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Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Endo-xylanase and endo-glucanase are supplemented to poultry diets in order to improve nutrient digestion and non-starch polysaccharide (NSP) fermentation. Here, the action of these enzymes on alcohol insoluble solids (AIS) from wheat and maize grains as well as its implications for starch digestion in milled grains were evaluated in vitro, under conditions mimicking the poultry digestive tract. For wheat AIS, GH11 endo-xylanase depolymerized soluble arabinoxylan (AX) during the gizzard phase, and proceeded to release insoluble AX under small intestine conditions. At the end of the in vitro digestion (480 min), the endo-xylanase, combined with a GH7 endo-β-1,4-glucanase, released 30.5 % of total AX and 18.1 % of total glucan in the form of arabinoxylo- and gluco-oligosaccharides, as detected by HPAEC-PAD and MALDI-TOF-MS. For maize AIS, the combined enzyme action released 2.2 % and 7.0 % of total AX and glucan, respectively. Analogous in vitro digestion experiments of whole grains demonstrated that the enzymatic release of oligomers coincided with altered grain microstructure, as examined by SEM. In the present study, cell wall hydrolysis did not affect in vitro starch digestion kinetics for cereal grains. This study contributes to understanding the action of feed enzymes on cereal NSP under conditions mimicking the poultry digestive tract., Competing Interests: Declaration of competing interest Natalia Soares is employed by the funder. All other authors declare that they have no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, β-1,3- and β-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae , a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6 Δ and knr4 Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of β-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3 - 10 μm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ∼25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the β-1,6-glucan content is decreased in kre6 Δ, while all glucan content is decreased in the knr4 Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Highland hulless barley has garnered attention as a promising economic product and a potential healthy food ingredient. The present study aimed to comprehensively investigate the molecular structure of extractable fibers obtained from a specific highland hulless barley. Water-soluble fiber (WSF) and alkaline-soluble fiber (ASF) were extracted using enzymatic digestion and an alkaline method, respectively. The purified fibers underwent a thorough investigation for their structural characterization., Results: The monosaccharide composition revealed that WSF primarily consisted of glucose (91.7%), whereas ASF was composed of arabinose (54.5%) and xylose (45.5%), indicating the presence of an arabinoxylan molecule with an A/X ratio of 1.2. The refined structural information was further confirmed through methylation, 1 H NMR and Fourier-transform infrared spectroscopy analyses. WSF fiber exclusively exhibited α-anomeric patterns, suggesting it was an α-glucan. It has a low molecular weight of 5 kDa, as determined by gel permeation chromatography. Conversely, ASF was identified as a heavily branched arabinoxylan with 41.55% of '→2,3,4)-Xylp-(1→' linkages. ASF and WSF exhibited notable differences in their morphology, water absorption capabilities and rheological properties., Conclusion: Based on these findings, molecular models of WSF and ASF were proposed. The deep characterization of these fiber structures provides valuable insights into their physicochemical and functional properties, thereby unlocking their potential applications in the food industry. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Introduction and Objective: Poultry house employees spend a significant part of their work shift being exposed to airborne particulate pollutants. The aim of this study was to assess their exposure at different stages of chicken production cycle, based on quantification of pro-inflammatory mediators (IL-1β, IL-6, IL-8, and TNFα) in nasal lavage (NAL) samples., Material and Methods: The concentrations of airborne dust at 3 different stages of the production cycle (i.e. empty poultry house, with 7- and 42-day-old chickens) were stationary measured using Grimm spectrometer, as well as CIS and Button samplers. The dust collected by the latter 2 samplers was analyzed for endotoxin and (1→3)-β-D-glucan content. NAL samples were collected from employees after their work shift to determine the pro-inflammatory mediator levels., Results: The maximum particulate aerosol, endotoxin, and (1→3)-β-D-glucan concentrations at workplaces reached the levels of 4.12 mg/m 3 , 45.21 ng/m 3 , and 56.54 ng/m 3 , respectively. The IL-1β, IL-6, and IL-8 concentrations in NAL samples ranged between 0.62-18.12 pg/mL, <0.70-25.37 pg/mL, and <3.50-259.5 pg/mL, respectively. All TNFα levels were below 4 pg/mL. There were no significant differences between these cytokine concentrations in NAL samples collected at different stages of chicken breeding in either 'winter' or 'summer' seasons., Conclusions: Inhalation stimulation with poultry dust containing endotoxins and (1→3)-β-D-glucans resulted in the production of pro-inflammatory mediators, which proves the course of immunological processes in the exposed employees that may lead to adverse effects. The use of nasal lavage fluid in the control of such exposure confirms that NAL analysis is a reliable laboratory tool for assessing the impact of poultry dust on exposed farm workers.

It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1→3)-β-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1→3)-β-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1→3)-β-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1→3)-β-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1→3)-β-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1→3)-β-D-glucans.

Aconitum septentrionale is known to contain toxic diterpene alkaloids, while other bioactive compounds in the plant remain unclear. The aim of this study was to explore the phenolic compounds and polysaccharides from the water extract of A. septentrionale roots. Fifteen phenolic compounds were isolated and identified by NMR and MS, including fourteen known and one new dianthramide glucoside (2-[[2-(β-D-glucopyranosyloxy)-5-hydroxybenzoyl]amino]-4,5-dihydroxybenzoic acid methyl ester, 14). One neutral (complex of glucans with minor amounts of mannans) and two acidic polysaccharide fractions (complexes of pectic polysaccharides and glucans) were also obtained. Hydroxytyrosol (1), hydroxytyrosol-1-O-β-glucoside (2) and bracteanolide A (7) inhibited the release of nitric oxide by dendritic cells. Magnoflorine (8) and 2-[[2-(β-D-glucopyranosyloxy)-5-hydroxybenzoyl]amino]-5-hydroxybenzoic acid methyl ester (12) inhibited 15-lipoxygenase, and bracteanolide A (7) was a moderate inhibitor of xanthine oxidase. This study is the first to describe the diversity of phenolics and polysaccharides from A. septentrionale and their anti-inflammatory and anti-oxidant activities., (© 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG, Zurich, Switzerland.)

