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International audience; Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant-specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography-mass spectrometry (LC-MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC-MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi-stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo-esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.

International audience; The pharmacologically active dichloromethane extracts of dried woad leaves (Isatis tinctoria L.), and the methanol extracts of comparable fresh leaves of the same plants, were analyzed by LC-MSn. The fresh leaf metabolite profile revealed a complex pattern of indolic compounds. Besides the known indigo precursors, isatan A, isatan B and indican, seven previously unreported indole derivatives were characterized: acetylindican, malonylindican, two dioxindole glucosides, dioxindole malonylglucoside, 6-hydroxyindole-3-carboxylic acid 6-O-glucoside and 6-hydroxyindole-3-carboxylic acid glucose ester. The integration of 122 compounds in fresh leaves and of five selected compounds (indoxyl, isatin, indigo, indirubin, and tryptanthrin) in dried leaves, formed the input data for a stepwise modelling procedure generating five predictive linear models. The structure of the predictive models and a cross validation provide evidence that the models could predict well or moderately well the accumulation of the selected lipophilic compounds, and were simple enough to be used in a woad cultivation program. PLS regression models relating each of the five selected dry leaf indolics to the fresh leaf metabolome were then fitted in order to deduct potential precursors and mechanisms leading to the formation of these lipophilic indolics in drying woad leaves. The models suggested glucobrassicin, isatan A and isatan B as the main candidate precursors of these compounds, besides a minor contribution of other fresh leaf indolics, including malonylindican, actylindican and dioxindole malonylglucoside. Dioxindole malonylglucoside was identified here as isatan C. The models further suggested that the accumulation of phenylpropanoid antioxidants in woad leaves has a negative impact on the formation of indoxyl, isatin, indigo, indirubin and tryptanthrin.

International audience; 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.

International audience; Pre-sowing treatment of seeds with cold plasma, a reactive mixture of charged particles, is an emerging, environmentally friendly method, shown to improve water absorption, germination and seedling growth in many crop species. We studied how plasma affects different structural layers in flax seed coats, and focused on the effects on some of the earliest germination events, directly related to imbibition: solute leakage and mucilage extrusion. We characterized the ultrastructure of flax seed coats using scanning electron microscopy (SEM) and observed mucilage extrusion in situ, using environmental SEM. Time-courses of water uptake and solute leakage during germination were recorded, respectively, by weighing the seeds and semi-quantitative metabolite profiling of the exudates using GC-MS, LC-MS and LC-UV. Plasma treatment perforated the cuticle of flax seeds but did not visibly etch the underlying mucilage secretory cell (MSC) walls. Plasma treated seeds took up more water than untreated seeds during imbibition. Mucilage expansion of untreated seeds detached the distal MSC walls from the radial walls and extrusion occurred, relatively slowly, via sparse cracks in the distal cell wall layer. In plasma treated seeds, mucilage extrusion occurred fast, via rupture of distal cell walls of almost every single MSC, and the junctions between distal and radial MSC walls remained intact. Initially, imbibitional solute leakage was higher in plasma treated seeds but the leakage had quasi stopped after one hour of imbibition. In untreated seeds, the leakage continued amply more than two hours after the start of imbibition. Fragments of the lignan macromolecule, stored in parenchymatous cells under the MCS, were released more abundantly in the spermosphere of plasma treated than untreated flax seeds. It is concluded that plasma treatment etches the cuticle of flax seeds and weakens the underlying MSC cell walls, and that such treatment leads to significantly faster recovery of membranes during imbibition, reducing leakage time in germinating seeds.

