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A current challenge in microbial pathogenesis is to identify biological control agents that may prevent and/or limit host invasion by microbial pathogens. In natura, hosts are often infected by multiple pathogens. However, most of the current studies have been performed under laboratory controlled conditions and by taking into account the interaction between a single commensal species and a single pathogenic species. The next step is therefore to explore the relationships between host–microbial communities (microbiota) and microbial members with potential pathogenic behavior (pathobiota) in a realistic ecological context. In the present study, we investigated such relationships within root-associated and leaf-associated bacterial communities of 163 ecologically contrasted Arabidopsis thaliana populations sampled across two seasons in southwest of France. In agreement with the theory of the invasion paradox, we observed a significant humped-back relationship between microbiota and pathobiota α-diversity that was robust between both seasons and plant organs. In most populations, we also observed a strong dynamics of microbiota composition between seasons. Accordingly, the potential pathobiota composition was explained by combinations of season-specific microbiota operational taxonomic units. This result suggests that the potential biomarkers controlling pathogen’s invasion are highly dynamic.

Polyphenol-rich fruits represent promising natural candidates for mitigating oxidative stress. We determined in dose–response manner the intracellular antioxidant activities of P. cerasus (tart cherry) extract in HepG2 cells using three different cellular assays targeting specific mechanisms of action: (1) the AOP1 assay, to assess intracellular ROS scavenging activity; (2) the CAA assay, to estimate ROS scavenging activity at the cell membrane; and (3), the HepG2-ARE-luc assay, to evaluate Antioxidant Response Element (ARE) pathway modulation. Tart cherry extract exhibited a high and concentration-dependent intracellular ROS scavenging activity with the AOP1 assay (EC50 of 72.02 µg/mL), whereas antioxidant efficacy measured via the CAA assay was much lower (EC50 of 6.975 mg/mL). Notably, P. cerasus extract did not activate the ARE-driven luciferase gene expression. Instead, the extract induced a clear dose-dependent repression of ARE-driven transcriptional activity, with a reduction in luciferase gene expression ranging from 20 to 70% across the sample tested concentrations (0.38–98 µg/mL). These findings suggest that, at concentrations where it functions as a potent intracellular ROS scavenger, P. cerasus extract exerts a negative regulation of the ARE pathway. Further investigations are warranted to elucidate the compounds underlying these effects.

Taking advantage of Light Up Cell System (LUCS) technology, which allows for fine monitoring of reactive oxygen species (ROS) production inside live cells, a new assay called Anti Oxidant Power 1 (AOP1) was developed to specifically measure ROS and/or free-radical scavenging effects inside living cells. This method is quantitative and EC50s obtained from AOP1 dose-response experiments were determined in order to classify the intracellular antioxidant efficacy of 15 well known antioxidant compounds with different hydrophilic properties. Six of them (epigallocatechin gallate, quercetin, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethoxyquin, resveratrol) gave EC50s in the range of 7–64 μM, four (Trolox, catechin, epicatechin, EUK134) in the range of 0.14 to 1 mM, and 5 (sulforaphane, astaxanthin, α- and γ-tocopherols, vitamin E acetate) showed only partial or no effect. Interestingly, effects with measurable EC50s were observed for compounds with hydrophilic properties (LogP ≤ 5.3), while all antioxidants known to act at the plasma membrane level (LogP ≥ 10.3) had partial or no effect. Sulforaphane, a hydrophilic but strict Keap1/Nrf2 pathway enhancer, did not show any effect either. Importantly, AOP1 assay captures both antioxidant and prooxidant effects. Taken together, these results led us to the conclusion that AOP1 assay measures antioxidant effect of compounds that selectively enter the cell, and act as free radical scavengers in the cytosol and/or nucleus level.