Objectives: Dust generated from wood processing comprises a heterogeneous mixture of inorganic and organic particles, including wood fragments, microorganisms, endotoxins, (1-3)-β-d-glucans, and allergens. This study characterized exposure to wood dust and its determinants in the Mozambiquan wood processing industry., Methods: A total of 124 personal inhalable samples, collected from a stratified random sample of 30 workers, were analysed for dust particulate, endotoxins, and (1-3)-β-d-glucans. Mixed-effects models were developed to investigate significant exposure determinants., Results: The geometric mean (GM) inhalable dust particulate concentrations were 3.29 mg m-3, 98 endotoxin units (EU) m-3, and 123 ng m-3 for (1-3)-β-d-glucans. Significant predictors for higher particulate levels included machinery (GMR = 1.93), sawing (GMR = 2.80), carpentry (GMR = 2.77), or painting (GMR = 3.03) tasks. Lebombo-ironwood species was associated with higher dust particulate levels (GMR = 1.97). Determinants of endotoxin concentrations included working with dry wood and damp cleaning methods, which were associated with lower levels. Working in closed buildings (GMR = 3.10) and dry sweeping methods were associated with higher (1-3)-β-d-glucan concentrations (GMR = 1.99)., Conclusions: Work tasks in certain exposure groups (machinery, sawing, carpentry, painting), processing certain wood species (Lebombo-ironwood) and working in closed buildings were associated with higher exposures, whilst using dry wood and damp cleaning practices reduced exposure levels., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

A novel reuteran-like glucan with low digestibility was fabricated using microbial glucanotransferase (GTase) treated maltodextrin. For GTase treated maltodextrin with DE 6, the molecular weight of reuteran-like glucan increased from 8.35 × 10 4 to 5.14 × 10 6  g/mol in the initial 6 h, increasing to 1.47 × 10 7  g/mol at 72 h. The short chain fraction (DP 3-12) of reuteran-like glucan increased from 45.2 % to 100.0 %, accompanied by an increase in α-1,6 glycosidic linkage percentage from 3.9 % to 33.3 %. This reaction promoted rearrangements in glycosidic chains, leading to a substantial increase in resistant starch content (13.4 % to 37.4 %) in the reuteran-like glucan. During in vitro fecal fermentation for 48 h, the reuteran-like glucan yielded large amounts of short-chain fatty acids (212.33 mM), especially butyric acid (12.64 mM). Thus, reuteran-like glucan could be used as a low-digestible and highly fermentable fiber for controlling blood glucose levels and prebiotic potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The fungal cell wall is essential for growth and survival, and is a key target for antifungal drugs and the immune system. The cell wall must be robust but flexible, protective and shielding yet porous to nutrients and membrane vesicles and receptive to exogenous signals. Most fungi have a common inner wall skeleton of chitin and β-glucans that functions as a flexible viscoelastic frame to which a more diverse set of outer cell wall polymers and glycosylated proteins are attached. Whereas the inner wall largely determines shape and strength, the outer wall confers properties of hydrophobicity, adhesiveness, and chemical and immunological heterogeneity. The spatial organization and dynamic regulation of the wall in response to prevailing growth conditions enable fungi to thrive within changing, diverse and often hostile environments. Understanding this architecture provides opportunities to develop diagnostics and drugs to combat life-threatening fungal infections., (© 2022. Springer Nature Limited.)

Airborne concentrations of organic dust on animal farms are known to be very high. This dust is partly composed of microorganisms such as bacteria, fungi and their components [endotoxins, (1→3)-β-d-glucans, mould allergens, mycotoxins], recognised as being responsible for numerous health effects. Several cross-sectional studies have measured levels of airborne bacteria, fungi and endotoxins on pig farms. However, the temporal dynamics of organic dust's components throughout the year have rarely been assessed, and airborne concentrations of (1→3)-β-d-glucans and mould allergens remain poorly understood in these work environments. This longitudinal, four-season study measured cultivable fungi, endotoxins, (1→3)-β-d-glucans, Aspergillus versicolor (AveX), Aspergillus fumigatus (Asp f1) and Alternaria sp (Alt a1) allergens on 31 pig farms in Switzerland. Results showed that exposure to AveX occurred in all four seasons. Total mean airborne concentration of endotoxins were between 3 and 4 times higher than the Swiss recommended limit value of 1000 EU m-3 and mean airborne concentrations of fungi were between 30 and 50 times higher than the Swiss recommended limit value of 1000 cfu m-3. Finally, accumulations of faecal matter on floors, humidity and dusty pathways were associated with increased concentrations of (1→3)-β-d-glucans. In conclusion, pig farmers require better information about biological occupational risks, and measures to improve air quality should be implemented, especially in winter., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by α-1,4 glucoside bonds. The latter are attached to each other by α-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, β-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The cell wall contains mannans and glucans that are recognized by the host immune system. In this chapter, we will describe the methods to isolate mannans and glucans from the C. albicans cell wall. In addition, we describe how to determine purity, molecular size, and structure of the mannans and glucans. We also detail how to prepare the carbohydrates for in vitro, ex vivo, or in vivo use by describing endotoxin removal (depyrogenation), derivatization, and labeling and evaluation of bioactivity., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Sewage treatment plant workers (STPs) are exposed to gaseous pollutants (H2S) and bioaerosols and their health is at risk., Objective: The aim of the study was to evaluate exposures to dust, 1,3 Beta D Glucans, endotoxins and gaseous pollutants in different process plants and to provide suitable recommendations., Methods: Gaseous pollutants and bioaerosols (inhalable dust, 1,3 beta D-glucans and endotoxins) were evaluated in two sewage treatment plants (STPs) of Ahmedabad city, India. The concentration of H2S, CO, CH4 and Cl2 were monitored in two process areas of STPs using real-time gas detectors. The dust, 1,3 beta D-glucans and endotoxins were evaluated as per standard methods., Results: The mean concentration of H2S exceeded the permissible exposure limit of 10 ppm, whereas the concentration of other gaseous pollutants (CO, Cl2 and CH4) were below the permissible exposure limits of Indian Factories Act, 1948. The inhalable dust concentration was also within the permissible exposure limit of 10 mg/m3 as per Indian Factories Act, 1948., Conclusions: Significant exposures to gaseous and bioaerosols were found in the work environment of STPs. The paired t-test result showed a significant difference between two STPs for H2S, 1,3 beta D-Glucans and endotoxins. STPs were advised to reduce the exposure to H2S and bioaerosols as per CPCB guidelines applicable to India to prevent health effects.

