An Aspergillus nidulans endo-ß-1,3-glucanase exhibited specific catalytic features and was used to prepare 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose with high antioxidant activity from barley ß-glucan and laminarin, respectively.

An endo-ß-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of ß-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-ß-gentiobiosyl-d-glucose and hydrolyzed barley ß-glucan to mainly produce 3-O-ß-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley ß-glucan and E. bicyclis laminarin. Further study reveals that 3-O-ß-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley ß-glucan and 3-O-ß-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that ß-1,4-linkage or -1,6-linkage at non-reducing end of ß-glucotrioses had enhancing effect on antioxidant activity compared to ß-1,3-linkage. Furthermore, 3-O-ß-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley ß-glucan, indicating that ß-1,4-linkage at reducing end of ß-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to ß-1,3-linkage.

A reassessment of flocculosin-mediated biocontrol activity of Pseudozyma flocculosa through CRISPR/Cas9 gene editing.

Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.

1,4-Disubstituted 1H-1,2,3-Triazoles for Renal Diseases: Studies of Viability, Anti-Inflammatory, and Antioxidant Activities.

Inflammation is a hallmark of many metabolic diseases. We previously showed that ferrocene-appended 1H-1,2,3-triazole hybrids inhibit nitric oxide (NO) production in in vitro models of lipopolysaccharide-induced inflammation in the BV-2 cell. In the present study, we explored the viability, anti-inflammatory, and antioxidant potential of ferrocene-1H-1,2,3-triazole hybrids using biochemical assays in rat mesangial cells (RMCs). We found that, among all the ferrocene-1H-1,2,3-triazole hybrids, X2-X4 exhibited an antioxidant effect on mitochondrial free radicals. Among all the studied compounds, X4 demonstrated the best anti-inflammatory effect on RMCs. These results were supplemented by in silico studies including molecular docking with human cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2) enzymes as well as absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling. Besides, two new crystal structures of the compounds have also been reported. In addition, combining the results from the inducible nitric oxide synthase (iNOS), cPLA2, COX-2, and matrix metalloproteinase-9 (MMP-9) enzymatic activity analysis and NO production also confirmed this argument. Overall, the results of this study will be a valuable addition to the growing body of work on biological activities of triazole-based compounds.

Preparation of collagen/cellulose nanocrystals composite films and their potential applications in corneal repair.

As the main component of the natural cornea, collagen (COL) has been widely applied to the construction of corneal repair materials. However, the applications of collagen are limited due to its poor mechanical properties. Cellulose nanocrystals (CNCs) possess excellent mechanical properties, optical transparency and good biocompatibility. Therefore, in this study, we attempted to introduce cellulose nanocrystals into collagen-based films to obtain corneal repair materials with a high strength. CNCs were incorporated at 1, 3, 5, 7 and 10 wt%. The physical properties of these composite films were characterized, and in vitro cell-based analyses were also performed. The COL/CNC films possessed better mechanic properties, and the introduction of CNCs did not affect the water content and light transmittance. The COL/CNC films demonstrated good biocompatibility toward rabbit corneal epithelial cells and keratocytes in vitro. Moreover, the collagen films with appropriate ration of CNCs effectively induced the migration of corneal epithelial cells and inhibited the myofibroblast differentiation of keratocytes. A collagen film with 7 wt% CNCs displayed the best combination of physical properties and biological performance in vitro among all the films. This study describes a nonchemical cross-linking method to enhance the mechanical properties of collagen for use in corneal repair materials and highlights potential application in corneal tissue engineering.

Notochordal Signals Establish Phylogenetic Identity of the Teleost Spine.

The spine is a defining feature of the vertebrate body plan. However, broad differences in vertebral structures and morphogenetic strategies occur across vertebrate groups, clouding the homology between their developmental programs. Analysis of a zebrafish mutant, spondo, whose spine is dysmorphic, prompted us to reconstruct paleontological evidence, highlighting specific transitions during teleost spine evolution. Interestingly, the spondo mutant recapitulates characteristics present in basal fishes, not found in extant teleosts. Further analysis of the mutation implicated the teleost-specific notochord protein, Calymmin, as a key regulator of spine patterning in zebrafish. The mutation in cmn results in loss of notochord sheath segmentation, altering osteoblast migration to the developing spine, and increasing sensitivity to somitogenesis defects associated with congenital scoliosis in amniotes. These data suggest that signals from the notochord define the evolutionary identity of the spine and demonstrate how simple shifts in development can revert traits canalized for about 250 million years.

Novel lncRNA XLOC_032768 alleviates cisplatin-induced apoptosis and inflammatory response of renal tubular epithelial cells through TNF-α.

The cellular and molecular mechanisms through which cisplatin induces nephrotoxicity have been investigated extensively. However, the role of long non-coding RNAs (lncRNAs) in cisplatin-induced nephrotoxicity is not well known. We explored the functions and underlying mechanisms of a novel lncRNA XLOC_032768 in cisplatin-induced nephrotoxicity. Cisplatin treatment resulted in the apoptosis of the renal tubular epithelial cells and inflammatory response in a mouse model and human renal proximal tubular epithelial cells (HK-2). The differentially expressed genes (DEGs) of the transcriptome data were determined, and the results showed that lncRNA XLOC_032768 expression was significantly repressed by cisplatin treatment. This result was validated by an RT-qPCR experiment on in vivo and in vitro models. The overexpression of XLOC_032768 significantly inhibited the cisplatin-induced apoptosis and inflammatory response in HK-2 cells and mouse exposed to cisplatin. RNA sequencing analysis further confirmed that XLOC_032768 could regulate tumor necrosis factor (TNF)-α in the cisplatin-induced apoptosis of HK-2 cells in trans-manner. TNF-α inhibition also ameliorated cisplatin-induced apoptosis of renal tubular epithelial cells and renal structural damage. As such, XLOC_032768 suppressed cisplatin-induced apoptosis and inflammatory response of renal tubular epithelial cells through TNF-α. LncRNA XLOC_032768 is a potential novel agent to reduce cisplatin-induced nephrotoxicity.

Transformation of cellobiose during the interaction of cellobiose dehydrogenase and ß-glucosidase of Cerrena unicolor.

This work investigated the regulatory role of the interaction between cellobiose dehydrogenase (CDH) and ß-glucosidase (ß-GLU) in the conversion of cellobiose into cellobionolactone or glucose in vitro. To study the regulation, the two enzymes were isolated from the culture medium of the fungus Cerrena unicolor grown on a medium with microcrystalline cellulose. The enzymes were obtained in an electrophoretically homogeneous state. Their properties were studied. Both enzymes had acidic pH optima and were more stable in the acidic pH range. CDH was moderately thermostable, while ß-GLU had a low thermostability. Both enzymes efficiently catalyzed the transformation of cellobiose. A mixture of CDH and ß-GLU transformed cellobiose to glucose or cellobionolactone in the presence of various concentrations of laccase and hydroquinone. Formation of glucose and cellobionolactone in vitro during the competition between CDH and ß-GLU for cellobiose depended on the availability of quinones, formed as a result of the interaction of laccase and hydroquinone, for CDH. At low laccase and hydroquinone concentrations, the formation of glucose was found to predominate over that of cellobionolactone. The possible physiological role of the enzymes' interaction is discussed.

Effectors involved in fungal-fungal interaction lead to a rare phenomenon of hyperbiotrophy in the tritrophic system biocontrol agent-powdery mildew-plant.

Tritrophic interactions involving a biocontrol agent, a pathogen and a plant have been analyzed predominantly from the perspective of the biocontrol agent. We have conducted the first comprehensive transcriptomic analysis of all three organisms in an effort to understand the elusive properties of Pseudozyma flocculosa in the context of its biocontrol activity against Blumeria graminis f.sp. hordei as it parasitizes Hordeum vulgare. After inoculation of P. flocculosa, the tripartite interaction was monitored over time and samples collected for scanning electron microscopy and RNA sequencing. Based on our observations, P. flocculosa indirectly parasitizes barley, albeit transiently, by diverting nutrients extracted by B. graminis from barley leaves through a process involving unique effectors. This brings novel evidence that such molecules can also influence fungal-fungal interactions. Their release is synchronized with a higher expression of powdery mildew haustorial effectors, a sharp decline in the photosynthetic machinery of barley and a developmental peak in P. flocculosa. The interaction culminates with a collapse of B. graminis haustoria, thereby stopping P. flocculosa growth, as barley plants show higher metabolic activity. To conclude, our study has uncovered a complex and intricate phenomenon, described here as hyperbiotrophy, only achievable through the conjugated action of the three protagonists.

Missense mutations in progranulin gene associated with frontotemporal lobar degeneration: study of pathogenetic features.

GRN, the gene coding for the progranulin (PGRN) protein, was recognized as a gene linked to frontotemporal lobar degeneration (FTLD). The first mutations identified were null mutations giving rise to haploinsufficiency. Missense mutations were subsequently detected, but only a small subset has been functionally investigated. We identified missense mutations (C105Y, A199V, and R298H) in FTLD cases with family history and/or with low plasma PGRN levels. The aim of this study was to determine their pathogenicity. We performed functional studies, analyzing PGRN expression, secretion, and cleavage by elastase. GRN C105Y affected both secretion and elastase cleavage, likely representing a pathogenic mutation. GRN A199V did not alter the physiological properties of PGRN and GRN R298H produced only moderate effects on PGRN secretion, indicating that their pathogenicity is uncertain. In the absence of strong segregation data and neuropathological examinations, genetic, biomarker, and functional studies can be applied to an algorithm to assess the likelihood of pathogenicity for a mutation. This information can improve our understanding of the complex mechanisms by which GRN mutations lead to FTLD.