Oenobiol Sun Expert, a food formulation designed to enhance skin health prior to sun exposure, has been optimized by incorporating the OenoGrape Advanced Complex, which includes grape pomace extract, increased selenium content and 10% lycopene-rich tomato extract, with these constituents exhibiting high antioxidant potential. To evaluate the effects of these individual ingredients and the overall formulation at the cellular level, the AOP1 cell antioxidant efficacy assay was employed to measure the intracellular free radical scavenging activity, while the Cell Antioxidant Assay (CAA or DCFH-DA) assay was used to assess peroxidation scavenging at the plasma membrane level. The indirect antioxidant activity was examined using stably transfected cell lines containing a luciferase reporter gene controlled by the Antioxidant Response Element (ARE), which activates the endogenous antioxidant system via the Nrf2/Keap1-ARE pathway. Our results indicate that among the individual components, grape pomace extract and sodium selenite possess high and complementary antioxidant properties. Grape pomace extract was particularly effective in inhibiting free radicals (AOP1 EC50 = 6.80 μg/mL) and activating the ARE pathway (ARE EC50 = 231.1 μg/mL), whereas sodium selenite exerted its effects through potent ARE activation at sub-microgram levels (EC50 = 0.367 μg/mL). In contrast, the lycopene-rich tomato extract did not show a notable contribution to the antioxidant effects. The antiradical activity of the OenoGrape Advanced Complex, comprising these three ingredients, was very efficient and consistent with the results obtained for the individual components (AOP1 EC50 = 15.78 µg/mL and ARE EC50 of 707.7 μg/mL). Similarly, the free radical scavenging activity still persisted in the Oenobiol Sun Expert formulation (AOP1 EC50 = 36.63 µg/mL). Next, in vitro intestinal transepithelial transfer experiments were performed. The basolateral compartments of cells exposed to the ingredients were collected and assessed using the same antioxidant cell assays. The direct and indirect antioxidant activities were measured on both hepatocytes and keratinocytes, demonstrating the bioavailability and bioactivity of grape pomace extract and sodium selenite. These finding suggest that the ingredients of this food supplement contribute to enhanced cytoprotection following ingestion.

Oxidative stress and chronic inflammation contribute to some chronic diseases. Aronia berries are rich in polyphenols. The aim of the present study was to characterize the cellular antioxidant effect of an aronia extract to reflect the potential physiological in vivo effect. Cellular in vitro assays in three cell lines (Caco-2, HepG2, and SH-SY5Y) were used to measure the antioxidant effect of AE, in three enriched polyphenolic fractions (A1: anthocyanins and phenolic acids; A2: oligomeric proanthocyanidins; A3: polymeric proanthocyanidins), pure polyphenols and microbial metabolites. Both direct (intracellular and membrane radical scavenging, catalase-like effect) and indirect (NRF2/ARE) antioxidant effects were assessed. AE exerted an intracellular free radical scavenging activity in the three cell lines, and A2 and A3 fractions showed a higher effect in HepG2 and Caco-2 cells. AE also exhibited a catalase-like activity, with the A3 fraction having a significant higher activity. Only A1 fraction activated the NRF2/ARE pathway. Quercetin and caffeic acid are the most potent antioxidant polyphenols, whereas cyanidin and 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone showed the highest antioxidant effect among polyphenol metabolites. AE rich in polyphenols possesses broad cellular antioxidant effects, and proanthocyanidins are major contributors. Polyphenol metabolites may contribute to the overall antioxidant effect of such extract in vivo.

As the use of antioxidant compounds in the domains of health, nutrition and well-being is exponentially rising, there is an urgent need to quantify antioxidant power quickly and easily, ideally within living cells. We developed an Anti Oxidant Power in Yeast (AOPY) assay which allows for the quantitative measurement of the Reactive Oxygen Species (ROS) and free-radical scavenging effects of various molecules in a high-throughput compatible format. Key parameters for Saccharomyces cerevisiae were investigated, and the optimal values were determined for each of them. The cell density in the reaction mixture was fixed at 0.6; the concentration of the fluorescent biosensor (TO) was found to be optimal at 64 µM, and the strongest response was observed for exponentially growing cells. Our optimized procedure allows accurate quantification of the antioxidant effect in yeast of well-known antioxidant molecules: resveratrol, epigallocatechin gallate, quercetin and astaxanthin added in the culture medium. Moreover, using a genetically engineered carotenoid-producing yeast strain, we realized the proof of concept of the usefulness of this new assay to measure the amount of β-carotene directly inside living cells, without the need for cell lysis and purification.

In this work, both direct and indirect cell-based antioxidant profiles were established for 27 plant extracts and 1 algal extract. To evaluate the direct antioxidant effects, fluorescent AOP1 cell assay was utilized, which measures the ability of different samples to neutralize intracellular free radicals produced by a cell-based photo-induction process. As the intestinal barrier is the first cell line crossed by the product, dose response curves obtained from Caco-2 cells were used to establish EC50 values for 26 out of the 28 natural extracts. Among them, 11 extracts from Vitis, Hamamelis, Syzygium, Helichrysum, Ilex and Ribes genera showed remarkable EC50s in the range of 10 µg/mL. In addition to this, a synergistic effect was found when combinations of the most potent extracts (S. aromaticum, H. italicum, H. virginiana, V. vinifera) were utilized compared to extracts alone. Indirect antioxidant activities (i.e., the ability of cells to trigger antioxidant defenses) were studied using the ARE/Nrf2 luminescence reporter-gene assay in HepG2 cells, as liver is the first organ crossed by an edible ingredient once it enters in the bloodstream. Twelve extracts were subjected to an intestinal epithelial barrier passage in order to partially mimic intestinal absorption and show whether basolateral compartments could maintain direct or indirect antioxidant properties. Using postepithelial barrier samples and HepG2 cells as a target model, we demonstrate that indirect antioxidant activities are maintained for three extracts, S. aromaticum, H. virginiana and H. italicum. Our experimental work also confirms the synergistic effects of combinations of post-intestinal barrier compartments issued from apical treatment with these three extracts. By combining cell-based assays together with an intestinal absorption process, this study demonstrates the power of cell systems to address the issue of antioxidant effects in humans.