Linkage patterns and relaxation dynamics of baobab (Adansonia digitata) polysaccharides have been investigated by means of linkage analysis and rheometry. The fruit polysaccharide was mostly xylogalacturonan, with co-extracted α-glucan. The leaf polysaccharide consists predominantly of two domains, one branched at O-4 of the →2)-Rhap-(1→ residues and another branched at O-3 of the →4)-GalpA-(1→ backbone to single GlcpA-(1→ residues. Master curves of viscoelasticity of fruit polysaccharides manifested strong pH-dependency. At pH below the dissociation constant of galacturonic acid, dispersions showed liquid-like behaviour. In contrast, at neutral pH, a weak gel network formation was observed that destabilised rapidly under the influence of flow fields. The present work identifies xylogalacturonans from baobab fruit as polysaccharides with unique rheological characteristics that may point to new directions in food and pharmaceutical formulation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Arca subcrenata Lischke is a seafood with high nutritional value. In this study, we purified and characterized a novel water-soluble polysaccharide (ASPG-2) from Arca subcrenata with significant immunoregulatory effects and no apparent cell toxicity. ASPG-2 is a class of mixed-linkage α,β-d-glucan backbones with α-linked side chains with a molecular weight of 4.39 × 10 5 Da. Its structure was characterized as a repeating unit consisting of (1 → 3)-β-d-Glcp, (1 → 4)-α-d-Glcp, (1 → 4,6)-α-d-Glcp and (1 → 6)-α-d-Glcp. Using mouse RAW264.7 macrophages, we demonstrated that ASPG-2 exerted marked immunoregulatory effects by promoting the secretion of NO and increasing the phagocytosis of RAW264.7 cells in vitro. Moreover, flow cytometry analysis of the expression of the cell surface molecule CD86 revealed that ASPG-2 could polarize RAW264.7 cells into the M1 type. The immunomodulatory mechanism of ASPG-2 in macrophages was associated with the activation of the TLR4-MAPK/Akt-NF-κB signalling pathways. These results indicated that ASPG-2 might be researched and developed as a potential immunomodulatory agent or health product from marine organisms., (Copyright © 2021. Published by Elsevier B.V.)

Euglena gracilis is widely utilized as food or supplement to promote human and animal health, as it contains rich nutrients. In this study, we administered spray-dried powder of E. gracilis and paramylon, β-glucan stored in E. gracilis cells, to A4gnt knockout (KO) mice. A4gnt KO mice are a mutant mouse model that spontaneously develops gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence in the antrum of the stomach, and we observed the effects of E. gracilis and paramylon on the early involvements of A4gnt KO mice. Male and female 10-week-old A4gnt KO mice and their age-matched wildtype C57BL/6J mice were orally administered with 50 mg of E. gracilis or paramylon suspended in saline or saline as a control. After 3-week administration, animals were euthanatized and the stomach was examined histopathologically and immunohistochemically. Gene expression patterns of the stomach, which have been reported to be altered with A4gnt KO, and IgA concentration in small intestine were also analyzed with real-time PCR and ELISA, respectively. Administration of Euglena significantly reduced the number of stimulated CD3-positive T-lymphocytes in pyloric mucosa of A4gnt KO mice and tend to reduce polymorphonuclear leukocytes infiltration. Euglena administration further downregulated the expression of Il11 and Cxcl1 of A4gnt KO mice. Euglena administration also affected IgA concentration in small intestinal contents of A4gnt KO mice. Paramylon administration reduced the number of CD3-positive lymphocytes in pyloric mucosa of A4gnt KO mice, and downregulated the expressions of Il11 and Ccl2 of A4gnt KO mice. Although we found no significant effects on gross and microscopic signs of gastric dysplasia and cell proliferation, the present study suggests that the administration of Euglena and paramylon may ameliorate the early involvements of A4gnt mice through the effects on inflammatory reactions in the gastric mucosa. The cancer-preventing effects should be studied with long-term experiments until actual gastric cancer formation.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 10 7 /mL in G. lingzhi and 5.57 ± 0.49 × 10 6 /mL in G. applanatum. Osmotic stabilizer NaCl (0.4 M) at pH 5.8 and enzymolysis time 3.5 h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, β and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.