International audience; Background: The use of the desirability function approach combined with the response surface methodology (RSM), also called Desirability Optimization Methodology (DOM), has been successfully applied to solve medical, chemical, and technological questions. It is particularly efficient for the determination of the optimal conditions in natural or industrial processes involving different factors leading to the antagonist responses. Objectives: Surprisingly, DOM has never been applied to the research programs devoted to the study of plant responses to the complex environmental changes, and thus to biotechnological questions. Materials and Methods: In this article, DOM is used to study the response of Datura stramonium hairy roots (HRs), obtained by genetic transformation with Agrobacterium rhizogenes A(4) strain, subjected to the jasmonate treatments. Results: Antagonist effects on the growth and tropane alkaloid biosynthesis arc confirmed. With a limited number of experimental conditions, it is shown that 0.06 mM jasmonic acid (JA) applied for 24 h leads to an optimal compromise. Hyoscyamine levels increase by up to 290% after 24 h and this treatment does not significantly inhibit biomass growth. Conclusions: It is thus demonstrated that the use of DOM can efficiently - with a minimized number of replicates - leads to the optimization of the biotechnological processes.

Collaboration with: Université d’Orléans, 28000 Chartres, France, Université de Picardie Jules Verne, F-80037 Amiens, France De Montfort University Open access article Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

Plant secondary metabolites play a major role in plant adaptation to stress. Species belonging to Solanaceae family and Datura genus produce tropane alkaloids: D. stramonium, D. tatula and D. innoxia. These species are cultivated for their hyoscyamine (HS) content, whence the interest of this study to induce transformed roots of these species with strain A4 of Agrobacterium rhizogenes. Hairy roots (HRs) of Datura were established at high frequency by infecting vitroplants. All HRs (343 independent lines) were next employed to study the production of HS and growth. A screening of HRs alkaloid content by GC/MS is performed; it reveals, for the first time, the production of 13 alkaloids by the selected root lines. The selection of high productive line offers an interesting option to enhance the productivity. As HS is the dominant compound, the lines of Datura species were selected for their characteristics for biomass and HS production. The elicitors salicylic acid (SA) and acetyl salicylic acid (ASA) were also used to increase HS production. The results showed that the optimal concentration of the two elicitors (AS and ASA) was 0.1 mM. The highest HS content (17.94 ± 0.14 mg g−1 D.W.) obtained in HRs of D. tatula treated with 0.1 mM of acetyl salicylic acid.

International audience; The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography - mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with (13)C6-glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts.

Fumaria agraria (Fumariaceae) is an annual plant used traditionally in Algeria for various medicinal purposes. Alkaloids extracts from areal parts were subjected to solvent fractionation and GCMS analysis. Rates of alkaloids recorded were respectively of 700 mg/100 g of Dry Weight of total alkaloids “TA”, 400 mg/100 g DWof neutral fraction “NF”, 40 mg/100 g DW of acid fraction “AF” and 250 mg/100 g DWof the basic fraction “BF”. Protopine was found to be the most potent alkaloid of the total extract (41.55%). Fractionation showed that Protopine was found essentially in the BF fraction (60.7%). Antiproliferative activities of total alkaloids extract and BF fraction were assessed on MCF-7 and MDAMB- 231 breast cancer and MCF10A mammary normal cell lines, by trypan blue exclusion essay. Both TA extract and BF fraction showed potent antiproliferative activities against MCF-7 breast cancer cells in varying degrees with respective IC50 of 47.8 ± 1.8 µg/ml and 17.6 ± 0.4 µM, without cell death induction but no impact was observed on normal MCF-10A cell growth. MCF7 Cell line was most sensitive (IC50 of 17,6 ± 0,4µM) to BF fraction effect than MDAMB-231 one (IC50 of 36,4 ± 0,6µM). Isoquinolein alkaloids of Fumaria agraria and specially the fraction which contain Protopine showed potent antiproliferative activity against breast cancer cells.

