understand the impact of elasticity, matrix metalloprotease-mediated degradation and integrin ligand density on cyst morphogenesis. We demonstrated that hydrogel stiffness regulates cyst formation. We found that controlling integrin ligand density was key in the establishment of large polarized cysts of cholangiocytes. The mechanism of lumen formation was found to rely on cell self-organization and proliferation. The formed cholangiocyte organoids showed a good MDR1 (multi drug resistance protein) transport activity. Our study highlights the advantages of fully synthetic scaffold as a tool to develop bile duct models.

Preharvest Application of Elicitors to Monastrell Grapes: Impact on Wine Polysaccharide and Oligosaccharide Composition.

This work studied the effect of preharvest application in Monastrell grapes of four different elicitors [methyl jasmonate (MeJ), benzothiadiazole (BTH), chitosan from fungi (CHSf), and chitosan from seafood (CHSs)] on wine polysaccharide and oligosaccharide fractions. The polysaccharide and oligosaccharide fractions were isolated and characterized. Neutral monosaccharides were released after hydrolysis of polysaccharides and quantified by gas chromatography (GC). Sugar composition of oligosaccharides was determined after solvolysis by GC of their per-O-trimethylsilylated methyl glycoside derivatives. MeJ, BTH, CHSf, and particularly CHSs decrease the polysaccharide content in wine. The oligosaccharide concentration was also reduced after both CHS treatments. These results pointed to a lower degradation of the skin cell wall from treated grapes. We suggested that the cause would be a reinforcement of the skin cell wall as a result of the action of these elicitors. In conclusion, the application of any of these four elicitors in the clusters of the vineyard affected the complex carbohydrate composition of elaborated wine.

Proteins that control the geometry of microtubules at the ends of cilia.

Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.

Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules.

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.

Polysaccharides, oligosaccharides and nitrogenous compounds change during the ageing of Tempranillo and Verdejo sparkling wines.

BACKGROUND: Verdejo and Tempranillo are traditional varieties for producing still wines; however, they could provide an alternative for the manufacturing of sparkling wines. Sparkling wines were elaborated by the traditional method, followed by ageing on lees for 9 months. A study on the changes that take place in polysaccharides, oligosaccharides and nitrogenous compounds during the ageing on lees of Tempranillo and Verdejo sparkling wines has been undertaken. RESULTS: Mannoproteins and the glucose residue of oligosaccharides were the major carbohydrates detected in all vinification stages. Yeast polysaccharides and glucan-like structures of the oligosaccharides increased after 3 months of ageing. The evolution of yeast polysaccharides and the composition of PRAG-like structure were different among grape varieties. A decrease in amino acids and biogenic amines was observed during the ageing. The contents of polysaccharides, oligosaccharides and nitrogenous compound were significantly higher in Tempranillo than in Verdejo sparkling wines at the end of the ageing period. CONCLUSION: Polysaccharides and oligosaccharides from yeast were more significant autolysis markers of sparkling wines than the nitrogenous compounds. Our data suggest a potential cultivar effect on the evolution of yeast polysaccharides and on the composition of PRAG, which may influence the physico-chemical and sensory properties of sparkling wines. © 2017 Society of Chemical Industry.

The role of wine polysaccharides on salivary protein-tannin interaction: A molecular approach.

Polysaccharides are described to inhibit aggregation between food polyphenols and salivary proteins (SP) and may hence lead to astringency modulation. In this work, the effect of two wine polysaccharides (arabinogalactan proteins-AGPs and rhamnogalacturonan II- RGII) on SP-polyphenol interaction was evaluated. In general, both polysaccharides were effective to inhibit or reduce SP-polyphenol interaction and aggregation. They can act by two different mechanisms (ternary or competitive) depending on the SP-tannin pair. In the case of salivary P-B peptide, AGPs and RGII seem to act by a ternary mechanism, in which they surround this complex, enhancing its solubility. Concerning acidic proline-rich proteins (aPRPs), it was possible to observe both mechanisms, depending on the tannin and the polysaccharide involved. Overall, this work point out for a specific property of wine polysaccharides important to modulate this and other beverages and food astringency perception.

Polysaccharides and Oligosaccharides Produced on Malvar Wines Elaborated with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 Native Yeasts from D.O. "Vinos de Madrid".

Polysaccharides and oligosaccharides released into Malvar white wines elaborated through pure, mixed, and sequential cultures with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 native yeasts from D.O. "Vinos de Madrid" were studied. Both fractions from different white wines were separated by high-resolution size-exclusion chromatography. Glycosyl composition and wine polysaccharide linkages were determined by GC-EI-MS chromatography. Molar-mass distributions were determined by SEC-MALLS, and intrinsic viscosity was determined by differential viscometer. Yeast species and type of inoculation have a significant impact on wine carbohydrate composition and structure. Mannose residues from mannoproteins were significantly predominant in those cultures where T. delbrueckii was present in the fermentation process in comparison with when pure cultures of S. cerevisiae were present in the fermenation process. Galactose residues from polysaccharides rich in arabinose and galactose presented greater values in pure cultures of S. cerevisiae, indicating that S. cerevisiae released fewer mannoproteins than T. delbrueckii. Moreover, we reported structural differences between mannoproteins released by T. delbrueckii CLI 918 and those released by S. cerevisiae CLI 889. These findings help to provide important information about the polysaccharides and oligosaccharides released from the cell walls of Malvar grapes and the carbohydrates released from each yeast species.

Effect of grape juice press fractioning on polysaccharide and oligosaccharide compositions of Pinot meunier and Chardonnay Champagne base wines.

Press fractioning is an important step in the production of sparkling base wines to segregate the grape juices with different qualities. Grape juice fractions were collected during the pressing cycle at industrial and laboratory scales. The Pinot meunier and Chardonnay Champagne base wines obtained from the free-run juice and the squeezed juices exhibited strong differences from the beginning to the last step of pressing cycle for numerous enological parameters. Significant changes in polysaccharide (PS) and oligosaccharide (OS) base wine composition and concentration were found as the pressing cycle progressed. During the pressing cycle, the total PS concentration decreased by 31% (from 244 to 167mg/L) and 32% (from 201 to 136mg/L) in the Pinot meunier and Chardonnay wines respectively. The wine OS amounts varied between 97 and 139mg/L. The polysaccharide rich in arabinose and galactose (39-54%) and mannoproteins (38-55%) were the major PS in the base wines.