Useful approach to the synthesis of aryl thio- and selenoglycosides in the presence of rongalite.

A simple, mild, and cost effective methodology has been developed for the synthesis of aryl thio-and selenoglycosides from glycosyl halides and diaryl dichalcogenides. Diaryl dichalcogenides undergo reductive cleavage in the presence of rongalite (HOCH2SO2Na) to generate a chalcogenide anion in situ followed by reaction with glycosyl halides to furnish the corresponding aryl thio- and selenoglycosides in excellent yields. Using this protocol, synthesis of 4-methyl-7-thioumbelliferyl-ß-D-cellobioside (MUS-CB), a fluorescent non-hydrolyzable substrate analogue for cellulases has been achieved.

Supramolecular stacking motifs in the solid state of amide and triazole derivatives of cellobiose.

1-Acetamido-1-deoxy-(4-O-ß-d-glucopyranosyl-ß-d-glucopyranose) (5) and 1-deoxy-1-(4-phenyl-1,2,3-triazolyl)-(4-O-ß-d-glucopyranosyl-ß-d-glucopyranose) (7) were synthesised from 1-azido-1-deoxy-(4-O-ß-d-glucopyranosyl-ß-d-glucopyranose) (2) and crystallised as dihydrates. Crystal structural analysis of 5·2H2O displayed an acetamide C(4) chain and stacked cellobiose residues. The structure of 7·2H2O featured π-π stacking and stacking of the cellobiose residues.

Synthesis of quercetin glycosides and their melanogenesis stimulatory activity in B16 melanoma cells.

4'-O-ß-d-Glucopyranosyl-quercetin-3-O-ß-d-glucopyranosyl-(1→4)-ß-d-glucopyra-noside (3) was isolated from Helminthostachys zeylanica root extract as a melanogenesis acceleration compound and was synthesized using rutin as the starting material. Related compounds were also synthesized to understand the structure-activity relationships in melanin biosynthesis. Melanogenesis activities of the glycosides were determined by measuring intracellular melanin content in B16 melanoma cells. Among the synthesized quercetin glycosides, quercetin-3-O-ß-d-glucopyranoside (1), quercetin-3-O-ß-d-glucopyranosyl-(1→4)-ß-d-glucopyranoside (2), and 3 showed more potent intracellular melanogenesis acceleration activities than theophyline used as positive control in a dose-dependent manner with no cytotoxic effect.

Synthesis of bis-cellobiose and bis-glucose derivatives of azacrown macrocycles as hosts in complexes with acetylsalicylic acid and 4-acetamidophenol.

Two new C2 symmetric bis-cellobiose and bis-glucose azacrown derivatives were prepared according to the one-step procedure using azacrown ethers and azidosaccharides. Their complexes with aspirin and paracetamol were studied with the use of proton NMR spectroscopy. It was found that these pseudocryptands bind aspirin and paracetamol but each one in a different manner.

Rational design, synthesis, evaluation and enzyme-substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases.

Methylumbelliferyl-ß-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain ß-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF-G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-ß-cellobiosides [6-chloro-4-methyl- (ClMUF); 6-chloro-4-trifluoromethyl- (ClF3MUF); 4-phenyl- (PhUF); 6-chloro-4-phenyl- (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10-150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of k(cat) and K(M) for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF- and ClPhUF-ß-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF-G2.

Redefining XynA from Penicillium funiculosum IMI 378536 as a GH7 cellobiohydrolase.

The secretome of Penicillium funiculosum contains two family GH7 enzymes, one of which (designated XynA) has been described as a xylanase. This is unusual because it is the only xylanase in family GH7, which is mainly composed of cellobiohydrolases and endoglucanases, and also because XynA is highly similar to the cellobiohydrolase I from Talaromyces emersonii and Trichoderma reesei (72 and 65 % identity, respectively). To probe this enigma, we investigated the biochemical properties of XynA, notably its activity on xylans and ß-D-glucans. A highly pure sample of XynA was obtained and used to perform hydrolysis tests on polysaccharides. These revealed that XynA is 100-fold more active on ß-1,4-glucan than on xylan. Likewise, XynA was active on both 4-nitrophenyl-ß-D-lactopyranoside (pNP-ß-D-Lac) and 4-nitrophenyl-ß-D-cellobioside (pNP-cellobiose), which shows that XynA is principally an exo-acting type 1 cellobiohydrolase enzyme that displays 5.2-fold higher performance on pNP-cellobiose than on pNP-ß-D-Lac. Finally, analyses performed using cellodextrins as substrate revealed that XynA mainly produced cellobiose (C2) from substrates containing three or more glucosyl subunits, and that C2 inhibits XynA at high concentrations (IC(50) (C2) = 17.7 µM). Overall, this study revealed that XynA displays typical cellobiohydrolase 1 activity and confirms that the description of this enzyme in public databases should be definitively amended. Moreover, the data provided here complete the information provided by a previous proteomics investigation and reveal that P. funiculosum secretes a complete set of cellulose-degrading enzymes.

