Antibacterial Compounds from Mushrooms: A Lead to Fight ESKAPEE Pathogenic Bacteria?

Infectious diseases are among the greatest threats to global health in the 21st century, and one critical concern is due to antibiotic resistance developed by an increasing number of bacterial strains. New resistance mechanisms are emerging with many infections becoming more and more difficult if not impossible to treat. This growing phenomenon not only is associated with increased mortality but also with longer hospital stays and higher medical costs. For these reasons, there is an urgent need to find new antibiotics targeting pathogenic microorganisms such as ESKAPEE bacteria. Most of currently approved antibiotics are derived from microorganisms, but higher fungi could constitute an alternative and remarkable reservoir of anti-infectious compounds. For instance, pleuromutilins constitute the first class of antibiotics derived from mushrooms. However, macromycetes still represent a largely unexplored source. Publications reporting the antibacterial potential of mushroom extracts are emerging, but few purified compounds have been evaluated for their bioactivity on pathogenic bacterial strains. Therefore, the aim of this review is to compile up-to-date data about natural products isolated from fruiting body fungi, which significantly inhibit the growth of ESKAPEE pathogenic bacteria. When available, data regarding modes of action and cytotoxicity, mandatory when considering a possible drug development, have been discussed in order to highlight the most promising compounds.

Cell geometry determines symmetric and asymmetric division plane selection in Arabidopsis early embryos.

Plant tissue architecture and organ morphogenesis rely on the proper orientation of cell divisions. Previous attempts to predict division planes from cell geometry in plants mostly focused on 2D symmetric divisions. Using the stereotyped division patterns of Arabidopsis thaliana early embryogenesis, we investigated geometrical principles underlying plane selection in symmetric and in asymmetric divisions within complex 3D cell shapes. Introducing a 3D computational model of cell division, we show that area minimization constrained on passing through the cell centroid predicts observed divisions. Our results suggest that the positioning of division planes ensues from cell geometry and gives rise to spatially organized cell types with stereotyped shapes, thus underlining the role of self-organization in the developing architecture of the embryo. Our data further suggested the rule could be interpreted as surface minimization constrained by the nucleus position, which was validated using live imaging of cell divisions in the stomatal cell lineage.

Pregnane glycosides from Cynanchum menarandrense.

Five new pregnane-type steroidal glycosides, named menarandrosides A-E (1-2, 5-7) were isolated from the aerial parts of Cynanchum menarandrense, together with three known compounds, carumbelloside I (3), carumbelloside II (4), and pregnenolone-3-O-gentiobioside (8). Their structures were determined on the basis of spectroscopic analyses including NMR and mass spectrometry, reporting C-21 steroids glycosylated only by one or two glucose moieties. Compounds were then investigated for their potential to stimulate glucagon-like peptide-1 (GLP-1) secretion in intestinal cells; although none of the pure compounds had any influence, the fraction enriched in pregnanes exhibited a significant activity, suggesting a possible synergistic effect. Furthermore, none of the purified compounds affected cell viability.

Pregnane Glycosides from Cynanchum marnierianum Stimulate GLP-1 Secretion in STC-1 Cells.

In the framework of the search for natural glucagon-like peptide-1 secretagogues, the bioassay-guided fractionation of the ethanolic extract from Cynanchum marnierianum led to the isolation of two new pregnane glycosides named marnieranosides A (1) and B (2). The structures were determined based on spectroscopic data and were established as 12ß,20 S-O-dibenzoyl-pregn-6-en-5α,8ß,14ß,17ß-tetraol-3-O-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranoside (1) and 12ß,20R-O-dibenzoyl-pregn-6-en-5α,8ß,14ß-triol-3-O-ß-D-oleandropyranosyl-(1 → 4)-ß-D-canaropyranosyl-(1 → 4)-ß-D-cymaropyranoside (2). They present structural analogies to pregnanes previously described in species known for their appetite suppressant and antihyperglycemic effects, such as P57 from Hoodia gordonii. Lupeol (3), a known dipeptidyl peptidase-4 inhibitor, and the insulinomimetic kaempferol-3-O-neohesperidoside (4) were also identified in C. marnierianum. In an in vitro assay on secretin tumor cell line-1 cells, compounds 1, 2, and P57 were found to stimulate the secretion of GLP-1 by 130 % (all tested at 100 µM). These results suggest that C. marnierianum could be of great interest in the treatment of type 2 diabetes, and that pregnane derivatives should be partly responsible via the stimulation of glucagon-like peptide-1 secretion.

Purification of vandaterosides from Vanda teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography.

Vandaterosides are polar glucosyloxybenzyl eucomate derivatives found in Vanda teres (Orchidaceae), which display biological activities that slow the skin ageing process. In order to obtain larger quantities to allow us to go further in the bioassays, the hydroalcoholic extract of aerial parts (leaves and stems) of V. teres were fractionated by centrifugal partition chromatography, combining isocratic, gradient, and dual elution modes. The first fractionation was performed on the extract maintained in the stationary phase as water saturated in butanol, while increasing the polarity of the mobile phase by changing the proportions of ethyl acetate/1-butanol/water, in order to obtain two enriched fractions. Vandateroside I was then purified by isocratic mode with ethyl acetate/ethanol/water (46:14:40), while vandateroside II was obtained by combining isocratic elution with ethyl acetate/isopropanol/water (30:20:50) followed by a multiple dual mode with ethyl acetate/ethanol/water (46:14:40). In this manner, hundreds of milligrams of vandateroside I and II were recovered from 10 g of V. teres extract.

Sequence-structure relationship study in all-α transmembrane proteins using an unsupervised learning approach.

Transmembrane proteins (TMPs) are major drug targets, but the knowledge of their precise topology structure remains highly limited compared with globular proteins. In spite of the difficulties in obtaining their structures, an important effort has been made these last years to increase their number from an experimental and computational point of view. In view of this emerging challenge, the development of computational methods to extract knowledge from these data is crucial for the better understanding of their functions and in improving the quality of structural models. Here, we revisit an efficient unsupervised learning procedure, called Hybrid Protein Model (HPM), which is applied to the analysis of transmembrane proteins belonging to the all-α structural class. HPM method is an original classification procedure that efficiently combines sequence and structure learning. The procedure was initially applied to the analysis of globular proteins. In the present case, HPM classifies a set of overlapping protein fragments, extracted from a non-redundant databank of TMP 3D structure. After fine-tuning of the learning parameters, the optimal classification results in 65 clusters. They represent at best similar relationships between sequence and local structure properties of TMPs. Interestingly, HPM distinguishes among the resulting clusters two helical regions with distinct hydrophobic patterns. This underlines the complexity of the topology of these proteins. The HPM classification enlightens unusual relationship between amino acids in TMP fragments, which can be useful to elaborate new amino acids substitution matrices. Finally, two challenging applications are described: the first one aims at annotating protein functions (channel or not), the second one intends to assess the quality of the structures (X-ray or models) via a new scoring function deduced from the HPM classification.

Identification of phenanthrene derivatives in Aerides rosea (Orchidaceae) using the combined systems HPLC-ESI-HRMS/MS and HPLC-DAD-MS-SPE-UV-NMR.