The mature basal stipe of mushroom Coprinopsis cinerea loses wall extensibility. We found that an endo-β-1,3-glucanase ENG from C. cinerea could restore mature basal stipe wall extensibility via pretreatment such that the ENG-pretreated basal stipe walls could be induced to extend by chitinase ChiIII. ENG pretreatment released glucose, laminaribiose, and 3-O-D-gentiobiose-D-glucose from the basal stipe walls, consistent with ENG-digested products of β-1,6-branched β-1,3-glucan. Different effects of endo-β-1,3-glucanase ENG and exo-β-1,3-glucanase EXG pretreatment on the structure, amount and ratio (β-1,3-glucoside bonds to β-1,6-glucoside bonds) of products from the basal stipe and the apical stipe cell walls, respectively, and on the cell wall extensibility and the cell wall ultra-architecture of the basal stipes were analyzed. All results demonstrate that the more accumulation and cross-linkage of β-1,6-branched β-1,3-glucan with wall maturation lead to loss of wall extensibility of the basal stipe regions compared to the apical stipe cell walls., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this research communication we evaluate the impact of the addition of prebiotic components (inulin, polydextrose, and modified starch, 40 g/l) as fat substitutes on the physicochemical characteristics, probiotic survival, and sensory acceptance of probiotic (Lacticaseibacillus casei 01, 108 CFU/ml) Greek yogurts during storage (7 °C, 28 d). All formulations had probiotic counts higher than 107 CFU/ml during storage and simulated gastrointestinal conditions (SGIC). The prebiotic components increased the probiotic survival to the enteric phase of the SGIC, with inulin producing the most pronounced effect. Inulin addition resulted in products with lower pH values and consistency and higher titratable acidity during storage, with negative impact on the sensory acceptance (flavor, texture, and overall impression) at the end of the storage period. Modified starch addition impacted negatively on the acceptance of the products (appearance, flavor, texture, and overall impression). Polydextrose addition resulted in products with lower consistency, but similar sensory acceptance to the full-fat yogurt. It can be concluded that it is possible to prepare potentially synbiotic Greek yogurts by desorption technique using L. casei as probiotic culture and inulin, polydextrose or modified starch as prebiotic components, with the utilization of polydextrose being advisable.

Pythium insidiosum is an oomycete that affects mammals, especially humans and horses, causing a difficult-to-treat disease. Typically, surgical interventions associated with antimicrobial therapy, immunotherapy, or both are the preferred treatment choices. PitiumVac® is a therapeutic vaccine prepared from the mycelial mass of P. insidiosum and is used to treat Brazilian equine pythiosis. To better understand how PitiumVac® works, we analyzed the composition of PitiumVac® and the immune response triggered by this immunotherapy in mice. We performed an enzymatic quantification that showed a total glucan content of 21.05% ± 0.94 (α-glucan, 6.37% ± 0.77 and (1,3)(1,6)-β-glucan, 14.68% ± 0.60) and mannose content of 1.39% ± 0.26; the protein content was 0.52 mg ml-1 ± 0.07 mg ml-1. Healthy Swiss mice (n = 3) were subcutaneously preimmunized with one, two, or three shots of PitiumVac®, and immunization promoted a relevant Th1 and Th17 responses compared to nonimmunization of mice. The highest cytokine levels were observed after the third immunization, principally for IFN-γ, IL-17A, IL-6, and IL-10 levels. Results of infected untreated (Pythiosis) and infected treated (Pythiosis + PVAC) mice (n = 3) showed that PitiumVac® reinforces the Th1/Th17 response displayed by untreated mice. The (1,3)(1,6)-β-glucan content can be, at least in part, related to this Th1/Th17 response., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The soluble α-polyglucan glycogen is a central metabolite enabling transient glucose storage to suit cellular energy needs. Glycogen storage diseases (GSDs) comprise over 15 entities caused by generalized or tissue-specific defects in enzymes of glycogen metabolism. In several, e.g. in Lafora disease caused by the absence of the glycogen phosphatase laforin or its interacting partner malin, degradation-resistant abnormally structured insoluble glycogen accumulates. Sensitive quantification methods for soluble and insoluble glycogen are critical to research, including therapeutic studies, in such diseases. This paper establishes methodological advancements relevant to glycogen metabolism investigations generally, and GSDs. Introducing a pre-extraction incubation method, we measure degradation-resistant glycogen in as little as 30 mg of skeletal muscle or a single hippocampus from Lafora disease mouse models. The digestion-resistant glycogen correlates with the disease-pathogenic insoluble glycogen and can readily be detected in very young mice where glycogen accumulation has just begun. Second, we establish a high-sensitivity glucose assay with detection of ATP depletion, enabling 1) quantification of α-glucans in cell culture using a medium-throughput assay suitable for assessment of candidate glycogen synthesis inhibitors, and 2) discovery of α-glucan material in healthy human cerebrospinal fluid, establishing a novel methodological platform for biomarker analyses in Lafora disease and other GSDs., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nitschke et al.)

The moisture sorption and desorption properties of hard capsules have a great influence on the quality of capsule products. However, studies on them have rarely been reported. Herein, we studied the moisture sorption and desorption properties of three kinds of hard capsules (gelatin, hydroxypropyl methyl cellulose (HPMC) and pullulan capsules) in terms of hygroscopicity, crystallinity, thermal behaviors and so on. It is found that HPMC capsules have weaker moisture sorption ability and moisture keeping ability than pullulan or gelatin capsules with lower moisture sorption rates, equilibrium moisture contents, moisture keeping rates and higher critical relative humidity. In comparison with gelatin capsules, pullulan capsules have weaker moisture sorption ability and comparable moisture keeping ability. HPMC or pullulan capsules can more effectively protect high, moderate and low hygroscopic capsule contents (chitosan, potato starch or ethyl cellulose) from outside moisture absorption. The diffraction peaks of the moisture equilibrated gelatin, HPMC and pullulan capsules are much smaller than those of their dried ones. The dried and the moisture equilibrated gelatin, HPMC or pullulan capsules all have smooth surface morphology. HPMC or pullulan capsules can be an attractive alternative to animal gelatin capsules due to their appropriate moisture sorption and desorption properties., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier B.V.)

α-Glucans that were enzymatically synthesized from sucrose using glucansucrase cloned from Leuconostoc mesenteroides NRRL B-1118 were found to have a glass transition temperature of approximately 80 °C. Using high-pressure homogenization (~70 MPa), the α-glucans were converted into nanoparticles of ~120 nm in diameter with a surface potential of ~-3 mV. Fluorescence measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the α-glucan nanoparticles have a hydrophobic core that remains intact from 10 to 85 °C. α-Glucan nanoparticles were found to be stable for over 220 days and able to form at three pH levels. Accelerated exposure measurements demonstrated that the α-glucan nanoparticles can endure exposure to elevated temperatures up to 60 °C for 6 h intervals.