Resume Introduction L’evolution du cancer de la prostate au stade metastatique avec resistance a la castration (mCPRC) est de mauvais pronostic avec une atteinte secondaire principalement osseuse et ganglionnaire. Le traitement par acetate d’abiraterone (AA), initialement approuve chez des patients presentant un mCPRC progressif apres traitement par docetaxel, a montre un avantage avec un gain en survie. Le taux de PSA (en ng/mL) et le performance status (PS) restent les examens de reference pour le suivi et l’evaluation de la reponse therapeutique chez ces patients. La TEP/TDM a la 18F-FCH a montre des resultats prometteurs pour le bilan metastatique et la recherche de la recidive chez les sujets atteints de cancer prostatique. Notre etude a pour objectif de preciser si cet examen peut permettre une evaluation precoce de la reponse therapeutique a un mois chez les patients presentant un mCPRC traites par AA. L’objectif secondaire est de preciser, chez les patients multi-metastatiques, s’il existe une heterogeneite de la reponse tumorale en fonction du type de localisations secondaires. Materiel et methodes Trente-huit patients presentant un mCPRC ont ete inclus dans un essai clinique multicentrique evaluant l’efficacite de l’acetate d’abiraterone avec mesure du taux de PSA (en ng/mL a M0, M1 et M3) et realisation d’examens TEP/TDM a la 18F-FCH avant l’instauration du traitement a M0 et apres un mois de traitement a M1 pour evaluer la reponse metabolique tumorale. La reponse TEP etant evaluee selon des criteres PERCIST modifies avec mesure du SUVpeak et des mesures de SUVmax, avec analyse de l’evolution metabolique globale pour tous les patients et en fonction du site tumoral pour les patients presentant une atteinte mixte. Cette reponse TEP a ete confrontee aux donnees de survie globale et a l’evolution du taux de PSA. Les patients ont ete classes « repondeurs metaboliques » s’ils presentaient a M1 une diminution de la moyenne des SUVmax ou des SUVpeak superieure a 30 % par rapport a M0 et « non repondeurs metaboliques » dans le cas contraire. Un classement similaire a ete effectue en fonction du site metastatique chez les patients presentant une atteinte mixte en differenciant l’atteinte osseuse et l’atteinte ganglionnaire. Resultats Vingt-neuf patients ont realise les examens TEP/TDM a la 18F-FCH a M0 et a M1 et ont ete retenus pour notre etude : 12 patients ont ete classes « repondeurs metaboliques » et 17 patients « non repondeurs metaboliques ». L’evolution metabolique globale etait heterogene chez 55 % des patients, majoritairement chez les patients « repondeurs metaboliques » : 11 patients sur 12. Il n’y avait pas de difference significative pour la survie globale entre les deux groupes. La diminution du taux de PSA au cours de l’etude etait plus importante chez les patients « repondeurs metaboliques » sans etre significative. Lors de l’analyse de l’evolution metabolique par sites metastatiques chez les 15 patients qui avaient une atteinte mixte ganglionnaire et osseuse il semblait y avoir un avantage en survie globale et une diminution plus marquee du taux de PSA chez les patients qui presentaient une reponse metabolique ganglionnaire seule ou associee a une reponse metabolique osseuse, cependant cette tendance n’etait pas statistiquement significative. Conclusion Cette etude a montre l’interet potentiel de la TEP/TDM a 18F-FCH pour l’evaluation de la reponse therapeutique a un mois chez les patients presentant un cancer prostatique metastatique resistant a la castration progressif apres docetaxel et traites par acetate d’abiraterone, elle a surtout permis de mettre en evidence l’heterogeneite de reponse tumorale chez les patients presentant une atteinte metastatique multiple.

The aim of this prospective study was to assess the predictive value of 18F-FDG PET/CT imaging for pathologic response to neoadjuvant chemotherapy (NACT) and outcome in inflammatory breast cancer (IBC) patients. Methods: Twenty-three consecutive patients (51 y ± 12.7) with newly diagnosed IBC, assessed by PET/CT at baseline (PET1), after the third course of NACT (PET2), and before surgery (PET3), were included. The patients were divided into 2 groups according to pathologic response as assessed by the Sataloff classification: pathologic complete response for complete responders (stage TA and NA or NB) and non–pathologic complete response for noncomplete responders (not stage A for tumor or not stage NA or NB for lymph nodes). In addition to maximum standardized uptake value (SUVmax) measurements, a global breast metabolic tumor volume (MTV) was delineated using a semiautomatic segmentation method. Changes in SUVmax and MTV between PET1 and PET2 (ΔSUV1–2; ΔMTV1–2) and PET1 and PET3 (ΔSUV1–3; ΔMTV1–3) were measured. Results: Mean SUVmax on PET1, PET2, and PET3 did not statistically differ between the 2 pathologic response groups. On receiver-operating-characteristic analysis, a 72% cutoff for ΔSUV1–3 provided the best performance to predict residual disease, with sensitivity, specificity, and accuracy of 61%, 80%, and 65%, respectively. On univariate analysis, the 72% cutoff for ΔSUV1–3 was the best predictor of distant metastasis-free survival (P = 0.05). On multivariate analysis, the 72% cutoff for ΔSUV1–3 was an independent predictor of distant metastasis-free survival (P = 0.01). Conclusion: Our results emphasize the good predictive value of change in SUVmax between baseline and before surgery to assess pathologic response and survival in IBC patients undergoing NACT.

