Generation of Antifungal Stilbenes Using the Enzymatic Secretome of Botrytis cinerea.

The protein secretome of Botrytis cinerea was used to perform the biotransformation of resveratrol, pterostilbene, and a mixture of both. Metabolite profiling by UHPLC-HRMS revealed the presence of compounds with unusual molecular formula, suggesting the existence of new products. To isolate these products, the reactions were scaled-up, and 21 analogues were isolated and fully characterized by NMR and HRESIMS analyses. The reaction with pterostilbene afforded five new compounds, while the reaction with a mixture of pterostilbene and resveratrol afforded seven unusual stilbene dimers. The antifungal properties of these compounds were evaluated using in vitro bioassays against Plasmopara viticola. The cytological effects of the isolated antifungal compounds on the ultrastructure of P. viticola were also evaluated.

Antifungal Quinoline Alkaloids from Waltheria indica.

Chemical investigation of a dichloromethane extract of the aerial parts of Waltheria indica led to the isolation and characterization of five polyhydroxymethoxyflavonoids, namely, oxyanin A (1), vitexicarpin (3), chrysosplenol E (4), flindulatin (5), 5-hydroxy-3,7,4'-trimethoxyflavone (6), and six quinolone alkaloids, waltheriones M-Q (2, 7, 8, 10, 11) and 5(R)-vanessine (9). Among these, compounds 2, 7, 8, 10, and 11 have not yet been described in the literature. Their chemical structures were established by means of spectroscopic data interpretation including (1)H and (13)C, HSQC, HMBC, COSY, and NOESY NMR experiments and UV, IR, and HRESIMS. The absolute configurations of the compounds were established by ECD. The isolated constituents and 10 additional quinoline alkaloids previously isolated from the roots of the plant were evaluated for their in vitro antifungal activity against the human fungal pathogen Candida albicans, and 10 compounds (7, 9, 11-16, 18, 21) showed growth inhibitory activity on both planktonic cells and biofilms (MIC ≤ 32 µg/mL). Their spectrum of activity against other pathogenic Candida species and their cytotoxicity against human HeLa cells were also determined. In addition, the cytological effect of the antifungal isolated compounds on the ultrastructure of C. albicans was evaluated by transmission electron microscopy.

Anti-Candida Cassane-Type Diterpenoids from the Root Bark of Swartzia simplex.

A dichloromethane extract of the roots from the Panamanian plant Swartzia simplex exhibited a strong antifungal activity in a bioautography assay against a genetically modified hypersusceptible strain of Candida albicans. At-line HPLC activity based profiling of the crude extract enabled a precise localization of the antifungal compounds, and dereplication by UHPLC-HRESIMS indicated the presence of potentially new metabolites. Transposition of the HPLC reversed-phase analytical conditions to medium-pressure liquid chromatography (MPLC) allowed an efficient isolation of the major constituents. Minor compounds of interest were isolated from the MPLC fractions using semipreparative HPLC. Using this strategy, 14 diterpenes (1-14) were isolated, with seven (5-10, 14) being new antifungal natural products. The new structures were elucidated using NMR spectroscopy and HRESIMS analysis. The absolute configurations of some of the compounds were elucidated by electronic circular dichroism spectroscopy. The antifungal properties of these compounds were evaluated as their minimum inhibitory concentrations in a dilution assay against both hypersusceptible and wild-type strains of C. albicans and by assessment of their antibiofilm activities. The potential cytological effects on the ultrastructure of C. albicans of the antifungal compounds isolated were evaluated on thin sections by transmission electron microscopy.

Changes in leaf stomatal conductance, petiole hydraulics and vessel morphology in grapevine (Vitis vinifera cv. Chasselas) under different light and irrigation regimes.

Hydraulic conductance and water transport in plants may be affected by environmental factors, which in turn regulate leaf gas exchange, plant growth and yield. In this study, we assessed the combined effects of radiation and water regimes on leaf stomatal conductance (gs), petiole specific hydraulic conductivity (Kpetiole) and anatomy (vessel number and size); and leaf aquaporin gene expression of field-grown grapevines at the Agroscope Research Station (Leytron, Switzerland). Chasselas vines were subjected to two radiation (sun and shade) levels combined with two water (irrigated and water-stressed) regimes. The sun and shade leaves received ~61.2 and 1.48molm-2day-1 of photosynthetically active radiation, respectively, during a clear-sky day. The irrigated vines were watered weekly from bloom to veraison whereas the water-stressed vines did not receive any irrigation during the season. Water stress reduced gs and Kpetiole relative to irrigated vines throughout the season. The petioles from water-stressed vines showed fewer large-sized vessels than those from irrigated vines. The shaded leaves from the irrigated vines exhibited a higher Kpetiole than the sun leaves at the end of the season, which was partially explained by a higher number of vessels per petiole and possibly by the upregulation of some of the aquaporins measured in the leaf. These results suggest that not only plant water status but also the light environment at the leaf level affected leaf and petiole hydraulics.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

Vitis vinifera canes, a new source of antifungal compounds against Plasmopara viticola, Erysiphe necator, and Botrytis cinerea.