In this study, Raman spectroscopy is employed for the characterization and comparison of two different classes of exo-polysaccharides including glucans and fructans which are produced by different bacteria. For this purpose, nine samples are used including five samples of glucans and four of fructans. Raman spectral results of all these polysaccharides show clear differences among various glucans as well as fructans showing the potential of this technique to identify the differences within the same class of the compounds. Moreover, these two classes are also compared on the basis of their Raman spectral data and can be differentiated on the basis of their unique Raman features. Multivariate data analysis techniques, Principle Component Analysis (PCA) is found very helpful for the comparison of the Raman spectral data of these classes of the carbohydrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Cranberry pomace obtained from industrial juice production was characterized by proximate composition analysis and monosaccharide profile of the dietary fiber. Extraction of carbohydrates from pomace was investigated using microwave-assisted alkaline method and five commercial biocatalysts (pure endo-galactanase and four multienzyme biocatalysts). The extracts obtained from microwave-assisted approach had average total sugars yield of 21.3% and contained mostly oligosaccharides in the degree of polymerization range of 7 to 10. All multienzyme biocatalysts led to yields similar or higher than microwave-assisted approach (23.4% to 42.0%), but mainly generated shorter oligosaccharides with a degree of polymerization of 2 to 5. Compared to cranberry pomace dietary fiber, microwave-assisted extracts were enriched in pectic oligosaccharides, whereas the enzymatic extracts were enriched in glucans and had less rhamnose and galactose. Pomace ground for 5 min or more by ball mill assumed a powdery consistence. Longer milling did not affect particle size but increased their roughness. Such physical changes had no effect on the efficiency of multienzymatic treatment. PRACTICAL APPLICATION: The increased production of cranberries and cranberry products will continuously generate more pomace, a potentially valuable material for the generation of added-value products. Up to 60% to 70% of cranberry pomace is composed of plant cell wall material. The properties of naturally occurring plant cell wall polysaccharides and their corresponding oligosaccharides have been of a great interest, and many of them find application as functional food ingredients. Despite the fact that the cranberry pomace is rich in plant cell polysaccharides, it has been mainly explored as a source of phenolic antioxidants. This study reveals the efficiency of cranberry pomace as a source of nondigestible oligosaccharides. The use of microwave-assisted extraction and different biocatalysts for the enzymatic extraction led to oligosaccharides with well-defined monosaccharide composition and molecular weight distribution. The study of the effects of these extraction techniques on the yield and the characteristics of generated oligosaccharides would allow the modulation of their properties. As an overall, the findings of this study would contribute to lay the scientific ground for the development of innovative process for the isolation of nondigestible oligosaccharides as functional ingredients from cranberry pomace by products., (© 2020 Institute of Food Technologists®.)

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the β-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation., Competing Interests: The authors declare that no competing interests exist. Embrapa, the employer of MMCC, is a not-for-profit, state-funded research organization. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: The observed deficit of lung cancer in farmers has been partly attributed to exposure to organic dusts and endotoxins based largely on surrogate metrics. To move beyond these surrogates for etiological studies, we characterized task-based and time-weighted average (TWA) exposure to inhalable endotoxin, (1 → 3)-β-D-glucan, and dust in Iowa farmers., Methods: We collected 320 personal inhalable dust samples from 32 farmers during 69 sample days in 2015 and 2016. Samples were collected using Button aerosol samplers and analyzed for endotoxin using a kinetic chromogenic amebocyte lysate assay, and for (1 → 3)-β-D-glucan using a Limulus endpoint assay. We assessed relationships between bioaerosol concentrations and selected tasks and farm characteristics using linear mixed-effects models., Results: Bedding work, hog handling, and working in barn/confinement buildings, grain bins, and grain elevators were associated with higher endotoxin exposure. We found a monotonic trend between higher endotoxin concentrations and increasing number of animals. Bedding work, cleaning, and feed/grain storage work were associated with higher (1 → 3)-β-D-glucan concentrations. The median concentrations by task spanned one order of magnitude for inhalable dust and two orders of magnitude for endotoxin and (1 → 3)-β-D-glucan. Pearson correlations between endotoxin and glucan concentrations were 0.22 for TWA exposure and 0.56 for task samples., Conclusions: This characterization of exposure factors that influence bioaerosol concentrations can support the development of refined bioaerosol exposure metrics for future etiologic analyses of cancer and other health outcomes in farmers., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier GmbH.)

