Cyril Jousse

Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease

Objective There is substantial inter-individual diversity in the susceptibility of alcoholics to liver injury. Alterations of intestinal microbiota (IM) have been reported in alcoholic liver disease (ALD), but the extent to which they are merely a consequence or a cause is unknown. We aimed to demonstrate that a specific dysbiosis contributes to the development of alcoholic hepatitis (AH). Design We humanised germ-free and conventional mice using human IM transplant from alcoholic patients with or without AH. The consequences on alcohol-fed recipient mice were studied. Results A specific dysbiosis was associated with ALD severity in patients. Mice harbouring the IM from a patient with severe AH (sAH) developed more severe liver inflammation with an increased number of liver T lymphocyte subsets and Natural Killer T (NKT) lymphocytes, higher liver necrosis, greater intestinal permeability and higher translocation of bacteria than mice harbouring the IM from an alcoholic patient without AH (noAH). Similarly, CD45+ lymphocyte subsets were increased in visceral adipose tissue, and CD4+T and NKT lymphocytes in mesenteric lymph nodes. The IM associated with sAH and noAH could be distinguished by differences in bacterial abundance and composition. Key deleterious species were associated with sAH while the Faecalibacterium genus was associated with noAH. Ursodeoxycholic acid was more abundant in faeces from noAH mice. Additionally, in conventional mice humanised with the IM from an sAH patient, a second subsequent transfer of IM from an noAH patient improved alcohol-induced liver lesions. Conclusions Individual susceptibility to ALD is substantially driven by IM. It may, therefore, be possible to prevent and manage ALD by IM manipulation.

Salicylic Acid, an Ambimobile Molecule Exhibiting a High Ability to Accumulate in the Phloem

The ability of exogenous salicylic acid (SA) to accumulate in castor bean (Ricinus communis) phloem was evaluated by HPLC and liquid scintillation spectrometry analyses of phloem sap collected from the severed apical part of seedlings. Time-course experiments indicated that SA was transported to the root system via the phloem and redistributed upward in small amounts via the xylem. This helps to explain the peculiarities of SA distribution within the plant in response to biotic stress and exogenous SA application. Phloem loading of SA at 1, 10, or 100 μm was dependent on the pH of the cotyledon incubating solution, and accumulation in the phloem sap was the highest (about 10-fold) at the most acidic pH values tested (pH 4.6 and 5.0). As in animal cells, SA uptake still occurred at pH values close to neutrality (i.e. when SA is only in its dissociated form according to the calculations made by ACD LogD suite software). The analog 3,5-dichlorosalicylic acid, which is predicted to be nonmobile according to the models of Bromilow and Kleier, also moved in the sieve tubes. These discrepancies and other data may give rise to the hypothesis of a possible involvement of a pH-dependent carrier system translocating aromatic monocarboxylic acids in addition to the ion-trap mechanism.

Salicylic Acid Transport in Ricinus communis Involves a pH-Dependent Carrier System in Addition to Diffusion

Despite its important functions in plant physiology and defense, the membrane transport mechanism of salicylic acid (SA) is poorly documented due to the general assumption that SA is taken up by plant cells via the ion trap mechanism. Using Ricinus communis seedlings and modeling tools (ACD LogD and Vega ZZ softwares), we show that phloem accumulation of SA and hydroxylated analogs is completely uncorrelated with the physicochemical parameters suitable for diffusion (number of hydrogen bond donors, polar surface area, and, especially, LogD values at apoplastic pHs and Δ LogD between apoplast and phloem sap pH values). These and other data (such as accumulation in phloem sap of the poorly permeant dissociated form of monohalogen derivatives from apoplast and inhibition of SA transport by the thiol reagent p-chloromercuribenzenesulfonic acid [pCMBS]) lead to the following conclusions. As in intestinal cells, SA transport in Ricinus involves a pH-dependent carrier system sensitive to pCMBS; this carrier can translocate monohalogen analogs in the anionic form; the efficiency of phloem transport of hydroxylated benzoic acid derivatives is tightly dependent on the position of the hydroxyl group on the aromatic ring (SA corresponds to the optimal position) but moderately affected by halogen addition in position 5, which is known to increase plant defense. Furthermore, combining time-course experiments and pCMBS used as a tool, we give information about the localization of the SA carrier. SA uptake by epidermal cells (i.e. the step preceding the symplastic transport to veins) insensitive to pCMBS occurs via the ion-trap mechanism, whereas apoplastic vein loading involves a carrier-mediated mechanism (which is targeted by pCMBS) in addition to diffusion.

Tropane alkaloid profiling of hydroponic Datura innoxia Mill. Plants inoculated with Agrobacterium rhizogenes.

INTRODUCTION Hydroponics has been shown as a possible way to produce high quality plant biomass with improved phytochemical levels. Nevertheless, effects of plant biotic and abiotic environment can lead to drastic changes and plant growth conditions must be optimised. OBJECTIVE To evaluate how much microbes and Agrobacterium rhizogenes TR7 wild strain may affect the tropane alkaloid profile in Datura innoxia Mill. plants cultivated in hydroponic conditions. METHODOLOGY Datura innoxia Mill. plants were cultivated in hydroponic with sterile or non-sterile conditions. For half of the non-sterile plants, Agrobacterium rhizogenes TR7 strain was added to the nutrient solution for hydroponics. The tropane alkaloid content of leaves and roots was analysed by UFLC/ESI-HRMS and MS/MS. The metabolite profiles were compared using partial least square-discriminant analysis. RESULTS In sterile conditions, aerial parts contained more scopolamine than the roots. However, the diversity of tropane alkaloids was greater in roots. Furthermore, 21 known compounds and four non-elucidated tropane alkaloids were found. The tropane alkaloid profile was shown to be statistically different between sterile and non-sterile hydroponic conditions. The levels of 3-acetoxy-6-hydroxytropane and 3-hydroxylittorine were higher in plants inoculated with A. rhizogenes. Five other tropane compounds were found in higher amounts in non-axenic control plants. Hyoscyamine and scopolamine total contents were much higher in the whole plant co-cultivated with A. rhizogenes TR7 than in controls. Furthermore, the leaves and roots of axenic plants contained more alkaloids than non-sterile ones. CONCLUSION In hydroponic conditions, microbes induced variations of the phytochemical levels. Addition of A. rhizogenes TR7 into the nutrient solutions improved the total hyoscyamine and scopolamine production.

Co-occurrence of microcystin and anatoxin-a in the freshwater lake Aydat (France): analytical and molecular approaches during a three-year survey.

Cyanobacterial mass occurrence is becoming a growing concern worldwide. They notably pose a threat to water users when cyanotoxins are produced. The aim of this study was to evaluate the occurrence and the dynamics of two cyanotoxins: microcystin (MC) and anatoxin-a (ANTX-a), and of two of the genes responsible for their production (respectively mcyA and anaC) during three consecutive bloom periods (2011, 2012 and 2013) in Lake Aydat (Auvergne, France). MC was detected at all sampling dates, but its concentration showed strong inter- and intra-annual variations. MC content did not correlate with cyanobacterial abundance, nor with any genera taken individually, but it significantly correlated with mcyA gene abundance (R2=0.51; p=0.042). MC content and mcyA gene abundance were maximal when cyanobacterial abundance was low, either at the onset of the bloom or during a trough of biomass. The LC-MS/MS analysis showed the presence of ANTX-a in the 2011 samples. To our knowledge, this is the first report of the presence of this neurotoxin in a French lake. The presence of ANTX-a corresponded to the only year for which Anabaena did not dominate the cyanobacterial community alone, and several cyanobacterial genera were present, including notably Aphanizomenon. anaC gene detection by PCR was not coherent with ANTX-a presence, both gene and toxin were never found for a same sample. This implies that molecular tools to study genes responsible for the production of anatoxin-a are still imperfect and the development of new primers is needed. This study also highlights the need for better monitoring practices that would not necessarily focus only on the peak of cyanobacterial abundance and that would take cyanotoxins other than MC into account.

Improved stability of TMS derivatives for the robust quantification of plant polar metabolites by gas chromatography-mass spectrometry.