Methanolic and ethanolic crude extracts of Vitis vinifera canes exhibited significant antifungal activity against the three major fungal pathogens affecting grapevines, Plasmopara viticola, Erysiphe necator and Botrytis cinerea. The active extracts were analyzed by LC-PDA-ESI-MS, and selected compounds were identified. Efficient targeted isolation using medium-pressure liquid chromatography afforded six pure constituents in one step. The structures of the isolated compounds were elucidated by NMR and HRMS. Six identified compounds (ampelopsin A, hopeaphenol, trans-resveratrol, ampelopsin H, ε-viniferin, and E-vitisin B) presented antifungal activities against P. viticola. ε-Viniferin also exhibited a low antifungal activity against B. cinerea. None of the identified compounds inhibited the germination of E. necator. The potential to develop a novel natural fungicide against the three major fungal pathogens affecting V. vinifera from viticulture waste material is discussed.

Protease inhibitors decrease the resistance of Vitaceae to Plasmopara viticola.

Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll).

Susceptibility to downy mildew in grape clusters: New microscopical and biochemical insights.

Grape clusters of different Vitis genotypes, including Vitis vinifera cvs Chasselas and Merlot, and two interspecific grape varieties, Solaris (cvs. Merzling* x (Saperavi severneyi x Muscat ottonel)) and 2091 (cvs. Gamaret x Bronner), are susceptible or resistant to downy mildew. These cultivars were inoculated with Plasmopara viticola at three developmental stages (BBCH stages 53, 69 and 75). Samples were examined by scanning electron microscopy and the synthesis of stilbenes was measured. Microscopical examinations of pedicels, rachis and calyptras showed important differences in stomatal structures within seasonal development. At BBCH 53, successful infections were observed on all tested cultivars and functional stomata were present, while no infections were observed after this stage. At BBCH 69 and 75, cracks were observed around the stomata and guard cells were unstructured or completely collapsed, leading to closed-like stomata. At BBCH 53, significant stilbene accumulation was quantified in 2091 and Solaris; pterostilbene and δ-viniferin were produced in large amounts. In the susceptible varieties, only piceid and resveratrol were induced. At the other two stages, the concentration of all measured stilbenes was undetectable. The critical roles of seasonal development and stilbenes in the resistance of grape clusters towards downy mildew are discussed.

The effectiveness of stilbenes in resistant Vitaceae: ultrastructural and biochemical events during Plasmopara viticola infection process.

Leaves of different Vitis vinifera L. cultivars, susceptible or resistant to downy mildew, Chasselas, Solaris, IRAC 2091 (cvs. Gamaret x Bronner) and Muscadinia rotundifolia were inoculated with Plasmopara viticola. Samples were then examined by scanning and transmission electron microscopy, by light microscopy and for their ability to synthesise stilbenes. These phytoalexins were strictly analysed at infection sites. In the susceptible Chasselas, P. viticola colonises, at 72h post-infection (hpi), all of the spongy mesophyll with functional haustoria and produces mainly the non toxic piceide. No necrotic zone was observed on Chasselas leaves. The ultrastructural response to downy mildew infection is different in each of the other three resistant grape cultivars. In Solaris, where leaf necrosis are rapidly induced, the infection is restricted to the upper part of the loose spongy mesophyll, and associated with a rapid cell wall disruption and the dispersion of cytoplasmic content along with the production of viniferins. In IRAC 2091, leaf necrosis are quite similar to those observed on Solaris but the infected plant cell, as well as the haustoria, show high electron dense cellular particles without any recognisable organelles, probably related to the effect of the toxic compound pterostilbene, which is synthesised in this grape cultivar. In M. rotundifolia leaf necrosis are much more scarce and smaller than in other cultivars, but pathogen and plant cells are both strongly affected, with concomitant expulsion of cytoplasmic materials through the stomata after P. viticola penetration. In this cultivar, the concentration of all identified stilbenes exceeds 1×10³ µmol mg(-1) FW. The critical role of stilbenes in the resistance of Vitis spp. is discussed.

Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are currently under development for the intracellular delivery of therapeutics. However, the mechanisms of cellular uptake and the cellular reaction to this uptake, independent of therapeutics, are not well defined. The interactions of biocompatible cationic aminoUSPIONs with human cells was studied in 2D and 3D cultures using biochemical and electron microscopy techniques. AminoUSPIONs were internalized by human melanoma cells in 2D and 3D cultures. Uptake was clathrin mediated and the particles localized in lysosomes, inducing activation of the lysosomal cathepsin D and decreasing the expression of the transferrin receptor in human melanoma cells and/or skin fibroblasts. AminoUSPIONs deeply invaded 3D spheroids of human melanoma cells. Thus, aminoUSPIONs can invade tumors and their uptake by human cells induces cell reaction.