Validity of particle size analysis techniques for measurement of the attrition that occurs during vacuum agitated powder drying of needle-shaped particles.

Analysis of needle-shaped particles of cellobiose octaacetate (COA) obtained from vacuum agitated drying experiments was performed using three particle size analysis techniques: laser diffraction (LD), focused beam reflectance measurements (FBRM) and dynamic image analysis. Comparative measurements were also made for various size fractions of granular particles of microcrystalline cellulose. The study demonstrated that the light scattering particle size methods (LD and FBRM) can be used qualitatively to study the attrition that occurs during drying of needle-shaped particles, however, for full quantitative analysis, image analysis is required. The algorithm used in analysis of LD data assumes the scattering particles are spherical regardless of the actual shape of the particles under evaluation. FBRM measures a chord length distribution (CLD) rather than the particle size distribution (PSD), which in the case of needles is weighted towards the needle width rather than their length. Dynamic image analysis allowed evaluation of the particles based on attributes of the needles such as length (e.g. the maximum Feret diameter) or width (e.g. the minimum Feret diameter) and as such, was the most informative of the techniques for the analysis of attrition that occurred during drying.

Beta hydroxylation of glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-dependent ß-hydroxylase.

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the ß position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the ß position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the ß-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for ß hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of ß-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.

Studies of particle drying using non-invasive Raman spectrometry and particle size analysis.

The evaporation of methanol from needle-shaped particles of cellobiose octaacetate (COA) has been studied directly in a jacketed vacuum drier using in situ measurements by Raman spectrometry. A design of experiments (DoE) approach was used to investigate the effects of three parameters (method of agitation, % solvent loss on drying and jacket temperature), with the intention of minimising the drying time and extent of particle attrition. Drying curves based on Raman signals for methanol and COA in the spectra of the wet particles indicated the end of drying and revealed three stages in the drying process that could be used to monitor the progress of solvent removal in real time. Off-line particle size measurements based on laser diffraction were made to obtain information on the extent of attrition, to compare with the trends revealed by the Raman drying curves. The study demonstrated that non-invasive Raman spectrometry can be used to study the progress of drying during agitation of particles in a vacuum drier, allowing optimisation of operating conditions to minimise attrition and reduce drying times. Although a correlation between particle size and off-line Raman measurements of COA was demonstrated, it was not possible to derive equivalent information from the in situ Raman spectra owing to the greater effects of particle motion or bulk density variations of the particles in the drier.

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa.

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O-glycosidically linked to 3,15,16-trihydroxypalmitic acid. The sugar moiety is acylated with 2-hydroxy-octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl-transferase and is lacking a gene homologous to the α-hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl-transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl-transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.

NMR spectroscopic studies of cellobiose solvation in EmimAc aimed to understand the dissolution mechanism of cellulose in ionic liquids.

The dissolution mechanism of cellulose in ionic liquids has been investigated by using cellobiose and 1-ethyl-3-methylimidazolium acetate (EmimAc) as a model system under various conditions with conventional and variable-temperature NMR spectroscopy. In DMSO-d(6) solution, NMR data of the model system clearly suggest that hydrogen bonding is formed between hydroxyls of cellobiose and both anion and cation of EmimAc. The CH(3)COO(-) anion favors the formation of hydrogen bonds with hydrogen atoms of hydroxyls, and the aromatic protons in bulky cation [Emim](+), especially the most acidic H2, prefer to associate with the oxygen atoms of hydroxyls with less steric hindrance, while after acetylation of all hydroxyls in cellobiose the interactions between cellobiose octaacetate and EmimAc become very weak, implying that hydrogen bonding is the major reason of cellobiose solvation in EmimAc. Meanwhile the stoichiometric ratio of EmimAc/hydroxyl is estimated to be between 3:4 and 1:1 in the primary solvation shell, suggesting that there should be one anion or cation to form hydrogen bonds with two hydroxyl groups simultaneously. In situ and variable-temperature NMR spectra suggest the above mechanism also works in the real system.