Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H 2 O 2 , followed by differential expression of nine defense-related genes and stimulation of chitinase and b-1,3-glucanase activities. Inhibition of OGAinduced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and b-1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55-65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.

We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-XL and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-XL expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations.

The fungal pathogen Botrytis cinerea is capable of developing on a wide variety of host plants that differ greatly in their pH values and biochemical defences. To evaluate whether the pH of the host tissue can regulate the production of pathogenicity factors by this fungus, we examined the ability of two isolates of B. cinerea that originated from different plant species to secrete putative virulence elements on synthetic media buffered at pH 2.0 to pH 7.0. Even though differing in the intensity of their responses, both isolates reacted similarly to their ambient pH. The production of extracellular polysaccharides and oxalic acid was detectable above pH 4.0 and pH 5.0 respectively. Conversely, the production of aspartic acid proteases could only be seen between pH 3.0 and 4.0. Finally, the secretion of polygalacturonase and laccase activity was found to exhibit two maxima, one around pH 3.1 and one around pH 6.0. Thus, pathogenicity factor production was found to be minimal between pH 4.5 and 5.5 and a different set of factors was produced at pH 3.1 and 6.0, two values that were found to correspond respectively to the average host fruit and leaf pH. These results demonstrate that ambient pH differentially regulates the synthesis of pathogenicity factors by Botrytis and may act as a novel regulatory element to assist this fungus in tuning its virulence machinery to the composition of its host tissue.

The lanthanide and Th4+ complexes with calix[4]arene ligands substituted either on the narrow or at the wide rim by four coordinating groups behave totally differently as shown by an NMR investigation of the dia- and paramagnetic complexes. Solutions of complexes were prepared by reacting anhydrous metal perchlorate salts with the ligands in dry acetonitrile (CAUTION). Relaxation time T1 titrations of acetonitrile solutions of Gd3+ by calixarenes indicate that ligands subsituted on the narrow rim form stable 1:1 complexes whether they feature four amide groups (1) or four phosphine oxide functions. In contrast, a ligand substituted by four (carbamoylmethyl)-diphenylphosphine oxide moieties on the wide rim (3) and its derivatives from polymeric species even at a 1:1 ligand/metal concentration ratio. Nuclear magnetic relaxation dispersion (NMRD) curves (relaxation rates 1/T1 vs magnetic field strength) of Gd3+, Gd3+.1 and Gd3+.3 perchlorates in acetonitrile are analyzed by an extended version of the Solomon-Bloembergen-Morgan equations. A comparison of the calculated rotational correlation times tau r shows that ligand 3 forms oligomeric Gd3+ species. The chelates of ligand 1 are axially symmetric (C4 symmetry), and the paramagnetic shifts induced by the Yb3+ ion are accounted for quantitatively. The addition of water or of nitrate ions does not modify the geometry of the complex. The metal chelates of 3 and its derivatives adopt a C2 symmetry, and the paramagnetic shifts are interpreted on a semiquantitative basis only. Water and NO3- ions completely labilize the complexes of the heavy lanthanides. The very high selectivity of ligand 3 through the lanthanide series stems from a complex interplay of factors.

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pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IV)-sensitive sites. Seven Fpg protein-sensitive sites per pBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.The Fpg protein of Escherichia coli was initially identified as a DNA glycosylase which excised the imidazole ringopened form of N7-methylguanine (2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine or Fapy) residues in DNA (2, 9, 13). This enzyme was shown also to liberate the imidazole ring-opened form of adenine residues from DNA treated with ionizing radiation (10) and of guanine residues modified at the N-7 position with bulky alkylating agents (12). The molecular cloning of the fpg+ gene of E. coli (8), the purification of the Fpg protein (9), and the isolation of a bacterial mutant defective in Fpg protein (4) were critical steps for studies of the biological significance of this protein.The Fpg protein exhibits a large substrate specificity, including imidazole ring-opened purines modified at the C-8 position by N-hydroxy-2-aminofluorene (3). The Fpg protein is a metalloprotein containing one zinc atom per monomer (9) and is endowed with a nicking activity which incises DNA at apurinic-apyrimidinic (AP) sites (38). The biological importance of this enzyme is suggested by the fact that Fapy-DNA glycosylase activities have been conserved in prokaryotes (7) and eukaryotes (29,33) and that the imidazole ringopened form of N7-methylguanine residues in DNA is a potentially lethal lesion (6, 37). However, the lack of sensitivity of the E. coli mutant defective in Fpg protein to methylating agents suggested that imidazole ring-opened N7-methylguanine is not a biological substrate and/or that these residues are excised ...

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross‐studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin® kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter‐sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT‐qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two‐fold lower inter‐sample variability than the widely used endogenous miR‐16‐5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up‐to 0.97 between the microarrays and individual RT‐qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice.