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International audience; Lactococcus cremoris and Lactococcus lactis are among the most extensively exploited species of lactic acid bacteria in dairy fermentations. The cell wall of lactococci, like other Gram-positive bacteria, possesses a thick peptidoglycan layer, which may incorporate cell wall polysaccharides (CWPS), wall teichoic acids (WTA), and/or lipoteichoic acids (LTA).In this study, we report the isolation, purification and structural analysis of the carbohydrate moieties of glycolipids (GL) and LTA of the L. cremoris model strain 3107. Chemical structures of these compounds were studied by chemical methods, NMR spectroscopy and positive and negative mode ESI MS. We found that the LTA of strain 3107 is composed of short chains of 1,3-polyglycerol phosphate (PGP), attached to O-6 of the non-reducing glucose of the kojibiose-Gro backbone of the glycolipid anchor. Extraction of cells with cold TCA afforded the detection of 1,3-glycerol phosphate chains randomly substituted at O-2 of glycerol by D-Ala. Unlike the LTA of L. lactis strains studied to date, the PGP backbone of the LTA of L. cremoris 3107 did not carry any glycosyl substitution. The deacylated glycolipid fraction contained the free kojibiose-Gro oligosaccharide, identical to the backbone of the GL anchor of LTA, and its shorter fragment α-Glc-1-Gro. These OS may have originated from the GL precursors of LTA biosynthesis.

International audience; Nanoplastics (NPLs), the presence in the environment of which was considered only “highly plausible” until recently, have become the focus of environmental and ecotoxicological studies. However, up to know, little is known about the potential risks of NPLs to human health. In this review, we provide an overview of the evidence of a specific impact of NPLs on human digestive health reported to date. We focus on the different sources of oral exposure to NPLs, including food packaging, food and beverages. We then summarize the toxicological effects of such exposure on the digestive ecosystem in vitro and in vivo in rodents. Importantly, gut toxicity results should be assessed with the type of NPL model materials employed, due to the critical influence of their chemical and physical properties. As a result, we thoroughly describe NPLs with their source, chemical composition and physicochemical behavior to emphasize the lack of NPL characterization and/or model materials. Finally, we propose avenues for interdisciplinary studies at the interface of toxicology and chemistry, with a view to achieving appropriate scientific assessments of the risks to gut health posed by NPLs, and improvements in their management.

Most organisms adjust their development according to the environmental conditions. For the majority, this implies the sensing of alterations to cell walls caused by different cues. Despite the relevance of this process, few molecular players involved in cell wall sensing are known and characterized. Here, we show that the wall-associated kinase-like protein RESISTANCE TO FUSARIUM OXYSPORUM 1 (RFO1) is required for plant growth and early defense against Fusarium oxysporum and functions by sensing changes in the pectin methylation levels in the cell wall. The RFO1 dwell time at the plasma membrane is affected by the pectin methylation status at the cell wall, regulating MITOGEN-ACTIVATED PROTEIN KINASE and gene expression. We show that the extracellular domain of RFO1 binds de-methylated pectin in vitro, whose distribution in the cell wall is altered during F. oxysporum infection. Further analyses also indicate that RFO1 is required for the BR-dependent plant growth alteration in response to inhibition of pectin de-methyl-esterase activity at the cell wall. Collectively, our work demonstrates that RFO1 is a sensor of the pectin methylation status that plays a unique dual role in plant growth and defense against vascular pathogens., Molecular Plant, 16 (5), ISSN:1752-9867, ISSN:1674-2052

International audience; Baillonella toxisperma is a medicinal plant used in northern Gabon to treat microbial diseases. It is a plant well-known by local populations, but very few studies have focused on the molecules responsible for the antibacterial activities of B. toxisperma. This study proposes a dereplication strategy based on molecular networking generated from HPLC-ESI-Q/TOF data, allowing investigation of the molecules responsible for the antibacterial activity of B. toxisperma. From this strategy, eighteen compounds were putatively identified. All of these compounds belonged mainly to five families of natural compounds, including phenylpropanolamines, stilbenes, flavonoids, lignans and phenolic glycosides. The chemical study carried out from the bark of B. toxisperma allowed us to identify, for the first time, compounds such as resveratrol and derivatives, epicatechin, epigallocatechin and epigallocatechin gallate. In addition, antibacterial activity (diffusion method and microdilution) and cytotoxicity (Cell Counting Kit-8 (CCK-8 Assay)) in vitro were evaluated. The crude ethanolic extract, as well as the fractions of B. toxisperma, showed significant antibacterial activity. However, the ethanolic fractions F2 and F4 presented high antibacterial activity compared to the crude extract. Cytotoxicity studies on colon-cancer cells (Caco-2) and human keratinocyte cells (HaCaT) showed moderate cytotoxicity in both cell types. This study clearly shows the therapeutic potential of the ethanolic extract of the bark of B. toxisperma and provides information on the phytochemical composition and bioactive compounds of the plant.

The aim of this study was to identify the chemical composition and sequential structure of alginate isolated from C. crinita harvested in the Bulgarian Black Sea, as well as its effects in histamine-induced paw inflammation in rats. The serum levels of TNF-α, IL-1β, IL-6, and IL-10 in rats with systemic inflammation, and the levels of TNF-α in a model of acute peritonitis in rats were also investigated. The structural characterization of the polysaccharide was obtained by FTIR, SEC-MALS, and 1H NMR. The extracted alginate had an M/G ratio of 1.018, a molecular weight of 7.31 × 104 g/mol, and a polydispersity index of 1.38. C. crinita alginate in doses of 25 and 100 mg/kg showed well-defined anti-inflammatory activity in the model of paw edema. A significant decrease in serum levels of IL-1β was observed only in animals treated with C. crinita alginate in a dose of 25 mg/kg bw. The concentrations of TNF-α and IL-6 in serum were significantly reduced in rats treated with both doses of the polysaccharide, but no statistical significance was observed in the levels of the anti-inflammatory cytokine IL-10. A single dose of alginate did not significantly alter the levels of the pro-inflammatory cytokine TNF-α in the peritoneal fluid of rats with a model of peritonitis.

International audience; Laurus nobilis belongs to the family Lauraceae and is characterized by its evergreen nature. It is well known because of its traditional use in cooking and folk medicine. Many studies have been performed regarding its chemical composition and the pharmacological potential it exhibits. It has been reported to have antioxidant, antibacterial, antifungal, antiviral, biocidal, antidiabetic, antiulcerogenic, neuroprotective, analgesic, anti-inflammatory, and anticholinergic properties, making this plant of great clinical importance. The main chemical compounds that show bioactive properties in L. nobilis are terpenoids, phenolics, and fatty acids. The purpose of this review is to describe the main use of L. nobilis, notably of the leaves and fruits, and to relate their metabolite content with their pharmacological and toxicological properties.

Sesquiterpene lactones (STLs) are a large group of terpenoids most commonly found in plants of the Asteraceae family, e.g., in chicory plants, displaying a wide range of interesting biological activities. However, further studies on the biological potential of chicory-derived STLs and analogues are challenging as only four of these molecules are commercially available (as analytical standards), and to date, there are no published or patented simple extraction–purification processes capable of large-scale STLs isolation. In this work, we describe a novel three-step large-scale extraction and purification method for the simultaneous purification of 11,13-dihydrolactucin (DHLc) and lactucin (Lc) starting from a chicory genotype rich in these STLs and the corresponding glucosyl and oxalyl conjugated forms. After a small-scale screening on 100 mg of freeze-dried chicory root powder, the best results were achieved with a 17 h water maceration at 30 °C. With these conditions, we managed to increase the content of DHLc and Lc, at the same time favoring the hydrolysis of their conjugated forms. On a larger scale, the extraction of 750 g of freeze-dried chicory root powder, followed by a liquid–liquid extraction step and a reversed-phase chromatography, allowed the recovery of 642.3 ± 76.3 mg of DHLc and 175.3 ± 32.9 mg of Lc. The two pure STLs were subsequently used in the context of semisynthesis to generate analogues for biological evaluation as antibacterial agents. In addition, other described chicory STLs that are not commercially available were also synthesized or extracted to serve as analytical standards for the study. In particular, lactucin-oxalate and 11,13-dihydrolactucin-oxalate were synthesized in two steps starting from Lc and DHLc, respectively. On the other hand, 11β,13-dihydrolactucin-glucoside was obtained after a MeOH/H2O (70/30) extraction, followed by a liquid–liquid extraction step and a reversed-phase chromatography. Together, this work will help facilitate the evaluation of the biological potential of chicory-derived STLs and their semisynthetic analogues.

(1) Background: Many human diseases are associated with oxidative stress, which is caused by reactive oxygen species and free radicals generated in living cells. Some biomass extracts derived from various types of plants can act as efficient drugs against pathological disorders related to oxidative stress. Numerous herbal blends have thus been shown to improve health. Cleistocalyx operculatus (Roxb.) Merr. and L.M.Perry teas have been considered in that way. Problem: Because of amertume, the taste of C. operculatus avoids or limits a large use of such alleged healthy leaf infusions. (2) Methods: The phytochemistry, oxygen, free radical scavenging activity, and antilipid peroxidation of C. operculatus teas were here studied in vitro. Then different mixes of C. operculatus and Mentha arvensis were infused together and tasted in a hedonic test. The chemical and biological properties of the best mix were then analyzed. (3) Results: The herbal blend of C. operculatus revealed significant scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) with IC50 values of 35.6 µg/mL. Induced by hydroxyl radicals, this beverage could significantly inhibit the lipid peroxidation of mouse brain homogenates. Our results demonstrate that the lipid peroxidation inhibition of dried leaves of C. operculatus might be related to its scavenging effects on oxygen free radicals. This C. operculatus bitter blend was then combined with various amounts of M. arvensis Linn. The taste was evaluated, and further biochemical analyses were conducted on the best 7/3 ratio. They showed that the healthy properties were maintained. (4) Conclusion: The optimized 7/3 ratio of the Cleistocalyx/Mentha mix led to the best results in terms of taste (sensory tests). It is demonstrated that the potential health benefits against oxidative stress remained high as compared with pure C. operculatus infusion.

Type 2 diabetes (T2D) is a metabolic disorder characterized by loss of pancreatic β- cell function, decreased insulin secretion and increased insulin resistance, that affects more than 400 million people worldwide. Although several treatments are proposed to patients suffering from T2D, long-term control of glycemia remains a challenge. Therefore, identifying new potential drugs and targets that positively affect β-cell function and insulin secretion remains crucial. Here, we developed an automated approach to allow the identification of new compounds or genes potentially involved in β-cell function in a 384-well plate format, using the murine β-cell model Min6. Using MALDI-TOF mass spectrometry, we have implemented a high-throughput screening (HTS) strategy based on the automation of a cellular assay allowing to detect insulin secretion in response to glucose, quantitative detection of insulin, in a miniaturized system. As a proof of concept, we screened siRNA targeting well-know β-cell genes and 1600 chemical compounds and identified several molecules as potential regulators of insulin secretion and/or synthesis, demonstrating that our approach allows HTS of insulin secretionin vitro.

Pistacia lentiscus L. is a medicinal plant that grows spontaneously throughout the Mediterranean basin and is traditionally used to treat diseases, including diabetes. The aim of this work consists of the evaluation of the α-glucosidase inhibitory effect (i.e., antidiabetic activity in vitro) of different extracts from the leaves, stem barks and fruits of P. lentiscus harvested on mountains and the littoral of Tizi-Ouzou in Algeria. Metabolomic profiling combined with a chemometric approach highlighted the variation of the antidiabetic properties of P. lentiscus according to the plant’s part and origin. A multiblock OPLS analysis showed that the metabolites most involved in α-glucosidase inhibition activity were mainly found in the stem bark extracts. The highest inhibitory activity was found for the stem bark extracts, with averaged inhibition percentage values of 84.7% and 69.9% for the harvested samples from the littoral and mountain, respectively. On the other hand, the fruit extracts showed a lower effect (13.6%) at both locations. The UHPLC-ESI-HRMS characterization of the metabolites most likely responsible for the α-glucosidase-inhibitory activity allowed the identification of six compounds: epigallocatechin(4a>8)epigallocatechin (two isomers), (epi)gallocatechin-3′-O-galloyl-(epi)gallocatechin (two isomers), 3,5-O-digalloylquinic acid and dihydroxy benzoic acid pentoside.

International audience; The hop plant (Humulus lupulus L.) has been exploited for a long time for both its brewing and medicinal uses, due in particular to its specific chemical composition. These last years, hop cultivation that was in decline has been experiencing a renewal for several reasons, such as a craze for strongly hopped aromatic beers. In this context, the present work aims at investigating the genetic and chemical diversity of fifty wild hops collected from different locations in Northern France. These wild hops were compared to ten commercial varieties and three heirloom varieties cultivated in the same sampled geographical area. Genetic analysis relying on genome fingerprinting using 11 microsatellite markers showed a high level of diversity. A total of 56 alleles were determined with an average of 10.9 alleles per locus and assessed a significant population structure (mean pairwise FST = 0.29). Phytochemical characterization of hops was based on volatile compound analysis by HS-SPME GC-MS, quantification of the main prenylated phenolic compounds by UHPLC-UV as well as untargeted metabolomics by UHPLC-HRMS and revealed a high level of chemical diversity among the assessed wild ac-cessions. In particular, analysis of volatile compounds revealed the presence of some minor but original com-pounds, such as aromadendrene, allo-aromadendrene, isoledene, fl-guaiene, alpha-ylangene and fl-pinene in some wild accessions; while analysis of phenolic compounds showed high content of fl-acids in these wild accessions, up to 2.37% of colupulone. Genetic diversity of wild hops previously observed was hence supported by their chemical diversity. Sample soil analysis was also performed to get a pedological classification of these different collection sites. Results of the multivariate statistical analysis suggest that wild hops constitute a huge pool of chemical and genetic diversity of this species.

Pistacia lentiscus (lentisk) is a plant species of the Anacardiaceae family. It is a medicinal plant that grows wild in the Mediterranean region. This review aims to update the existing knowledge regarding P. lentiscus polyphenols by consulting references dated from 1996 to 2022. The data are organized and analyzed as follows: (i) to show the chemical diversity of phenolic products from P. lentiscus; (ii) to summarize the variability in phenolic composition and quantity; this could be attributed to plant origin, environmental conditions, phenological stage, and the polarity of the extraction solvents; (iii) to present the pharmacological properties in agreement with the traditional uses of this plant; and (iv) to demonstrate the correlation between the chemical profile and the pharmacological effect. Various compositions were observed, including phenolic acids, flavonoid glycosides, anthocyanins, catechins, and their derivatives. The biological and therapeutic potentials of lentisk extracts have been evaluated in terms of antioxidant, antimicrobial, and anti-inflammatory activities. Most of these activities are related to the phenolic composition of this plant. The content of this review will undoubtedly contribute to the choice of techniques for isolating the different bioactive molecules contained in the P. lentiscus. It is also of significance for the potential development of a micro-industrial sector based on the valorization of lentisk polyphenols.