Introduction Hairy root cultures of Linum sp. are an alternative for the high production of lignans. Linum perenne is known to produce arylnaphthalene-type lignans such as justicidin B, isojusticidin and diphyllin. Objective To elucidate the presence of aryltetralin-type lignan diastereoisomers, besides the known arylnaphthalene-type lignans, in hairy roots of Linum perenne, and to determine the configurations of one diastereoisomer of 6-methoxypodophyllotoxin (6-MPTOX). Methods Lignans from hairy root cultures of Linum perenne were extracted and separated by HPLC. Arylnaphthalene-type lignans were identified by LC-MS, according to the literature. Two diastereoisomers of aryltetralin-type lignans were analysed by mass spectrometry and NMR spectroscopy. Results Numerous arylnaphthalene-type lignans (diphyllin-2-hexose-pentose, diphyllin-3-pentose and diphyllin-hexose) were identified in hairy root cultures. Methoxypodophyllotoxin, an aryltetralin-type lignan, was also identified, as well as one diastereoisomer. This aryltetralin-type lignan could be derived via 7-hydroxymatairesinol as a hypothetical biosynthetic pathway. The stereochemical configurations of aryltetralin isomers were determined. Conclusion Arylnaphthalene and two diastereoisomers of aryltetralin-type lignans are produced in Linum perenne hairy root cultures. Matairesinol, the precursor of justicidin B, also seems to be converted into 6-MPTOX via 7-hydroxymatairesinol. This is the first report of the stereochemical configurations of an aryltetralin-type lignan other than podophyllotoxin (PTOX). Copyright © 2015 John Wiley & Sons, Ltd.

223Ra is a radiopharmaceutical used as unsealed source in nuclear medicine. In the case of staff inhalation contamination of 223Ra, methods to estimate the committed effective dose should be chosen with care. Three methods are available: whole-body measurement and gamma spectrometry for urine or faeces samples. Considering the analytical performances and uncertainties of these three methods, we propose recommendations for special dose assessment. As a first choice, due to its rapidity and its non-invasiveness, an in vivo analysis (with HPGe detector) is the most appropriate method. However, after 24 h, whole-body counting is not sensitive enough to detect a minimum effective dose of 1 mSv. Sufficient sensitivity can only be reached up to 8 days after contamination by true 24 h faeces samples analyses. Thus, despite its main drawbacks, this method appears to be more appropriate than urine to estimate the committed effective dose in addition to whole-body counting.

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.

Throughout the VEGETATION programme, the Flemish Institute for Technological Research (VITO) uninterruptedly hosted the prime user segment of both VEGETATION 1 and VEGETATION 2 multispectral instruments on board the Satellite Pour l’Observation de la Terre 4 (SPOT 4) and SPOT 5 satellites. Operational since the launch of SPOT 4 in March 1998, and foreseen to continue at least until the end of the SPOT 5 mission (anticipated in spring 2014), this user segment comprises a processing facility (PF), actively receiving, processing, correcting, archiving, and distributing the VEGETATION data and derived added-value products. First and foremost, the VEGETATION programme has been serving the needs of operational users – both institutional and commercial – requesting data in near-real time. However, scientific and educational users too benefited significantly, in particular from VEGETATION’s unique time series of the Earth’s land cover, and more specifically the vegetation cover. Over the years, the centralized ar...