INTRODUCTION: In our continued efforts to contribute to the general knowledge on the chemical diversity of orchids, we have decided to focus our investigations on the Aeridinae subtribe. Following our previous phytochemical study of Vanda coerulea, which has led to the identification of phenanthrene derivatives, a closely related species, Aerides rosea Lodd. ex Lindl. & Paxton, was chosen for investigation. OBJECTIVE: To identify new secondary metabolites, and to avoid isolation of those already known, by means of the combined systems HPLC-DAD(diode-array detector) with high-resolution tandem mass spectrometry (HRMS/MS) and HPLC-DAD-MS-SPE(solid-phase extraction)-UV-NMR. METHODS: A dereplication strategy was developed using a HPLC-DAD-HRMS/MS targeted method and applied to fractions from A. rosea stem extract. Characterisation of unknown minor compounds was then performed using the combined HPLC-DAD-MS-SPE-UV-NMR system. RESULTS: The dereplication method allowed the characterisation of four compounds (gigantol, imbricatin, methoxycoelonin and coelonin), previously isolated from Vanda coerulea stem extract. The analyses of two fractions permitted the identification of five additional minor constituents including one phenanthropyran, two phenanthrene and two dihydrophenanthrene derivatives. The full set of NMR data of each compound was obtained from microgram quantities. CONCLUSION: Nine secondary metabolites were characterised in A. rosea stems, utilising HPLC systems combined with high-resolution analytical systems. Two of them are newly described phenanthrene derivatives: aerosanthrene (5-methoxyphenanthrene-2,3,7-triol) and aerosin (3-methoxy-9,10-dihydro-2,5,7-phenanthrenetriol).

Quercetin and kaempferol 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]-7-O-α-L-rhamnopyranosides from Anthyllis hermanniae: structure determination and conformational studies.

The study reports the isolation and structural identification of two new flavonol triglycosides from the methanolic extract of Anthyllis hermanniae, exhibiting the same glycosylation pattern: quercetin 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]-7-O-α-L-rhamnopyranoside (1) and kaempferol 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]-7-O-α-L-rhamnopyranoside (2). A conformational study related to the central arabinoside moiety was carried out including the analysis of the contribution of NOE effects and acetylation to the elucidation of the 2-O-linked arabinoside configuration of the anomeric carbon. We also report the total synthesis of a model compound, quercetin 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (3), which verifies the structures of the isolated compounds.

One-step isolation of γ-oryzanol from rice bran oil by non-aqueous hydrostatic countercurrent chromatography.

The value-added γ-oryzanol was purified in one step from crude rice bran oil (RBO) using a preparative hydrostatic countercurrent chromatography (hydrostatic CCC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane-acetonitrile-butanol (1.8:1.4:0.7, v/v/v), leading rapidly to the target compounds. Transfer of the analytical CCC method to large-scale isolation was also carried out yielding a high quantity-high purity fraction of γ-oryzanol. In addition, a fraction of hydroxylated triterpene alcohol ferulates (polar γ-oryzanol) was clearly separated and obtained. Furthermore, a fast HPLC-APCI(±)-HRMS method was developed and applied for the identification of γ-oryzanol as well as the polar γ-oryzanol in RBO and the resulting fractions. The purity of γ-oryzanol fraction was estimated as 97% based on HPLC-APCI-HRMS analysis.

A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.

• We focused on a developmentally regulated growth acceleration in the dark-grown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. • To this end, precise transcriptome analysis on dissected dark-grown hypocotyls, Fourier transform infrared (FT-IR) microspectroscopy and kinematic analysis were used. • Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT-IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. • This study shows that the acceleration of cell elongation marks a developmental transition in dark-grown hypocotyl cells and supports a role for pectin de-methylesterification in the timing of this event.

High nitrogen fertilization and stem leaning have overlapping effects on wood formation in poplar but invoke largely distinct molecular pathways.