Polyphenol-rich fruits represent promising natural candidates for mitigating oxidative stress. We determined in dose–response manner the intracellular antioxidant activities of P. cerasus (tart cherry) extract in HepG2 cells using three different cellular assays targeting specific mechanisms of action: (1) the AOP1 assay, to assess intracellular ROS scavenging activity; (2) the CAA assay, to estimate ROS scavenging activity at the cell membrane; and (3), the HepG2-ARE-luc assay, to evaluate Antioxidant Response Element (ARE) pathway modulation. Tart cherry extract exhibited a high and concentration-dependent intracellular ROS scavenging activity with the AOP1 assay (EC[sub.50] of 72.02 µg/mL), whereas antioxidant efficacy measured via the CAA assay was much lower (EC[sub.50] of 6.975 mg/mL). Notably, P. cerasus extract did not activate the ARE-driven luciferase gene expression. Instead, the extract induced a clear dose-dependent repression of ARE-driven transcriptional activity, with a reduction in luciferase gene expression ranging from 20 to 70% across the sample tested concentrations (0.38–98 µg/mL). These findings suggest that, at concentrations where it functions as a potent intracellular ROS scavenger, P. cerasus extract exerts a negative regulation of the ARE pathway. Further investigations are warranted to elucidate the compounds underlying these effects.

A current challenge in microbial pathogenesis is to identify biological control agents that may prevent and/or limit host invasion by microbial pathogens. In natura, hosts are often infected by multiple pathogens. However, most of the current studies have been performed under laboratory controlled conditions and by taking into account the interaction between a single commensal species and a single pathogenic species. The next step is therefore to explore the relationships between host-microbial communities (microbiota) and microbial members with potential pathogenic behavior (pathobiota) in a realistic ecological context. In the present study, we investigated such relationships within root and leaf associated bacterial communities of 163 ecologically contrasted Arabidopsis thaliana populations sampled across two seasons in South-West of France. In agreement with the theory of the invasion paradox, we observed a significant humped-back relationship between microbiota and pathobiota -diversity that was robust between both seasons and plant organs. In most populations, we also observed a strong dynamics of microbiota composition between seasons. Accordingly, the potential pathobiota composition was explained by combinations of season-specific microbiota OTUs. This result suggests that the potential biomarkers controlling pathogen's invasion are highly dynamic.

Phytopathogens are a continuous threat for global food production and security. Emergence or re-emergence of plant pathogens is highly dependent on the environmental conditions affecting pathogen spread and survival. Under climate change, a geographic expansion of pathogen distribution poleward has been observed, potentially resulting in disease outbreaks on crops and wild plants. Therefore, estimating the adaptive potential of plants to novel epidemics and describing its underlying genetic architecture, is a primary need to propose agricultural management strategies reducing pathogen outbreaks and to breed novel plant cultivars adapted to pathogens that might spread in novel habitats under climate change. To address this challenge, we inoculated Pseudomonas syringae strains isolated from Arabidopsis thaliana populations located in south-west of France on the highly genetically polymorphic TOU-A A. thaliana population located east-central France. While no adaptive potential was identified in response to most P. syringae strains, the TOU-A population displays a variable disease response to the P. syringae strain JACO-CL belonging to the phylogroup 7 (PG7). This strain carried a reduced T3SS characteristic of the PG7 as well as flexible genomic traits and potential novel effectors. GWA mapping on 192 TOU-A accessions inoculated with JACO-CL revealed a polygenic architecture. The main QTL region encompasses two R genes and the AT5G18310 gene encoding for ubiquitin hydrolase, a target of the AvrRpt2 P. syringae effector. Altogether, our results pave the way for a better understanding of the genetic and molecular basis of the adaptive potential in an ecologically relevant A. thaliana – P. syringae pathosystem.