International audience; Panax vietnamensis Ha et Grushv. is an important source of majonoside R2, a dammarane saponin with an ocotillol side-chain, which is an interesting pharmacological active compound. Thirty-one transformed root lines were obtained after the inoculation of in vitro shoot explants of P. vietnamensis with Rhizobium rhizogenes ATCC 15834 strain. The transgenic nature of the hairy roots was confirmed by PCR amplification. The established hairy root lines displayed four morphological phenotypes when cultured on hormone-free medium. Most of the cultures showed the characteristic traits of hairy roots with primary roots, an extensive lateral branching and rapid growth. Kinetic growth of these hairy root lines was measured for 90 days and a biomass production of about 10 g L-1 fresh weight was obtained. The ginsenoside content of hairy roots was identified by LC-MS and compared with that of 6 year-old rhizomes of P. vietnamensis. Majonoside R2 was recovered in trace amounts, but other ocotillol saponins, pseudoginsenoside F11 and vinaginsenoside R1, were recovered in larger proportions. These results indicate that hairy roots of P. vietnamensis present an interesting potential to produce ginsenosides and, in particular, ocotillol saponins.

Seeds of oil flax (Linum usitatissimum) are rich in polyunsaturated fatty acids and their composition is one of the major criteria for flax selection.Conventional breeding schemes take many years and need a lot of space in greenhouses and/or in fields to achieve genotypes with the desiredtraits. We have improved the half-seed method that allows flax line selection from their oil seed composition. Cotyledon tips were analysed byGC to determine their FAME profiles while the remaining seed was sown to regenerate flax plant. An optimisation of the GC enabled a sample tobe analysed every 4 minutes. Such a technique could be applied without loss of germination rate for excised seeds. Further experiments showedthat both seed parts had the same fatty acid profile as cotyledon tips. This non-destructive procedure based on the half-seed method and ultrafastGC analysis provides an efficient screening of flax genotypes according to their seed fatty acid composition over only one generation.

A better understanding of gliomas biology is now leading to a combined histo-molecular classification of these tumors. In anaplastic gliomas ongoing studies depend on 1p/19q codeletion status and in glioblastomas on MGMT methylation status. Advanced brain tumor imaging elicits a better identification of gliomas evolutive potential of. In low-grade gliomas, the importance of maximal resection and the role of chemotherapy are being increasingly recognized. In anaplastic gliomas, phase III studies have clarified the respective roles of chemotherapy and radiotherapy. In glioblastomas concomitant chemoradiotherapy is the standard. Most targeted therapies, namely anti-EGFR therapies have failed to demonstrate efficacy but anti-angiogenics are promising. The aim of this review is to discuss the main advances in adults' gliomas biology, imaging and treatment.

Secondary metabolites are mostly involved in the relationship of the organism with its environment, e.g. in plant defence mechanisms or as signal compound; their synthesis is often induced by different kinds of biotic and/or abiotic stresses. In general the major strategies to improve their productivity are: the optimization of growth and production media or induction of secondary metabolite pathways by elicitors and metabolic engineering. From this point of view, elicitation is largely employed as a strategy to mimic stress effects, which then lead to a physiological response involving secondary metabolite synthesis and accumulation into the hairy root (HR). Thus, studies on the response to elicitation for HR in accordance with nutritional status are necessary. In the present study, which focuses on tropane alkaloid production in Datura stramonium L. hairy roots, the effects of minerals in the B5 culture medium [O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158] and elicitor (jasmonic acid) are studied using a mathematical model approach (screening analysis) and factorial design 23 was carried out to study the response to elicitation in accordance with nutritional status of HR for [NO3−], [H2PO4−] and [Ca2+]. The modelization of different minerals effects showed a response to elicitation only after the 8th day of cultivation. The study of models coefficients revealed that nitrate [NO3−], phosphorus [H2PO4−] and calcium [Ca2+] levels and combinations [NO3−/Ca2+] and [NO3−/H2PO4−/Ca2+] were positive with elicitation; however, interaction effects [NO3−/H2PO4−] and [H2PO4−/Ca2+] were negative. The coefficients position on the 28th day of cultivation, with the Pareto chart allowed to adjust the mathematical model by eliminating the no significant effects [NO3−/H2PO4−], [H2PO4−/Ca2+] and [NO3−/H2PO4−/Ca2+]. As a result, we show that simulation with the adjusted model allows predicting the response to elicitation in accordance with the nutritional state of HR in nitrate and calcium. The measured values on the 28th day were statistically identical with calculated values obtained by mathematical models. These results revealed that HR, with limited nitrate, showed lower response to elicitation by jasmonic acid. Moreover, the adjusted model, of first degree, was not predictably beyond the experimental domain. As a general conclusion, NO3− (62.5 mM) must be added to the B5 medium at the beginning of the culture. The lack of these compounds reduced alkaloid metabolism, even in combination with elicitor treatments like jasmonic acid. The same trend was observed with Ca2+ (11.1 mM), H2PO4− (6.06 mM) and nitrate/calcium ratio (62.5 mM/11.1 mM), which also increased alkaloid production when combined with jasmonic acid 10 mM. These positive effects appeared at the 2nd week after initiation of the culture.