Fish consumption in France has increased by 20% in 20 years, from 19.1 kg/inhabitant/year in 2000 to 23.9 kg/inhabitant/year in 2020. This increase leads to an overexploitation of traditional fishing areas that are no longer sufficient to supply the fresh market. To overcome this supply problem, the share of aquaculture has grown considerably over the last 20 years, from 25.7% to 46%. The exploitation of new fishing areas that are increasingly far from consumption sites is also another alternative to these supply shortages, which increases travel from fishing to consumer. As a consequence, fraudulent practices of selling frozen/ thawed aquatic products in the fresh market often occur. The development of a reliable tool for distinguishing fresh and frozen/thawed products remains a major challenge for the seafood sector. The thesis aimed to explore the use of mitochondria as a marker of freshness and freezing of fish. Mitochondria are mostly present in red muscle tissue. Therefore, our study focused on this tissue for the three fish species studied: sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss). We have found that freezing leads to profound structural changes in mitochondria, with an alteration of the outer and inner membranes (visible in electron microscopy). The alteration of mitochondrial membranes contributes to the total loss of mitochondrial potential of isolated mitochondria. This loss of potential was analyzed in flow cytometry using two potential probes: TMRM and Rhodamine 123. The loss of potential was observed on fresh fish fillet on Day 9, which limits the use of the loss of potential as a marker of freezing. Mitochondrial permeabilization by freezing/thawing shock was first studied by measuring the NADH permeability of isolated mitochondria. The level of permeabilization was normalized by comparison to permeabilization by a known channel-forming antibiotic alamethicin. This permeabilization was followed by the spectrophotometric measurement of the rate of consumption of NADH by mitochondria and the activation of oxygen consumption by oxygraphy. Freezing/thawing was shown to lead to NADH permeabilization of mitochondria and activation of enzyme complexes which results in increased oxygen consumption. The results obtained on isolated mitochondria were confirmed on entire muscle fibres, thus allowing to skip the mitochondrial extraction steps. Freezing leads to rupture of mitochondrial membranes and plasma membranes. Muscle fibres from frozen muscle are more permeable to NADH and therefore consume it faster than fresh fibres. A greater stimulation of oxygen consumption by thawed fibers was also observed. Maintaining a certain integrity of mitochondria in red muscle makes it possible to explore mitochondrial functions and use them as a marker for freezing/thawing fish fillets. This work showed that mitochondria isolated or present in the fibers of fresh fillets were sensitive to the action of a permeabilizer. Conversely, mitochondria and fibres isolated from frozen fillets were insensitive to alamethicin. This reflects a high level of alteration of mitochondrial membranes in these products. This technique, which uses red muscle, appears to be effective in discriminating between frozen aquatic products and fresh products. It is necessary to define whether it can be adapted to the white muscle of fish fillets.; La consommation de poisson en France a augmenté de 20 % en 20 ans, passant de 19,1 kg/hab/an en 2000 à 23,9 kg/hab/an en 2020. Cette hausse se traduit par une surexploitation de zones traditionnelles de pêche qui ne sont plus suffisantes pour alimenter le marché du frais. Pour pallier ce problème d’approvisionnement, la part de l’aquaculture s’est considérablement développée ces 20 dernières années en passant de 25,7 % à 46 %. L’exploitation de nouvelles zones de pêche de plus en plus éloignées des sites de consommation est aussi une autre alternative à ces problèmes d’approvisionnement. Si de nombreux produits aquatiques sont connus pour être commercialisés à l’état décongelé, l’alimentation du marché du frais par des produits décongelés constitue une fraude. La mise au point d’un outil fiable de détection des produits congelés/décongelés reste un enjeu majeur pour la filière des produits de la mer. Elle permettrait de détecter les fraudes, la congélation/décongélation pouvant entraîner des modifications au niveau du produit. La thèse avait pour but d’explorer l’utilisation des mitochondries comme marqueurs de fraîcheur et de décongélation. Les mitochondries sont particulièrement présentes dans le tissu musculaire rouge. C’est pourquoi notre étude s’est concentrée sur ce tissu pour les trois espèces de poissons étudiées : le bar (Dicentrarchus labrax), la daurade royale (Sparus aurata) et la truite arc en ciel (Oncorhynchus mykiss). La congélation va entraîner de profondes modifications structurales des mitochondries, avec une altération des membranes externes et internes (visible en microscopie électronique). L’altération des membranes mitochondriales va participer à la perte totale du potentiel mitochondrial des mitochondries isolées. Cette perte du potentiel a été analysée en cytométrie de flux grâce à deux sondes de potentiel : le TMRM et la Rhodamine 123. La perte de potentiel peut cependant être observée sur du filet de poisson frais à J9, ce qui limite l’utilisation de la perte du potentiel comme marqueur de décongélation. La perméabilisation des mitochondries par le choc congélation/décongélation a été étudiée en mesurant la perméabilité au NADH de mitochondries isolées. Le niveau de perméabilisation a été normalisé en utilisant un perméabilisant : l’alaméthicine. Cette perméabilisation a été suivie par la mesure de la vitesse de consommation du NADH par les mitochondries en spectrophotométrie et l’activation de la consommation de d’oxygène en oxygraphie. La congélation/décongélation conduit à une perméabilisation au NADH des mitochondries et à une activation des complexes enzymatiques qui se traduit par un regain de consommation d’oxygène. Pour s’affranchir des étapes d’extraction des mitochondries, ces résultats ont été confirmés sur fibres musculaires. La congélation entraîne une rupture des membranes mitochondriales et des membranes plasmiques. Les fibres musculaires issues du muscle décongelé sont plus perméables au NADH et le consomment donc plus rapidement que les fibres fraîches. Une stimulation plus importante de la consommation d’oxygène par les fibres décongelées est aussi observée. Le maintien d’une certaine intégrité des mitochondries dans le muscle rouge permet d’explorer les fonctions mitochondriales et de les utiliser comme marqueur de congélation /décongélation des filets de poisson. Ces travaux ont montré que les mitochondries isolées ou présentes dans les fibres de filets frais étaient sensibles à l’action d’un perméabilisant. A l‘inverse, les mitochondries et fibres isolées de filets décongelés étaient insensibles à l’alaméthicine. Ceci traduit un niveau élevé d’altération des membranes mitochondriales dans ces produits. Cette technique, qui utilise le muscle rouge, semble efficace pour discriminer les produits aquatiques décongelés des produits frais. Il reste à définir si celle-ci peut s’adapter au muscle blanc des filets de poisson.

Fish consumption in France has increased by 20% in 20 years, from 19.1 kg/inhabitant/year in 2000 to 23.9 kg/inhabitant/year in 2020. This increase leads to an overexploitation of traditional fishing areas that are no longer sufficient to supply the fresh market. To overcome this supply problem, the share of aquaculture has grown considerably over the last 20 years, from 25.7% to 46%. The exploitation of new fishing areas that are increasingly far from consumption sites is also another alternative to these supply shortages, which increases travel from fishing to consumer. As a consequence, fraudulent practices of selling frozen/thawed aquatic products in the fresh market often occur. The development of a reliable tool for distinguishing fresh and frozen/thawed products remains a major challenge for the seafood sector.The thesis aimed to explore the use of mitochondria as a marker of freshness and freezing of fish.Mitochondria are mostly present in red muscle tissue. Therefore, our study focused on this tissue for the three fish species studied: sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss). We have found that freezing leads to profound structural changes in mitochondria, with an alteration of the outer and inner membranes (visible in electron microscopy). The alteration of mitochondrial membranes contributes to the total loss of mitochondrial potential of isolated mitochondria. This loss of potential was analyzed in flow cytometry using two potential probes: TMRM and Rhodamine 123. The loss of potential was observed on fresh fish fillet on Day 9, which limits the use of the loss of potential as a marker of freezing.Mitochondrial permeabilization by freezing/thawing shock was first studied by measuring the NADH permeability of isolated mitochondria. The level of permeabilization was normalized by comparison to permeabilization by a known channel-forming antibiotic alamethicin. This permeabilization was followed by the spectrophotometric measurement of the rate of consumption of NADH by mitochondria and the activation of oxygen consumption by oxygraphy. Freezing/thawing was shown to lead to NADH permeabilization of mitochondria and activation of enzyme complexes which results in increased oxygen consumption.The results obtained on isolated mitochondria were confirmed on entire muscle fibres, thus allowing to skip the mitochondrial extraction steps. Freezing leads to rupture of mitochondrial membranes and plasma membranes. Muscle fibres from frozen muscle are more permeable to NADH and therefore consume it faster than fresh fibres. A greater stimulation of oxygen consumption by thawed fibers was also observed.Maintaining a certain integrity of mitochondria in red muscle makes it possible to explore mitochondrial functions and use them as a marker for freezing/thawing fish fillets. This work showed that mitochondria isolated or present in the fibers of fresh fillets were sensitive to the action of a permeabilizer. Conversely, mitochondria and fibres isolated from frozen fillets were insensitive to alamethicin. This reflects a high level of alteration of mitochondrial membranes in these products. This technique, which uses red muscle, appears to be effective in discriminating between frozen aquatic products and fresh products. It is necessary to define whether it can be adapted to the white muscle of fish fillets.; La consommation de poisson en France a augmenté de 20 % en 20 ans, passant de 19,1 kg/hab/an en 2000 à 23,9 kg/hab/an en 2020. Cette hausse se traduit par une surexploitation de zones traditionnelles de pêche qui ne sont plus suffisantes pour alimenter le marché du frais. Pour pallier ce problème d’approvisionnement, la part de l’aquaculture s’est considérablement développée ces 20 dernières années en passant de 25,7 % à 46 %. L’exploitation de nouvelles zones de pêche de plus en plus éloignées des sites de consommation est aussi une autre alternative à ces problèmes d’approvisionnement.Si de nombreux produits aquatiques sont connus pour être commercialisés à l’état décongelé, l’alimentation du marché du frais par des produits décongelés constitue une fraude. La mise au point d’un outil fiable de détection des produits congelés/décongelés reste un enjeu majeur pour la filière des produits de la mer. Elle permettrait de détecter les fraudes, la congélation/décongélation pouvant entraîner des modifications au niveau du produit.La thèse avait pour but d’explorer l’utilisation des mitochondries comme marqueurs de fraîcheur et de décongélation. Les mitochondries sont particulièrement présentes dans le tissu musculaire rouge. C’est pourquoi notre étude s’est concentrée sur ce tissu pour les trois espèces de poissons étudiées : le bar (Dicentrarchus labrax), la daurade royale (Sparus aurata) et la truite arc en ciel (Oncorhynchus mykiss). La congélation va entraîner de profondes modifications structurales des mitochondries, avec une altération des membranes externes et internes (visible en microscopie électronique). L’altération des membranes mitochondriales va participer à la perte totale du potentiel mitochondrial des mitochondries isolées. Cette perte du potentiel a été analysée en cytométrie de flux grâce à deux sondes de potentiel : le TMRM et la Rhodamine 123. La perte de potentiel peut cependant être observée sur du filet de poisson frais à J9, ce qui limite l’utilisation de la perte du potentiel comme marqueur de décongélation.La perméabilisation des mitochondries par le choc congélation/décongélation a été étudiée en mesurant la perméabilité au NADH de mitochondries isolées. Le niveau de perméabilisation a été normalisé en utilisant un perméabilisant : l’alaméthicine. Cette perméabilisation a été suivie par la mesure de la vitesse de consommation du NADH par les mitochondries en spectrophotométrie et l’activation de la consommation de d’oxygène en oxygraphie. La congélation/décongélation conduit à une perméabilisation au NADH des mitochondries et à une activation des complexes enzymatiques qui se traduit par un regain de consommation d’oxygène.Pour s’affranchir des étapes d’extraction des mitochondries, ces résultats ont été confirmés sur fibres musculaires. La congélation entraîne une rupture des membranes mitochondriales et des membranes plasmiques. Les fibres musculaires issues du muscle décongelé sont plus perméables au NADH et le consomment donc plus rapidement que les fibres fraîches. Une stimulation plus importante de la consommation d’oxygène par les fibres décongelées est aussi observée.Le maintien d’une certaine intégrité des mitochondries dans le muscle rouge permet d’explorer les fonctions mitochondriales et de les utiliser comme marqueur de congélation/décongélation des filets de poisson. Ces travaux ont montré que les mitochondries isolées ou présentes dans les fibres de filets frais étaient sensibles à l’action d’un perméabilisant. A l‘inverse, les mitochondries et fibres isolées de filets décongelés étaient insensibles à l’alaméthicine. Ceci traduit un niveau élevé d’altération des membranes mitochondriales dans ces produits. Cette technique, qui utilise le muscle rouge, semble efficace pour discriminer les produits aquatiques décongelés des produits frais. Il reste à définir si celle-ci peut s’adapter au muscle blanc des filets de poisson.

Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species.

International audience; The pomegranate fruit is made of white to deep purple seeds that are enclosed in a white, spongy, astringent membrane, also known as pericarp, covered by a thick red skin and a crown-shaped calyx. It contains a variety of beneficial ingredients, including flavonoids, ellagitannin, punicalagin, ellagic acid, vitamins, and minerals. Pomegranates possess numerous health benefits, and their use in disease treatment has been widely recognized since antiquity. This fruit was known to exhibit several biological properties, including antibacterial, anti-inflammatory, antioxidant, and anticancer activities. Pomegranate has been used in a variety of medical systems for the treatment and therapy of a wide range of diseases and illnesses. This review summarizes studies highlighting the potential role of pomegranate in the prevention and treatment of diseases related to respiratory and digestive systems.

Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by β-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB fromVerticillium dahliae, a plant pathogen. We first solved the crystal structure of VdPelB at 1.2Å resolution showing that it is a right-handed parallel β-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. VdPelB was most active on non-methylesterified pectins, at pH 8 in presence of Ca2+ions. VdPelB-G125R mutant was most active at pH 9 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released fromVerticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

The aim of this study was to evaluate the effects of fucoidan isolated from C. crinita on histamine-induced paw inflammation in rats, and on the serum levels of TNF-α, IL-1β, IL-6, and IL-10 in rats during systemic inflammation response. The levels of TNF-α in a model of acute peritonitis in rats were also investigated. The isolated crude fucoidan was identified as a sulfated xylogalactofucan with high, medium, and low molecular weight fractions and a content of fucose of 39.74%, xylose of 20.75%, and galactose of 15.51%. Fucoidan from C. crinita showed better anti-inflammatory effects in the rat paw edema model, and this effect was present during all stages of the experiment. When compared to controls, a commercial fucoidan from F. vesiculosus, the results also displayed anti-inflammatory activity on the 60th, 90th, and 120th minute of the experiment. A significant decrease in serum levels of IL-1β in rats treated with both doses of C. crinita fucoidan was observed in comparison to controls, whereas TNF-α concentrations were reduced only in the group treated with fucoidan from C. crinita at the dose of 25 mg/kg bw. In the model of carrageenan-induced peritonitis, we observed a tendency of decrease in the levels of the pro-inflammatory cytokine TNF-α in peritoneal fluid after a single dose of C. crinita fucoidan, but this did not reach the statistical significance margin. Single doses of C. crinita fucoidan did not alter serum levels of the anti-inflammatory cytokine IL-10 in animals with lipopolysaccharide-induced systemic inflammation.

International audience; The lack of one standardized method to evaluate microplastic pollution in different aquatic environments worldwide represent a gap to fill for the scientist's community. To help overcome this challenge, we adapted an aquatic drone, named Jellyfishbot®, to sample microplastics. The aquatic drone has been compared with the actual most used method for sampling MPs in surface waters: the Manta net. In order to test the reliability of the aquatic drone in different environments, samples were collected in a river and coastal waters sites. The results obtained with the two methods were similar in term of MPs abundances, shapes and colors. It provides also a better reproducibility and more accurate sampling of MPs located in the surface waters mainly the lighter and smaller ones. This sampling method has the advantage of combining the benefits of Manta net sampling (i.e. a representative surface water sampling method that covers a large sampling area and volume (several tens m3) with those of pump filtration and grab sampling (easy access to confined and hard-to-reach areas). This new sampling method could be applied in different aquatic environments making it possible to compare the data and hence become a new standardized approach to evaluate microplastic pollution levels.

Plasmodesmata (PD) pores connect neighbouring plant cells and enable direct transport across the cell wall. Understanding the molecular composition of these structures is essential to address their formation and later dynamic regulation. Here we provide a biochemical characterisation of the cell wall co-purified with primary PD of Arabidopsis thaliana cell cultures. To achieve this result we combined subcellular fractionation, polysaccharide analyses and enzymatic fingerprinting approaches. Relative to the rest of the cell wall, specific patterns were observed in the PD fraction. Most xyloglucans, although possibly not abundant as a group, were fucosylated. Homogalacturonans displayed short methylated stretches while rhamnogalacturonan I species were remarkably abundant. Full rhamnogalacturonan II forms, highly methyl-acetylated, were also present. We additionally showed that these domains, compared to the broad wall, are less affected by wall modifying activities during a time interval of days. Overall, the protocol and the data presented here open new opportunities for the study of wall polysaccharides associated with PD.

International audience; The properties of recombinant human gastric lipase produced in Arabidopsis thaliana roots have been investigated with the goal of determining the potential of the enzyme. This enzyme is stably bound to roots and can be extracted using a buffer at pH 2.2. This enzyme retains over 75% of its activity after two weeks at room temperature when stored in a pH 2.2 buffer. Some of this activity loss was due to the adsorption of the enzyme to the surface of the container. There was no loss of lipase activity in dehydrated roots stored at room temperature for 27 months. The half-life of the enzyme was approximately 15 min when stored in solution at 60 ° C whereas dried roots retained 90% lipase activity after one hour at 80 ° C. In vitro binding assays using different root cell wall extracts suggested that the lipase was bound to pectin in the roots. Lipase released from the root powder hydrolyzed tributyrin. The high stability of the recombinant human gastric lipase makes this enzyme a good candidate to be tested as a catalyst, whether in solution or bound to roots.

Leaves of Pistacia lentiscus were collected from two Algerian sites in the mountains and the littoral of the Tizi-Ouzou region. The harvest was conducted in four consecutive seasons on the same selected set of trees. Essential oils (EOs) were extracted by hydrodistillation; then, they were analyzed by gas chromatography coupled mass spectrometry (GC-MS). Forty-seven constituents could be detected and quantified, including α-pinene (2–13%), β-caryophyllene (8–25%), β-myrcene (0.3–19%), bornyl acetate (0.8–7%), δ-cadinene (3–8%), bisabolol (1–9%), β-pinene (0.9–7%), caryophyllene oxide (4–9%), and α-cadinol (3–11%). Antioxidant (AOx) activities of the EOs were assessed by ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assays. Significant differences in EO composition and AOx activities appeared dependent on the season and the site. Variations of AOx activities were significant for the FRAP and ABTS tests but not for DPPH. Characterization of the leaf fatty acyl (FA) profiles was performed by GC-MS. Variability appeared according to season and altitude. Polyunsaturated fatty acids levels were high (27–55%) at the coldest date and place. The levels of linolenic acyl in the leaves were significantly correlated with bisabolol levels in the EOs (Spearman’s correlation coefficient: 0.818). Such results will be useful for the sustainable local valorization of wild P. lentiscus. These data also open new routes for further studies on terpenoid biosynthesis using correlation networks and fluxomic approaches.

International audience; Erismadelphus exsul Mildbr bark is widely used in Gabonese folk medicine. However, little is known about the active compounds associated with its biological activities. In the present study, phytochemical profiling of the ethanolic extract of Erismadelphus exsul was performed using a de-replication strategy by coupling HPLC-ESI-Q/TOF with a molecular network approach. Eight families of natural compounds were putatively identified, including cyclopeptide alkaloids, esterified amino acids, isoflavonoid- and flavonoid-type polyphenols, glycerophospholipids, steroids and their derivatives, and quinoline alkaloids. All these compounds were identified for the first time in this plant. The use of molecular networking obtained a detailed phytochemical overview of this species. Furthermore, antioxidant (2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and ferric reducing capacity (FRAP)) and in vitro antimicrobial activities were assessed. The crude extract, as well as fractions obtained from Erismadelphus exsul, showed a better reactivity to FRAP than DPPH. The fractions were two to four times more antioxidant than ascorbic acid while reacting to FRAP, and there was two to nine times less antioxidant than this reference while reacting to DPPH. In addition, several fractions and the crude extract exhibited a significant anti-oomycete activity towards the Solanaceae phytopathogen Phytophthora infestans in vitro, and, at a lower extent, the antifungal activity against the wheat pathogen Zymoseptoria tritici had growth inhibition rates ranging from 0 to 100%, depending on the tested concentration. This study provides new insights into the phytochemical characterization and the bioactivities of ethanolic extract from Erismadelphusexsul bark.

International audience; Fusarium oxysporum is the one of the most common and impactful pathogens of flax. Cultivars of flax that show resistance to this pathogen have previously been identified. To better understand the mechanisms that are responsible for this resistance, we conducted time-lapse analysis of one susceptible and one resistant cultivar over a two-week period following infection. We also monitored changes in some metabolites. The susceptible cultivar showed a strong onset of symptoms from 6 to 8 days after inoculation, which at this time point, was associated with changes in metabolites in both cultivars. The resistant cultivar maintained its height and normal photosynthetic capacity but showed a reduced growth of its secondary stems. This resistance was correlated with the containment of the pathogen at the root level, and an increase in some metabolites related to the phenylpropanoid pathway.

International audience; There is no comprehensive method for differentiating between fresh and frozen-thawed fish fillets. This is an ongoing problem, particularly in relation to regulations. In this study, we showed the relevance of using the properties of mitochondria to discriminate fresh fish fillets from frozen-thawed fish fillets. The use of red muscle fibres of Gilthead sea bream allowed us to leave mitochondria in their physiological environment and to avoid possible alterations of mitochondrial membranes during isolation steps. The impact of freezing on fillets was evaluated by measuring the permeability of fibres and mitochondrial membranes to nicotinamide adenine dinucleotide + hydrogen (NADH). NADH permeability of fresh fillet fibres stored at 4°C was compared to the permeability of fibres extracted from frozen-thawed fillets. Two approaches were used to measure permeability changes: a spectrophotometric method that measured consumption of NADH by complex I, and an oxygraphic approach that measured stimulation of O 2 consumption by NADH. Fibres from frozen-thawed fillets were more permeable to NADH and were less sensitive to the permeabilizer alamethicin. The 2 sensitivity of this method allowed us to clearly detect red muscle fibres from frozen-thawed fish versus fresh fish fillets.

In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve element specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long distance transport as the plant grows – a defect that is aggravated when combined with another sieve element specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the sub36 cellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.

International audience; Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by β-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel β-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Reproducibility in organ size and shape is a fundamental trait of living organisms. The mechanisms underlying such robustness remain, however, to be elucidated. Taking the sepal of Arabidopsis as a model, we investigated whether variability of gene expression plays a role in variation of organ morphology. To address this question, we produced a dataset composed of both transcriptomic and morphological information obtained from 27 individual sepals from wild-type plants. Although nearly identical in their genetic background, environment, and developmental stage, these sepals exhibited appreciable variability in both morphology and transcriptome. We identified modules of co-expressed genes in sepals, three of which correlated significantly with morphology, revealing biologically relevant gene regulatory networks. Interestingly, cell-wall related genes were overrepresented in two of these three modules. Additionally, we found that highly variable genes were unexpectedly enriched in cell-wall related processes. We then analyzed sepal morphology from 16 cell-wall mutants and found that the more a gene is expressed in wild-type, the more variable the morphology of the corresponding mutant. Altogether, our work unravels the contribution of cell-wall related genes to the robustness of sepal morphology. More generally, we propose that canalizing traits during development could rely on the modulation of highly expressed genes.

International audience; Industrial chicory has been the subject of numerous studies, most of which provide clinical observations on its health effects. Whether it is the roasted root, the flour obtained from the roots or the different classes of molecules that enter into the composition of this plant, understanding the molecular mechanisms of action on the human organism remains incomplete. In this study, we were interested in three molecules or classes of molecules present in chicory root: fructose, chlorogenic acids, and sesquiterpene lactones. We conducted experiments on the murine model and performed a nutrigenomic analysis, a metabolic hormone assay and a gut microbiota analysis, associated with in vitro observations for different responses. We have highlighted a large number of effects of all these classes of molecules that suggest a pro-apoptotic activity, an anti-inflammatory, antimicrobial, antioxidant, hypolipidemic and hypoglycemic effect and also an important role in appetite regulation. A significant prebiotic activity was also identified. Fructose seems to be the most involved in these activities, contributing to approximately 83% of recorded responses, but the other classes of tested molecules have shown a specific role for these different effects, with an estimated contribution of 23–24%.

International audience; The exploration of certain microbial resources such as beneficial endophytic microorganisms is considered a promising strategy for the discovery of new antimicrobial compounds for the pharmaceutical industries and agriculture. Thirty-one endophytic bacterial strains affiliated with Bacillus, Janthinobacterium, Yokenella, Enterobacter, Pseudomonas, Serratia, and Microbacterium were previously isolated from vetiver (Chrysopogon zizanioides (L.) Roberty) roots. These endophytes showed antifungal activity against Fusarium graminearum and could be a source of antimicrobial metabolites. In this study, in particular, using high-throughput screening, we analyzed their antagonistic activities and those of their cell-free culture supernatants against three species of Fusarium plant pathogens, a bacterial strain of Escherichia coli, and a yeast strain of Saccharomyces cerevisiae, as well as their capacity to produce lipopeptides. The results showed that the culture supernatants of four strains close to B. subtilis species exhibited antimicrobial activities against Fusarium species and E. coli. Using mass spectrometry analyses, we identified two groups of lipopeptides (surfactins and plipastatins) in their culture supernatants. Whole-genome sequencing confirmed that these bacteria possess NRPS gene clusters for surfactin and plipastatin. In vitro tests confirmed the inhibitory effect of plipastatin alone or in combination with surfactin against the three Fusarium species.

Four b acteriophage i nsensitive m utants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following a challenge with the Brussowvirus SW13. The BIMs displayed an altered, sedimentation phenotype. Whole genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family-2-glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated r hamnose- g lucose p olymer (Rgp) biosynthesis locus (designated here as the rgp gene cluster). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis p oly s accharide p ellicle (PSP) initiator, WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescent labelling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide which is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. Importance: Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements which specify the surface receptor(s) is apparent. Our research identifies a single gene which is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

International audience; To address the issues related to high perishability and limited shelf life of food proteins from muscle origin, different innovative processing, and preservation techniques, as well as analytical methodological approaches have been developed to meet environmental challenges and consumer demand for food of high quality and sustainable production supported by circular economy principles. This development has been enhanced and increased during the ongoing age of the fourth industrial revolution (Industry 4.0), which has been gaining momentum since 2015, coming up with a range of automated and digitized technologies. This review provides an updated overview of the recently developed thermal and nonthermal processing and preservation technologies, along with selected advanced analytical techniques used in the industry of muscle foods. Particular attention has been given to Industry 4.0 technologies and their role in achieving smart production with high automation and digitalization rates. As in other industry sectors, technology breakthroughs are reshaping the food industry, including the muscle food sector. Emerging technologies, such as pulsed electric field, high-pressure processing, ohmic heating, nanotechnology, advanced mass spectrometry and hyperspectral imaging sensors are among the key elements in the current food revolution 4.0. Although Industry 4.0 offers countless possibilities, more studies are still needed to capture its full potential and further harness its technologies to solve current challenges and move forward toward Industry 5.0.

International audience; Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha-L-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4-D + 20.5 g/l KNO(3) ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A >150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming. This article is protected by copyright. All rights reserved.

International audience; In vitro culture of flax (Linum usitatissimum L.) was exposed to chitosan oligosaccharides (COS) in order to investigate the effects on the growth and secondary metabolites content in roots and shoots. COS are fragments of chitosan released from the fungal cell wall during plant-pathogen interactions. They can be perceived by the plant as pathogen-associated signals, mediating local and systemic innate immune responses. In the present study, we report a novel COS oligosaccharide fraction with a degree of polymerization (DP) range of 2-10, which was produced from fungal chitosan by a thermal degradation method and purified by an alcohol-precipitation process. COS was dissolved in hydroponic medium at two different concentrations (250 and 500 mg/L) and applied to the roots of growing flax seedlings. Our observations indicated that the growth of roots and shoots decreased markedly in COS-treated flax seedlings compared to the control. In addition, the results of a metabolomics analysis showed that COS treatment induced the accumulation of (neo)lignans locally at roots, flavones luteolin C-glycosides, and chlorogenic acid in systemic responses in the shoots of flax seedlings. These phenolic compounds have been previously reported to exhibit a strong antioxidant and antimicrobial activities. COS oligosaccharides, under the conditions applied in this study (high dose treatment with a much longer exposure time), can be used to indirectly trigger metabolic response modifications in planta, especially secondary metabolism, because during fungal pathogen attack, COS oligosaccharides are among the signals exchanged between the pathogen and host plant.

International audience; Plants are an everlasting inspiration source of biologically active compounds. Among these medicinal plants, the biological activity of extracts from some species of the Tillandsia genus has been studied, but the phytochemistry of the hardy species Tillandsia bergeri remains unknown. The aim of the present study was to perform the first phytochemical study of T. bergeri and to identify the compounds responsible for the antibacterial activity of T. bergeri extracts. Soxhlet extraction of predried and grinded leaves was first performed using four increasing polarity solvents. A bio-guided fractionation was performed using agar overlay bioautography as a screening method against 12 Gram-positive, Gram-negative, sensitive, and resistant bacterial strains. The results showed the inhibition of Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 29213 (MSSA), methicillin-resistant S. aureus N-SARM-1 (MRSA), and Staphylococcus caprae ATCC 35538 by the dichloromethane fraction. A phytochemical investigation led to the isolation and identification by high-resolution mass spectrometry and nuclear magnetic resonance of the two flavones penduletin and viscosine, responsible for this antibacterial activity. For viscosine, the minimum inhibitory concentration (MIC) value is equal to 128 μg/mL against MSSA and is equal to 256 μg/mL against MRSA and S. caprae. The combination of these compounds with vancomycin and cloxacillin showed a decrease in MICs of the antibiotics. Penduletin showed synergistic activity when combined with vancomycin against MSSA (FICI < 0.258) and S. caprae (FICI < 0.5). Thus, unexplored Tillandsia species may represent a valuable source for potential antibiotics and adjuvants.

International audience; Maize (Zea mays L.) is one of the major cereal crops in the world and is highly sensitive to low temperature. Here, changes in photosynthetic and cell wall metabolisms were investigated during a long chilling exposure in inbred line F2 and a low-lignin near-isogenic brown midrib3 mutant (F2bm3), which has a mutation in the caffeic acid O-methyltransferase (COMT) gene. Results revealed that the plant biomass was reduced, and this was more pronounced in F2bm3. Photosynthesis was altered in both lines with distinct changes in photosynthetic pigment content between F2bm3 and F2, indicating an alternative photoprotection mechanism between lines under chilling. Starch remobilization was observed in F2bm3 while concentrations of sucrose, fructose and starch increased in F2, suggesting a reduced sugar partitioning in F2. The cell wall was altered upon chilling, resulting in changes in the composition of glucuronorabinoxylan and a reduced cellulose level in F2. Chilling shifted lignin subunit composition in F2bm3 mutant to a higher proportion of p-hydroxyphenyl (H) units, whereas it resulted in lignin with a higher proportion of syringyl (S) residues in F2. On average, the total cell wall ferulic acid (FA) content increased in both genotypes, with an increase in ether-linked FA in F2bm3, suggesting a greater degree of cross-linking to lignin. The reinforcement of the cell wall with lignin enriched in H-units and a higher concentration in cell-wall-bound FA observed in F2bm3 as a response to chilling, could be a strategy to protect the photosystems.