Previous studies indicated that high nitrogen fertilization may impact secondary xylem development and alter fibre anatomy and composition. The resulting wood shares some resemblance with tension wood, which has much thicker cell walls than normal wood due to the deposition of an additional layer known as the G-layer. This report compares the short-term effects of high nitrogen fertilization and tree leaning to induce tension wood, either alone or in combination, upon wood formation in young trees of Populus trichocarpa (Torr. & Gray) × P. deltoides Bartr. ex Marsh. Fibre anatomy, chemical composition and transcript profiles were examined in newly formed secondary xylem. Each of the treatments resulted in thicker cell walls relative to the controls. High nitrogen and tree leaning had overlapping effects on chemical composition based on Fourier transform infrared analysis, specifically indicating that secondary cell wall composition was shifted in favour of cellulose and hemicelluloses relative to lignin content. In contrast, the high-nitrogen trees had shorter fibres, whilst the leaning trees had longer fibres that the controls. Microarray transcript profiling carried out after 28 days of treatment identified 180 transcripts that accumulated differentially in one or more treatments. Only 10% of differentially expressed transcripts were affected in all treatments relative to the controls. Several of the affected transcripts were related to carbohydrate metabolism, secondary cell wall formation, nitrogen metabolism and osmotic stress. RT-qPCR analyses at 1, 7 and 28 days showed that several transcripts followed very different accumulation profiles in terms of rate and level of accumulation, depending on the treatment. Our findings suggest that high nitrogen fertilization and tension wood induction elicit largely distinct and molecular pathways with partial overlap. When combined, the two types of environmental cue yielded additive effects.

TRICHOME BIREFRINGENCE and its homolog AT5G01360 encode plant-specific DUF231 proteins required for cellulose biosynthesis in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) trichome birefringence (tbr) mutant has severely reduced crystalline cellulose in trichomes, but the molecular nature of TBR was unknown. We determined TBR to belong to the plant-specific DUF231 domain gene family comprising 46 members of unknown function in Arabidopsis. The genes harbor another plant-specific domain, called the TBL domain, which contains a conserved GDSL motif known from some esterases/lipases. TBR and TBR-like3 (TBL3) are transcriptionally coordinated with primary and secondary CELLULOSE SYNTHASE (CESA) genes, respectively. The tbr and tbl3 mutants hold lower levels of crystalline cellulose and have altered pectin composition in trichomes and stems, respectively, tissues generally thought to contain mainly secondary wall crystalline cellulose. In contrast, primary wall cellulose levels remain unchanged in both mutants as measured in etiolated tbr and tbl3 hypocotyls, while the amount of esterified pectins is reduced and pectin methylesterase activity is increased in this tissue. Furthermore, etiolated tbr hypocotyls have reduced length with swollen epidermal cells, a phenotype characteristic for primary cesa mutants or the wild type treated with cellulose synthesis inhibitors. Taken together, we show that two TBL genes contribute to the synthesis and deposition of secondary wall cellulose, presumably by influencing the esterification state of pectic polymers.

Hydrostatic countercurrent chromatography and ultra high pressure LC: Two fast complementary separation methods for the preparative isolation and the analysis of the fragrant massoia lactones.

Using a one-step preparative hydrostatic countercurrent chromatography method, the fragrant massoia lactones were purified from the crude massoia bark oil, in less than 3 h. The fractionation was performed with the biphasic solvent system c-hexane-methanol-water (10:9:1, v/v/v), leading to target compounds with purity over 96%, as determined by GC-MS and ultra high pressure LC-MS analyses. Together with C-10, C-12 and C-14 massoia lactones, two other aromatic compounds used in perfumes, benzyl benzoate and benzyl salicylate, were also obtained as pure compounds. In parallel, an easy and efficient ultra high pressure LC method was developed for the ultra-fast analysis of massoia lactones, as an alternative to long GC-MS methods.

Ultra-performance liquid chromatography/time-of-flight mass spectrometry as a chemotaxonomic tool for the analysis of Gentianaceae species.