Recemment introduite dans la pratique clinique, la tomographie par emission de positons (TEP) au 18F-fluorodeoxyglucose (FDG) a trouve sa place dans le diagnostic et le bilan d’extension des affections cancereuses en raison de son aptitude a la caracterisation tissulaire des lesions. Son utilisation evolue vers l’evaluation de l’effet anti-tumoral des traitements anticancereux, en raison de la correlation entre la fixation d’un traceur metabolique et la viabilite des cellules malignes. Le fait que les effets metaboliques soient les premiers a etre induits par une chimiotherapie explique la precocite de l’information TEP quant a l’activite d’un traitement. Cependant, l’evaluation de l’efficacite therapeutique par TEP-FDG necessite une methodologie rigoureuse et doit prendre en compte les limites de la methode de quantification par le standardized uptake value (SUV). Par ailleurs, afin de depasser les limites du FDG, des traceurs plus specifiques sont developpes, et leur principale indication pourrait justement concerner l’evaluation de l’efficacite therapeutique. Les proprietes des emetteurs de positons permettent d’envisager le marquage des molecules therapeutiques elles-memes afin de tester in vivo les produits avant leur utilisation chez un patient donne. Ces perspectives constituent les bases d’une veritable personnalisation des traitements en cancerologie. Elles ouvrent un champ de recherche clinique immense, mais contribuer a ce mouvement impose de disposer des moyens necessaires pour l’imagerie et surtout pour la production des traceurs radioactifs. Compte tenu des performances actuelles des systemes TEP, le point reellement critique est la disponibilite des equipements permettant la conception et la synthese des radiopharmaceutiques de l’avenir.

Key points FDG-PET is now an established diagnostic tool in oncology. Fluorodeoxyglucose is not a specific tracer for malignant lesions but rather for elevated glucose metabolism, present not only in cancer but also in inflammatory and infectious lesions. FDG-PET has thus been suggested for diagnosis of fevers of unknown origin, deep bone or visceral infectious foci, inflammatory vasculitis or sarcoidosis and unknown primary tumors, all frequent situation in internal medicine. The main characteristics of FDG-PET are its ability to rule out focal inflammation or infection with a high degree of certainty when the examination is negative because of its good negative predictive value and its usefulness as an early marker of therapeutic response, compared with anatomy-based or conventional scintigraphic imaging. Large-scale prospective studies are necessary, however, before FDG-PET is integrated into routine clinical use. It should be compared with different techniques already validated (biology, radiology, conventional scintigraphic imaging) and its cost-effectiveness should be evaluated.

The spermosphere is the zone surrounding seeds where interactions between the soil, microbial communities and germinating seeds take place. The concept of the spermosphere is usually only applied during germination sensu stricto. Despite the transient nature of this very small zone of soil around the germinating seed, the microbial activities which occur there may have long-lasting impacts on plants. The spermosphere is indirectly characterized by either (i) seed exudates, which could be inhibitors or stimulators of micro-organism growth or (ii) the composition of the microbiome on and around the germinating seeds. The microbial communities present in the spermosphere directly reflect that of the germination medium or are host-dependent and influenced quantitatively and qualitatively by host exudates. Despite its strong impact on the future development of plants, the spermosphere remains little studied. This can be explained by the technical difficulties related to characterizing this concept due to its short duration, small size and biomass, and the number and complexity of the interactions that take place. However, recent technical methods, such as metabolite profiling, combining phenotypic methods with DNA- and RNA-based methods, could be used to investigate seed exudates, microbial communities and their interactions with the soil environment.