Background: Breast cancer (BC) is the second most common cancer overall. In women, BC is the most prevalent cancer and the leading cause of cancer-related mortality. Triple-negative BC (TNBC) is the most aggressive BC, being resistant to hormonal and targeted therapies. Hypothesis/Purpose: The medicinal plant Origanum syriacum L. is a shrubby plant rich in bioactive compounds and widely used in traditional medicine to treat various diseases. However, its therapeutic potential against BC remains poorly investigated. In the present study, we screened the phytochemical content of an ethanolic extract of O. syriacum (OSEE) and investigated its anticancer effects and possible underlying mechanisms of action against the aggressive and highly metastatic human TNBC cell line MDA-MB-231. Methods: MTT, trans-well migration, and scratch assays were used to assess cell viability, invasion, or migration, respectively. Antioxidant potential was evaluated in vitro using the DPPH radical-scavenging assay and levels of reactive oxygen species (ROS) were assessed in cells in culture using DHE staining. Aggregation assays were used to determine cell-cell adhesion. Flow cytometry was used to analyze cell cycle progression. Protein levels of markers of apoptosis (BCL-2, pro-Caspase3, p53), proliferation (p21, Ki67), cell migration, invasion, or adhesion (FAK, E-cadherin), angiogenesis (iNOS), and cell signaling (STAT3, p38) were determined by immunoblotting. A chorioallantoic Membrane (CAM) assay evaluated in ovo angiogenesis. Results: We demonstrated that OSEE had potent radical scavenging activity in vitro and induced the generation of ROS in MDA-MB-231 cells, especially at higher OSEE concentrations. Non-cytotoxic concentrations of OSEE attenuated cell proliferation and induced G0/G1 cell cycle arrest, which was associated with phosphorylation of p38 MAPK, an increase in the levels of tumor suppressor protein p21, and a decrease of proliferation marker protein Ki67. Additionally, only higher concentrations of OSEE were able to attenuate inhibition of proliferation induced by the ROS scavenger N-acetyl cysteine (NAC), indicating that the anti-proliferative effects of OSEE could be ROS-dependent. OSEE stimulated apoptosis and its effector Caspase-3 in MDA-MB-231 cells, in correlation with activation of the STAT3/p53 pathway. Furthermore, the extract reduced the migration and invasive properties of MDA-MB-231 cells through the deactivation of focal adhesion kinase (FAK). OSEE also reduced the production of inducible nitric oxide synthase (iNOS) and inhibited in ovo angiogenesis. Conclusion: Our findings reveal that OSEE is a rich source of phytochemicals and has robust anti-breast cancer properties that significantly attenuate the malignant phenotype of MD-MB-231 cells, suggesting that O. syriacum may not only act as a rich source of potential TNBC therapeutics but may also provide new avenues for the design of novel TNBC drugs.

International audience; The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

Efficient design of functional proteins with higher thermal stability remains challenging especially for highly diverse sequence variants. Considering the evolutionary pressure on protein folds, sequence design optimizing evolutionary fitness could help designing folds with higher stability. Using a generative evolution fitness model trained to capture variation patterns in natural sequences, we designed artificial sequences of a proteinaceous inhibitor of pectin methylesterase enzymes. These inhibitors have considerable industrial interest to avoid phase separation in fruit juice manufacturing or reduce methanol in distillates, averting chromatographic passages triggering unwanted aroma loss. Six out of seven designs with up to 30 % divergence to other inhibitor sequences are functional and two have improved thermal stability. This method can improve protein stability expanding functional protein sequence space, with traits valuable for industrial applications and scientific research.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction. ispartof: MBIO vol:12 issue:6 ispartof: location:United States status: accepted

National audience; En dix ans, les recherches sur les particules plastiques, nano- (NP) et microplastiques (MP), et leurs contaminants associés se sont largement développées. L’amélioration des connaissances de ces familles de contaminant s’étant accrue, les pratiques analytiques ont dû évoluer et être adaptées. La garantie de la qualité des résultats générés ne peut être obtenue que par la mise en commun des expertises, ainsi que la mise en place d’infrastructures et l’utilisation d’appareillages complémentaires.Dans le cadre du CPER Marco, différents laboratoires (Anses, LOG et ULCO) ont mis en place la plateforme 3PAC de façon à partager leurs équipements analytiques, méthodes, expertises et connaissances.En terme d’infrastructure, de nombreuses possibilités existent. Tout d’abord des systèmes d’aquarium avec un environnement contrôlé sont disponibles pour réaliser des études d’exposition. Une pièce « microplastique » permet de réaliser des manipulations (digestion, filtration) sous atmosphère maîtrisée et ainsi limiter les risques de contamination. La caractérisation des particules (taille, forme) peut être réalisée à l’aide de divers équipements : loupes binoculaires ou directement par microscopie via l’utilisation des capteurs optiques du µ-Raman ou du µ-FT-IR. Enfin, l’identification des particules ou des contaminants associés est rendue possible par l’utilisation de techniques complémentaires soit spectroscopiques (µ-Raman, µ-FT-IR), soit spectrométriques (Py-GC/MS et Py-GC/Orbitrap). L’acquisition de nouveaux équipements, complémentaires, est programmée de façon à venir enrichir les possibilités analytiques (microscopie confocale, technologie light scattering).Dès aujourd’hui, l’expertise des différents partenaires permet de couvrir un large champ de recherches allant des études en laboratoire, aux prélèvements sur le terrain. A titre d’exemple, des travaux récents ont porté sur l’étude de sédiments et organismes prélevés in situ, la réalisation d’études d’encagement d’organismes marins, ou encore l’exposition de bivalves en conditions contrôlées.Désormais, l’ambition de la plateforme 3PAC est de répondre aux appels à projets nationaux et internationaux portant sur l’étude des particules plastiques et de leurs conséquences. Ces projets seront menés au sein d’un écosystème analytique idoine, regroupant expertises, technologies et infrastructures.

International audience; This paper describes an innovative way of using environmental scanning electron microscopy (ESEM) and the development of a suitable accessory to perform in situ observation of living seedlings in the ESEM. We provide details on fabrication of an accessory that proved to be essential for such experiments but inexpensive and easy to build in the laboratory, and present our in situ observations of the tissue and cell surfaces. Sample-specific configurations and optimized tuning of the ESEM were defined to maintain Arabidopsis and flax seedlings viable throughout repetitive exposure to the imaging conditions in the microscope chamber. This method permitted us to identify cells and tissues of the live plantlets and characterize their surface morphology during their early stage of growth and development. We could extend the application of this technique, to visualize the response of living cells and tissues to exogenous enzymatic treatments with polygalacturonase in Arabidopsis, and their interaction with hyphae of the wilt fungus Verticillium dahliae during artificial infection in flax plantlets. Our results provide an incentive to the use of the ESEM for in situ studies in plant science and a guide for researchers to optimize their electron microscopy observation in the relevant fields.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of these integrative biology approach, suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

International audience; The objective of this study is to determine the lighting conditions for an optimal production of Arthrospira (Spirulina) platensis quantitatively and qualitatively for the process most commonly used by producers. A design of experiments made it possible to obtain surface modelling of responses which were validated under pilot conditions. Spirulina growth, protein and pigment content were modelled as a function of light intensity and duration of illumination. For each of them, the optima have been determined: 14 h (hours light exposure)/10 Klux (light intensity) for the growth rate; 11.8 h/7.2 Klux for phycocyanin rate; 6.2Klux/12.6 h for chlorophyll rate; 6.6Klux/12.6 h for carotene rate. The protein rate is the lowest when lighting conditions are of 10.6 Klux and 14.4 h. It is then possible for the producer to partially obscure the ponds and/or increase the light intensity using additional lighting.