Plant metabolite profiling is commonly carried out by GC-MS of methoximated trimethylsilyl (TMS) derivatives. This technique is robust and enables a library search for spectra produced by electron ionization. However, recent articles have described problems associated with the low stability of some TMS derivatives. This limits the use of GC-MS for metabolomic studies that need large sets of qualitative and quantitative analyses. The aim of this work is to determine the experimental conditions in which the stability of TMS derivatives could be improved. This would facilitate the analysis of the large-scale experimental designs needed in the metabolomics approach. For good repeatability, the sampling conditions and the storage temperature of samples during analysis were investigated. Multiple injections of one sample from one vial led to high variations while injection of one sample from different vials improved the analysis. However, before injection, some amino acid TMS derivatives were degraded during the storage of vials in the autosampler. Only 10% of the initial quantity of glutamine 3 TMS and glutamate 3 TMS and 66% of α-alanine 2 TMS was detected 48 h after derivatization. When stored at 4 °C until injection, all TMS derivatives remained stable for 12 h; at -20 °C, they remained stable for 72 h. From the integration of all these results, a detailed analytical procedure is thus proposed. It enables a robust quantification of polar metabolites, useful for further plant metabolomics studies using GC-MS.

Chapter Six - Exploring Metabolome with GC/MS

Abstract The rapidly expanding field of metabolomics has been driven by major advances in analytical tools such as mass spectrometry and the corresponding hyphenated methods such as gas chromatography (GC–MS) but also in chemometrics and bioinformatics. The metabolomic field has been mainly developed in plant sciences and GC/MS was applied very early as it allowed determining a large number of metabolites in a wide range of samples. A typical metabolomic experiment includes (i) sample preparation, (ii) derivatization, (iii) metabolic profiling by GC/MS, (iv) treatment of data set, (v) identification of key or discriminatory metabolites and eventually (vi) their quantification. Each of these steps needs to be optimized and technical choices are determined depending on the scientific objective. This chapter gives a review of technologies and methods dedicated to GC/MS-based metabolomics, as well as the description of major advances in this area in recent years and perspectives for the future. Finally, the main application area of plant metabolomics, linked to phytochemistry, gene modifications and environmental disorders, are briefly presented.

Datura innoxia plants hydroponically-inoculated with Agrobacterium rhizogenes display an enhanced growth and alkaloid metabolism

BACKGROUND The production of secondary metabolites through the culture of entire plants is of great interest. Soilless culture, such as hydroponics, enables the control of plant growth and metabolism. Specific environmental conditions must be developed to maximize the productivity of medicinal plants used as efficient natural bioreactors. METHODS The nutrient solution of newly established hydroponic cultures ofDatura innoxia Mill. were inoculated with Agrobacterium rhizogenes (A.r.) wild strains (TR7, TR107, 11325 or 15834). Growth and the alkaloid contents of roots and aerial parts were analyzed. Axenic cultures were also performed with modified TR7 strains containing the egfp or gus reporter gene. In vitro isolated root cultures enabled the phenological and molecular demonstration of gene transfer. RESULTS A.r.TR 7 led to a greater improvement in plant secondary metabolism and growth. Positive expression of the reporter genes occurred. Isolation and subculture of some of the roots of these plants showed a hairy root phenotype; molecular tests proved the transfer of bacterial genes into the roots isolated from the plants. CONCLUSIONS Hyoscyamine and scopolamine productivity is enhanced after A.r. inoculation in the nutrient solution of hydroponic plants. Transformation events occur in the original roots of the plants. This leads to chimeric plants with a part of their roots harboring a hairy root phenotype. Such semi-composite plants could be used for successful specialized metabolite bioproduction in greenhouses.

Quality of antiepileptic drugs in sub-Saharan Africa: A study in Gabon, Kenya, and Madagascar

Epilepsy is a major public health issue in low‐ and middle‐income countries, where the availability and accessibility of quality treatment remain important issues, the severity of which may be aggravated by poor quality antiepileptic drugs (AEDs). The primary objective of this study was to measure the quality of AEDs in rural and urban areas in 3 African countries.