To elucidate the role of long alkyl group in sulfated poly- and oligosaccharides on anti-HIV activity, the interaction between sulfated 3-O-octadecyl-(1→6)-α-d-glucopyranan with potent anti-HIV activity and liposomes with diameters of 58 ± 20 nm and 107 ± 28 nm as models of HIV were investigated. SPR measurements of sulfated 3-O-octadecyl-(1→6)-α-d-glucopyranans bearing 2.8 mol% of the octadecyl group and the liposome (diameter = 58 ± 20.0 nm and ζ=0 mV) resulted in an apparent association- k a  = 6 × 10 5 1/M, a dissociation-rate k d  = 4 × 10 -4 1/s, and a dissociation constants K D  = 8 × 10 -10 M. The particle size of the sulfated 3-O-octadecyl-(1→6)-α-d-glucopyranan (67 ± 14 nm) measured by DLS increased to 104 ± 25 nm, whereas the ζ potential (-29 mV) was unchanged (-33 mV). For dextran sulfate without an alkyl group, no interaction was observed. These results suggest that the long octadecyl group penetrated into the liposome and sulfated glucopyranan was covered on the liposome to increase the anti-HIV activity. The 107 nm liposome exhibited similar results., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dietary fibre (DF) profiles of natural hazelnut, roasted hazelnut and hazelnut skin were analyzed. Insoluble (IDF) and soluble (SDF) DFs were examined for monosaccharide and glycosyl-linkage compositions using gas chromatography-mass spectrometry (GC-MS). Total DF contents of natural hazelnut, roasted hazelnut, and hazelnut skin were 17.8, 15.4, and 69.8%, respectively; majority of which (>96%) were water-insoluble. IDFs of natural and roasted hazelnuts were composed of cellulose (~49%), pectic polysaccharides (~30%), and xyloglucans (~15%), whereas that of hazelnut skin made up lignin (~55%) and fibre polysaccharides (cellulose, pectic polysaccharides, and xyloglucans, ~45%). Unlike the ones from other sources, pectic polysaccharides in IDFs had lower proportion of smooth region and higher proportion of hairy region that is heavily branched with arabinan and galactan side chains. Xyloglucans were also densely branched with monomeric and/or dimeric side chains. SDFs of the samples were composed of heavily branched heteromannans (~60%), slightly branched pectic polysaccharides (~25%), and xyloglucans possessing monomeric side chains (~5%). These results suggest that hazelnut is rich in DFs that have potential to improve large bowel function and hazelnut skin, a byproduct of hazelnut roasting process, could be utilized for the production of functional carbohydrates having prebiotic capacities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The design of scaffolds for solubilizing/dispersing poorly water-soluble bioactive molecules in neutral aqueous media is a major challenge of functional food, pharmaceuticals, and cosmetics development, as highlighted by the plethora of corresponding solubilization/dispersion strategies. Herein, renatured β-1,3-1,6-glucan (r-glucan) nanoparticles prepared by neutralization of alkali-denatured β-1,3-1,6-glucan and subsequent centrifugation are used as a host to disperse water-insoluble bioactive molecules (curcumin, all-trans-retinoic acid, and rebamipide) by simple mixing of host and guest solutions. Curcumin in the r-glucan cavity is found to be stacked in the form of J-aggregates and twisted along the helix, and is demonstrated to be retained for significantly longer than curcumin in the corresponding γ-cyclodextrin (γ-CD) complex. Specifically, curcumin incorporated in γ-CD is released within 5.5 hours, whereas that in the r-glucan complex is released very slowly, with 12% of curcumin in the latter complex retained after 31-day incubation at 37°C. Thus, inclusion protocol simplicity and slow release ability make r-glucan nanoparticles a potential carrier scaffold for various applications., (© 2020 Wiley Periodicals, Inc.)

Marine microalgae sequester as much CO 2 into carbohydrates as terrestrial plants. Polymeric carbohydrates (i.e., glycans) provide carbon for heterotrophic organisms and constitute a carbon sink in the global oceans. The quantitative contributions of different algal glycans to cycling and sequestration of carbon remain unknown, partly because of the analytical challenge to quantify glycans in complex biological matrices. Here, we quantified a glycan structural type using a recently developed biocatalytic strategy, which involves laminarinase enzymes that specifically cleave the algal glycan laminarin into readily analyzable fragments. We measured laminarin along transects in the Arctic, Atlantic, and Pacific oceans and during three time series in the North Sea. These data revealed a median of 26 ± 17% laminarin within the particulate organic carbon pool. The observed correlation between chlorophyll and laminarin suggests an annual production of algal laminarin of 12 ± 8 gigatons: that is, approximately three times the annual atmospheric carbon dioxide increase by fossil fuel burning. Moreover, our data revealed that laminarin accounted for up to 50% of organic carbon in sinking diatom-containing particles, thus substantially contributing to carbon export from surface waters. Spatially and temporally variable laminarin concentrations in the sunlit ocean are driven by light availability. Collectively, these observations highlight the prominent ecological role and biogeochemical function of laminarin in oceanic carbon export and energy flow to higher trophic levels., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Various (semi)-synthetic non-digestible glucans (NDG) (resistant (malto)dextrin, isomaltooligosaccharide, soluble corn fiber, polydextrose) were analyzed by methylation analysis, 1 H NMR spectroscopy, ESI-MS n , TLC, and capillary electrophoresis (CE/UV). Partly complementary and partly corroborative results were obtained by these methods yielding a comprehensive overview about the various degrees of complexity and special structural features. Methylation analysis enabled the most detailed quantitative evaluation of glycosidic linkage positions and branching pattern, and detection of furanosyl residues. 1 H NMR spectroscopy provided additional information on anomeric configuration. By ESI-IT-MS up to DP 13, for doubly charged up to DP 22 was detected. Glucitol residues in polydextrose as well as anhydrosugar formation in thermally treated glucans could also be recognized from the mass spectra. Furthermore, MS 2 of the disaccharide portion gave insights into the linkage positions present in the glucan mixture. Electrophoresis of ANTS-labeled oligosaccharides showed the DP-resolved differences of complexity and was the most powerful method to identify the type of glucan by its CE-fingerprint. The aim was to present complementary analytical data obtained by application of identical conditions and methods to a wide range of NDG with increasing complexity to allow direct structure comparison., Competing Interests: Declaration of competing interest The authors gratefully acknowledge financial support for this project from Beneo GmbH, Germany, which had no role in the study design, data collection, analysis, interpretation or manuscript preparation. This financial support did not confine the authors from publishing their results. Publishing this research was possible without the prior approval of the sponsor. The authors declare no competing financial interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Samples of granular corn starch were treated with 1,4-α-glucan branching enzyme (GBE) for 20 h using three different methods. These GBE modification methods all increased glycosidic linkage ratio, cyclic glucan content, and proportion of short chains while reducing weight mean molecular weight. The in vitro digestion rates of the modified starches were suppressed. Among these methods, a novel two-stage modification method comprising a 10-h GBE treatment, gelatinization, and a second 10-h GBE treatment, produced samples with the lowest in vitro digestibility. The rapidly digestible starch content was 34.2% lower than that of the control and 18.0% lower than that of the product of one-stage modification with the same duration. Fine structure characterization showed that more cluster structures were proved during the two-stage modification. This two-stage method suppressed the digestibility of corn starch and increased the substrate concentration, showing great potential for the industrial processing of slowly-digestible starchy foods., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The functional ingredients of microalgal biomass are receiving substantial recognition as the global demands for health supplements produced from natural sources are on the rise. Paramylon, a conglomerate of β-1,3-glucans, is one of the major valuable sources derived from Euglena gracilis having multiple applications, thus necessitating the development of an efficient quantification method. Here, we employed a DNA aptamer to quantify the amount of paramylon produced by E. gracilis . Paramylon-specific aptamers were isolated by the systematic evolution of ligands by exponential enrichment (SELEX) process. To evaluate the potential aptamers, the binding affinity between aptamer candidates and paramylon granules was confirmed by a confocal laser scanning microscope and the dissociation constants of the selected aptamers were determined by nonlinear regression analysis. The selected DNA aptamer was successfully used for the quantification of paramylon, and the results were compared to those obtained by the standard methods. The new approach was also used for quantification of paramylon from E. gracilis cells cultured to different cell stages and physiologies. It can be concluded that the aptamer-based protocol for the measurement of paramylon proposed in this study is highly accurate and comparatively less time-consuming.