International audience; The interest of studying organic plastic additives (OPAs) is growing since many of them are considered as toxic for human beings, marine organisms and have been found in the ecosystem. The impact and the extent of this pollution is not fully understood because of the lack of methods able to comprehensively analyze the wide diversity of OPAs. This study aims at offering a quick and efficient way to identify specific harmful plastic additives in polymer materials. For this purpose, a general method, for identifying 49 OPAs, has been successfully developed using pyrolyzer/thermal desorption-gas chromatography coupled to a high-resolution mass spectrometer (Py/TD-GC-HRMS). The retention indices were calculated for each compound and a high-resolution mass spectra database for plastic additives was implemented. This method was then applied to characterize the OPAs included in food containers that are frequently found in the environment. Four samples made of polypropylene (PP) and polylactic acid (PLA) coming from two distinct suppliers were then selected. A total of 10 additives, which were plasticizers (phthalates and adipates), antioxidants (bisphenol B) and phosphorous flames retardants, were found in these food packaging. Generally, higher peak signals were detected in the PP samples in comparison to PLA samples, except for tributyl phosphate and bisphenol B that were exclusively found in PLA samples. Moreover, the experimentations pointed out additives differences between suppliers, for products intended for the same use and made of the same polymer, for both PP and PLA samples highlighting the complexity of identifying and generalizing the additive content of plastic materials.

Copepods represent an interesting alternative or a complement live food to brine shrimps and rotifers commonly used in aquaculture. They constitute the natural prey of many fish species and therefore do not require a potential nutritional enrichment. But an optimization of the microalgal diets used to feed copepods is essential to improve their mass culture. This study examined the effects of seven microalgal diets, namely single-species diets of Rhodomonas salina (R), Tisochrysis lutea (T), and Pavlova lutheri (=Diacronema lutheri) (P), two-species diets (R + T, T + P, and R + P), and a three-species diet (R + T + P), on the fatty acid and monosaccharide composition of the cyclopoid copepod Paracyclopina nana as well as its reproductive investment. Experiments were run during 15 days in 10-L beakers; starting with nauplii collected from a large 300-L batch culture. Copepods fatty acid contents were studied, particularly the relative amounts of docosahexaenoic acid (DHA) and eicosa-pentaenoic acid (EPA). The R + T, R, and T diets induced the highest total fatty acid amount in copepods. R + T and R also generated the lowest DHA/EPA ratios in copepods due to high EPA contents. The highest value of total monosaccharides was found in copepods fed with R + T + P. Diets R + T and R induced the greatest prosome volumes and clutch volumes in ovigerous females. Both prosome volume and clutch volume in P. nana ovigerous females were correlated to the individual EPA amount. The results demonstrated that all diets including R. salina enhanced the productivity of P. nana in mass culture, particularly when combined with T. lutea. R. salina, and T. lutea induced complementary fatty acid and monosaccharide profiles, confirming that R + T represents the best microalgae combination for productive culture of P. nana. Conversely, P. lutheri did not enhance the nutritional profile nor the fecundity of P. nana in the culture. This study is the first to demonstrate that R. salina is a suitable microalga for productive mass culture of P. nana for use as live food in aquaculture.

Purpose In this study, a combinatory approach was undertaken to assay the efficiency of fungal enzymatic cocktails from different fermentation conditions to degrade different lignocellulosic biomasses with the aim of finely characterizing fungal enzymatic cocktails. Methods Enzymatic assays (AZO and pNP-linked substrates and ABTS) were used to assess the composition of the fungal enzymatic cocktails for cellulase, xylanase and laccase activities. Comparisons were made with a new range of chromogenic substrates based on complex biomass (CBS substrates). The saccharification efficiency of the cocktails was evaluated as a quantification of the sugar monomers released from the different biomasses after incubation with the enzymatic cocktails. Results The results obtained showed striking differences between the AZO and pNP-linked substrates and the CBS substrates for the same enzymatic cocktails. On AZO and pNP-linked substrates, different hydrolysis profiles were observed between the different fungi species with Aspergillus oryzae being the most efficient. However, the results on CBS substrates were more contrasted depending on the biomass tested. Altogether, the results highlighted that assessing laccase activities and taking into account the complexity of the biomass to degrade were key in order to provide the best enzymatic cocktails. Conclusion The complementary experiments performed in this study showed that different approaches needed to be taken in order to accurately assess the ability of an enzymatic cocktail to be efficient when it comes to lignocellulosic biomass degradation. The saccharification assay proved to be essential to validate the data obtained from both simple and complex substrates.

Salinity affects plant growth and development as shown with the glycophyte model plant, Arabidopsis thaliana (Arabidopsis). Two Arabidopsis accessions, Wassilewskija (Ws) and Columbia (Col-0), are widely used to generate mutants available from various Arabidopsis seed resources. However, these two ecotypes are known to be salt-sensitive with different degrees of tolerance. In our study, 3-week-old Col-0 and Ws plants were treated with and without 150 mM NaCl for 48, 72, or 96 h, and several physiological and biochemical traits were characterized on shoots to identify any specific traits in their tolerance to salinity. Before salt treatment was carried out, a different phenotype was observed between Col-0 and Ws, whose main inflorescence stem became elongated in contrast to Col-0, which only displayed rosette leaves. Our results showed that Col-0 and Ws were both affected by salt stress with limited growth associated with a reduction in nutrient uptake, a degradation of photosynthetic pigments, an increase in protein degradation, as well as showing changes in carbohydrate metabolism and cell wall composition. These traits were often more pronounced in Col-0 and occurred usually earlier than in Ws. Tandem Mass Tags quantitative proteomics data correlated well with the physiological and biochemical results. The Col-0 response to salt stress was specifically characterized by a greater accumulation of osmoprotectants such as anthocyanin, galactinol, and raffinose; a lower reactive oxygen detoxification capacity; and a transient reduction in galacturonic acid content. Pectin degradation was associated with an overaccumulation of the wall-associated kinase 1, WAK1, which plays a role in cell wall integrity (CWI) upon salt stress exposure. Under control conditions, Ws produced more antioxidant enzymes than Col-0. Fewer specific changes occurred in Ws in response to salt stress apart from a higher number of different fascilin-like arabinogalactan proteins and a greater abundance of expansin-like proteins, which could participate in CWI. Altogether, these data indicate that Col-0 and Ws trigger similar mechanisms to cope with salt stress, and specific changes are more likely related to the developmental stage than to their respective genetic background.

Arabidopsis has become a powerful model to study morphogenesis, plant growth, development but also plant response to environmental conditions. Over 1000 Arabidopsis genomes are available and show natural genetic variations. Among them, the main reference accessions Wassilewskija (Ws) and Columbia (Col-0), originally growing at contrasted altitudes and temperatures, are widely studied, but data contributing to their molecular phenotyping are still scarce. A global quantitative proteomics approach using isobaric stable isotope labeling (Tandem Mass Tags, TMT) was performed on Ws and Col-0. Plants have been hydroponically grown at 16 h/8 h (light/dark cycle) at 23°C day/19°C night for three weeks. A TMT labeling of the proteins extracted from their shoots has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several proteins families possibly involved in the differential responses observed for Ws and Col-0 during plant development and upon environmental changes. In particular, Ws and Col-0 mainly differ in photosynthesis, cell wall-related proteins, plant defense/stress, ROS scavenging enzymes/redox homeostasis and DNA/RNA binding/transcription/translation/protein folding. This article is protected by copyright. All rights reserved.