INTRODUCTION: The segregation between the genera Gentiana and Gentianella among the Gentianaceae family is poorly defined. In order to clarify the classification of these genera, some researchers have tried to incorporate data about the chemical constitution, but this has not yet been achieved in a comprehensive way. OBJECTIVE: To develop a fast and reproducible analytical method for the observation of characteristic fingerprints of secondary metabolites of each genus. METHODOLOGY: Seven species were investigated, three Gentianella and four Gentiana selected for their close taxonomic links within each genus. Ten xanthones previously isolated from one of these species were used as chemotaxonomic markers. A UPLC/ESI-TOF-MS method was developed to analyse the methanolic extracts. RESULTS: The UPLC/TOF-MS provided clear metabolic fingerprints and elemental composition of the compounds. The profiles of the three Gentianella species were strikingly similar. On the contrary, metabolic profiles of Gentiana species were very different from the Gentianella chromatograms and also from each other. Several compounds were unique to each genus and therefore could be used as biomarkers. CONCLUSION: UPLC/TOF-MS can be applied as a chemotaxonomic tool for the rapid screening of Gentianaceae species and for the distinction between closely related taxa from the Gentianaceae family.

Preparative isolation of closely-related xanthones from Gentianella amarella ssp. acuta by High-speed countercurrent chromatography.

INTRODUCTION: A methanolic extract from Gentianella amarella ssp. acuta was shown to contain several xanthones exhibiting acetylcholinesterase inhibitory activity. These xanthones were difficult to separate by conventional LC techniques, which prevented the isolation of pure compounds in sufficient amounts to perform in-depth biological testing. OBJECTIVE: To develop a suitable preparative method for the separation of closely related xanthones. METHODOLOGY: The methanolic extract was first partitioned with solvents of increasing polarity, in order to separate glycosides from xanthone aglycones. High-speed countercurrent chromatography (HSCCC) methods were then optimised for the fractionation of both polar and non-polar extracts. RESULTS: The use of HSCCC enabled the separation of xanthones which co-eluted by HPLC. Ten closely related xanthones--three of which were isomeric--were successfully isolated by developing suitable solvent systems. All compounds were obtained in sufficient amounts to allow further biological assays (e.g. up to 250 mg), including even minor compounds that were not detectable by analytical HPLC. CONCLUSION: The orthogonality of HSCCC with HPLC and the absence of solid-phase supports enabled the detection, separation and preparative isolation of closely related compounds which were difficult to resolve by other techniques.

In vitro screening assays to identify natural or synthetic acetylcholinesterase inhibitors: thin layer chromatography versus microplate methods.

Acetylcholinesterase inhibitors (AChEI) are currently still the best available pharmacotherapy for Alzheimer patients. Successful screening for new AChEI relies on effective and fast assays. Two colorimetric screening assays frequently used to search for new AChEI, namely a thin layer chromatography (TLC) assay with Fast Blue B salt as reagent and a 96-well plate assay based on Ellman's method, were compared. For the majority (83%) of the 138 test compounds of natural and synthetic origin, the results obtained with the two assays converged and both screening assays were considered suitable for the generation of new hits. Fifteen percent of investigated compounds were classified as active with the microplate assay but were shown to be inactive by TLC and about 2% were measured active by TLC but showed to be inactive with the microplate assay. These divergences were not due to the main differences between the experimental protocols of the two screening assays, namely the different colorimetric methods and pre-incubation of test compounds with acetylcholinesterase (AChE). They might be explained by the interaction of either AChE or test compounds with the silica of the TLC plates, resulting in an altered affinity of the enzyme for the compounds.

Xanthones from Gentiana campestris as new acetylcholinesterase inhibitors.

In order to discover new acetylcholinesterase (AChE) inhibitors, different plant extracts were screened by a previously established TLC bioautographic method. The methanol extract of Gentiana campestris leaves exhibited significant inhibition of AChE activity. A bioactivity-guided fractionation approach was undertaken to isolate the active components. Four xanthones, bellidin, bellidifolin, bellidin 8-O-beta-glucopyranoside (norswertianolin), and bellidifolin 8-O-beta-glucopyranoside (swertianolin), were found to be responsible for the anti-AChE activity effects. Bellidifolin showed similar activity to galanthamine in this enzyme assay.