To achieve fertilization, pollen tubes have to protect and properly deliver sperm cells through the pistil to the ovules. Pollen tube growth is a representative example of polarized growth where new components of the cell wall and plasma membrane are continuously deposited at the tip of the growing cell. The integrity of the cell wall is of fundamental importance to maintain apical growth. For this reason, pollen tube growth has become an excellent model to study the role of polysaccharides and structural cell wall proteins involved in polar cell expansion. However, quantification of structural polysaccharides at the pollen tube cell wall has been challenging due to technical complexity and the difficulty of finding specific dyes. Here, we propose simple methods for imaging and quantification of callose, pectin , and cellulose using specific dyes such as Aniline Blue, Propidium Iodide, and Pontamine Fast Scarlet 4B.

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in (β1→3, β1→4)-glucans, (α1→3)-, (α1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The extensive characterization of glycosidic linkages in carbohydrates remains a challenge because of the lack of known standards and limitations in current analytical techniques. This study encompasses the construction of an extensive glycosidic linkage library built from synthesized standards. It includes an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of glycosidic linkages derived from disaccharides, oligosaccharides, and polysaccharides present in complicated matrices. We present a method capable of the simultaneous identification of over 90 unique glycosidic linkages using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ MS) operated in dynamic multiple reaction monitoring (dMRM) mode. To build the library, known monosaccharides commonly found in plants were subjected to partial methylation to yield partially derivatized species representing trisecting, bisecting, linear, and terminal structures. The library includes glycosidic linkage information for three hexoses (glucose, galactose, and mannose), three pentoses (xylose, arabinose, and ribose), two deoxyhexoses (fucose and rhamnose), and two hexuronic acids (glucuronic acid and galacturonic acid). The resulting partially methylated monosaccharides were then labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by separation and analysis by UHPLC/dMRM MS. Validation of the synthesized standards was performed using disaccharide, oligosaccharide, and polysaccharide standards. Accuracy, reproducibility, and robustness of the method was demonstrated by analysis of xyloglucan (tamarind) and whole carrot root. The synthesized standards represent the most comprehensive group of carbohydrate linkages to date.

Mass spectrometry (MS) has become the primary method for high-sensitivity structural determination of oligosaccharides. Fragmentation in the negative-ion MS can provide a wealth of structural information and these can be used for sequence determination. However, although negative-ion MS of neutral oligosaccharide using the deprotonated molecule [M-H] - as the precursor has been very successful for electrospray ionization (ESI), it has only limited success for matrix-assisted laser desorption/ionization (MALDI). In the present study, the features of negative-ion MALDI primary spectra were investigated in detail and the product-ion spectra using [M-H] - and [M+Cl] - as the precursors were carefully compared. The formation of [M-H] - was the main difficulty for MALDI while [M+Cl] - was proved to be useful as alternative precursor anion for MALDI-MS/MS to produce similar fragmentation for sequencing of neutral oligosaccharides. N-(1-naphthyl)ethylenediamine dihydrochloride was then used as both the matrix and the Cl - dopant to evaluate the extent of structural information that can be obtained by negative-ion fragmentation from [M+Cl] - using laser-induced dissociation (LID)-MS/MS for linkage assignment of gluco-oligosaccharides and for typing of blood-group ABO(H) and Lewis antigens on either type 1 or type 2 backbone-chains., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Since its introduction, matrix-assisted laser desorption/ionization (MALDI) has been widely used for the mass analysis of biomolecules. The "soft ionization" of MALDI enables accurate mass determination of intact biomolecules. However, the ionization and desorption processes of MALDI are not adequately soft as many labile biomolecules, such as glycoconjugates containing sialic acid or the sulfate functional group, easily dissociate into fragments and sometimes, no intact molecules are observed. In this study, we compared the conventional matrix of MALDI, namely 2,5-dihydroxybenzoic acid, to various soft matrices of MALDI-specifically, 5-methoxysalicylic acid, diamond nanoparticle trilayers, HgTe nanostructures, ionic liquid, and droplets of frozen solutions-by using three labile glycoconjugates as analytes: gangliosides, heparin, and pullulan. We demonstrated that droplets of frozen solution are the softest matrices for gangliosides and heparin. In particular, droplets of frozen solution do not generate fragments for gangliosides and can be used to determine the relative abundance of various gangliosides, whereas ionic liquid 2,5-dihydroxybenzoic acid butylamine is the most suitable matrix for pullulan mass analysis. Graphical Abstract.

Enzymatic degradation of the β-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-β-D-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis β-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light&#95;m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.

Objectives: To determine the accuracy of Fungitell, a β-d-glucan (BDG) test, for the diagnosis of invasive fungal infection (IFI) among cancer patients., Methods: For this meta-analysis, MEDLINE and EMBASE were searched for references related to BDG testing. Study quality was evaluated using QUADAS-2. Statistical analysis was performed using Stata 14., Results: We screened 12,426 references and identified 189 studies for full-text review. Nineteen studies were included in the final meta-analysis. There was moderate heterogeneity between studies. Nine studies had a high risk of bias, which significantly elevated the overall specificity estimate. Restricting to only low-bias studies, the sensitivity and specificity were 80% and 63%, respectively., Conclusions: The overall sensitivity and specificity of Fungitell as a diagnostic test for IFI is moderate, and there is substantial heterogeneity between studies. Limiting studies to only low-bias risk reduced heterogeneity but also lowered the overall specificity estimate.

Introduction: Lafora disease is autosomal recessive progressive myoclonus epilepsy with late childhood-to teenage-onset caused by loss-of-function mutations in either EPM2A or EPM2B genes encoding laforin or malin, respectively., Development: The main symptoms of Lafora disease, which worsen progressively, are: myoclonus, occipital seizures, generalized tonic-clonic seizures, cognitive decline, neuropsychiatric syptoms and ataxia with a fatal outcome. Pathologically, Lafora disease is characterized by the presence of polyglucosans deposits (named Lafora bodies), in the brain, liver, muscle and sweat glands. Diagnosis of Lafora disease is made through clinical, electrophysiological, histological and genetic findings. Currently, there is no treatment to cure or prevent the development of the disease. Traditionally, antiepileptic drugs are used for the management of myoclonus and seizures. However, patients become drug-resistant after the initial stage., Conclusions: Lafora disease is a rare pathology that has serious consequences for patients and their caregivers despite its low prevalence. Therefore, continuing research in order to clarify the underlying mechanisms and hopefully developing new palliative and curative treatments for the disease is necessary.

Marine macroalgae are considered as promising sustainable alternatives to conventional terrestrial animal feed resources. The advantages include high growth rate, potential cultivation in saltwater, and no occupation of arable land. Macroalgae are broadly classified as brown (Phaeophyta), red (Rhodophyta) and green (Chlorophyta) algae, and are a diverse group of marine organisms. The nutritional value of macroalgae is highly variable. The protein and essential amino acid content can be low, especially in brown species, and indigestible polysaccharides adversely affect the energy value. Optimal use of macroalgae in feeds requires suitable processing, and biorefinery approaches may increase protein content and improve nutrient availability. Macroalgae are rich in unique bioactive components and there is a growing interest in the potentially beneficial health effects of compounds such as laminarin and fucoidan in different macroalgal and macroalgal products. This review summarizes current literature on different aspects of the use of macroalgae as sources of protein and health-promoting bioactive compounds in feed for monogastric animal species. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Development of aluminium (Al) resistant genotypes through molecular breeding is a major approach for increasing seed yield under acidic conditions. There are no available reports on mapping of Al resistance loci and molecular breeding for Al resistant varieties in lentil. The present study reports a major quantitative trait loci (QTL) for Al resistance using simple sequence repeat (SSR) markers in F 2 and F 3 mapping populations derived from contrasting parents. Phenotypic response to Al was measured on the bases of root re-growth (RRG), fluorescent signals (callose accumulation) and Al contents in hydroponic assay. After screening 495 SSR markers to search polymorphism between two contrasting parents, 73 polymorphic markers were used for bulk segregation analysis. Two major QTLs were identified using seven trait linked markers, one each for fluorescent signals and RRG mapped on linkage group (LG) 1 under Al stress conditions in F 2 mapping population of cross BM-4 × L-4602. One major QTL (qAlt&#95;fs) was localised between PLC&#95;88 and PBA&#95;LC&#95;373, covering 25.9 cM with adjacent marker PLC&#95;88 at a distance of 0.4 cM. Another major QTL (qAlt&#95;rrg) for RRG was in the marker interval of PBA&#95;LC&#95;1247 and PLC&#95;51, covering a distance of 45.7 cM with nearest marker PBA&#95;LC&#95;1247 at a distance of 21.2 cM. Similarly, in F 3 families of BM-4 × L-4602 and BM-4 × L-7903, LG-1 was extended to 285.9 and 216.4 cM respectively, having four newly developed genic-SSR markers. These QTLs had a logarithm of odd (LOD) value of 140.5 and 28.8 along with phenotypic variation of 52% and 11% for fluorescent signals and RRG respectively, whereas, qAlt&#95;rrg had LOD of 36 and phenotypic variance of 25% in F 3 population of BM-4 × L-4602. Two major QTLs identified in the present study can be further dissected for candidate gene discovery and development of molecular markers for breeding improved varieties with high Al resistance.

The objectives of this study were to employ response surface methodology (RSM) to investigate and optimize the effect of ultrasound-assisted extraction (UAE) variables, temperature, time and amplitude on the yields of polysaccharides (fucose and total glucans) and antioxidant activities (ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picryl-hydrazyl radical scavenging activity (DPPH)) from Laminaria digitata , and to explore the suitability of applying the optimum UAE conditions for L. digitata to other brown macroalgae ( L. hyperborea and Ascophyllum nodosum ). The RSM with three-factor, four-level Box-Behnken Design (BBD) was used to study and optimize the extraction variables. A second order polynomial model fitted well to the experimental data with R² values of 0.79, 0.66, 0.64, 0.73 for fucose, total glucans, FRAP and DPPH, respectively. The UAE parameters studied had a significant influence on the levels of fucose, FRAP and DPPH. The optimised UAE conditions (temperature = 76 °C, time = 10 min and amplitude = 100%) achieved yields of fucose (1060.7 ± 70.6 mg/100 g dried seaweed (ds)), total glucans (968.6 ± 13.3 mg/100 g ds), FRAP (8.7 ± 0.5 µM trolox/mg freeze-dried extract (fde)) and DPPH (11.0 ± 0.2%) in L. digitata . Polysaccharide rich extracts were also attained from L. hyperborea and A. nodosum with variable results when utilizing the optimum UAE conditions for L. digitata ., Competing Interests: The authors declare no conflict of interest.