National audience; Aim: We studied whether the characterization of tumor texture in FDopa-PET/CT could assist in the identification of tumor grades in both primitive and recurrent gliomas. Materials and Methods: Eighty one patients with gliomas were studied, including 52 newly diagnosed tumors and 29 recurrent tumors. For each tumor, the SUVpeak and metabolic volume (MV) were measured, as well as 32 textural indices (TI). The ability of SUVpeak, MV and TI was investigated by using each index alone first (with ROC analyses), and then by using couples consisting of one TI with SUVpeak in a binomial model (with ROC analyses and a reclassification method). The pathological examination was assumed to provide the gold standard grade. Results: Neither SUVpeak nor MV could discriminate low-grade tumors (LG) from high-grade tumors (HG) in newly-diagnosed tumors, while SUVpeak alone could discriminate LG from HG in recurrent tumors (p=0.02). Combining a TI with SUVpeak led to a significant LG / HG discrimination for newly-diagnosed tumors (p = 0.01). Among all TI, entropy led to the best reclassification performance. Conclusion: The co-analysis of FDopa-PET/CT SUVpeak and well-selected TI (such as entropy) made it possible to improve the classification of newly-diagnosed gliomas.

The tropane alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the alkaloid spectrum, or about the metabolic dynamics induced by tropane alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the tropane alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among tropane alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to tropane alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the tropane alkaloids. Remodeling of the tropane alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxytropane, and 3-methylbutyryloxytropane was controlled independently of the majority of tropane alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of tropane alkaloids may be far more complex and finely controlled than previously expected.

L’acide α-linolenique (ALA) fait partie de la famille des acides gras dits omega 3. Il est dit essentiel parce qu’il ne peut pas etre synthetise dans notre organisme et doit donc etre fourni par l’alimentation. Les plantes, sous forme de fruits, de legumes verts, de graines ou d’huiles issues de ces graines constituent une source majeure d’ALA. Cette synthese a pour but de rappeler l’importance relative des vegetaux quant a leur capacite a nous fournir des acides gras essentiels. Elle vise a faire le point sur les differentes voies developpees par l’industrie agroalimentaire et a envisager les developpements futurs.

Hairy root cultures of Linum sp. are an alternative for the high production of lignans. Linum perenne is known to produce arylnaphthalene-type lignans such as justicidin B, isojusticidin and diphyllin.To elucidate the presence of aryltetralin-type lignan diastereoisomers, besides the known arylnaphthalene-type lignans, in hairy roots of Linum perenne, and to determine the configurations of one diastereoisomer of 6-methoxypodophyllotoxin (6-MPTOX).Lignans from hairy root cultures of Linum perenne were extracted and separated by HPLC. Arylnaphthalene-type lignans were identified by LC-MS, according to the literature. Two diastereoisomers of aryltetralin-type lignans were analysed by mass spectrometry and NMR spectroscopy.Numerous arylnaphthalene-type lignans (diphyllin-2-hexose-pentose, diphyllin-3-pentose and diphyllin-hexose) were identified in hairy root cultures. Methoxypodophyllotoxin, an aryltetralin-type lignan, was also identified, as well as one diastereoisomer. This aryltetralin-type lignan could be derived via 7-hydroxymatairesinol as a hypothetical biosynthetic pathway. The stereochemical configurations of aryltetralin isomers were determined.Arylnaphthalene and two diastereoisomers of aryltetralin-type lignans are produced in Linum perenne hairy root cultures. Matairesinol, the precursor of justicidin B, also seems to be converted into 6-MPTOX via 7-hydroxymatairesinol. This is the first report of the stereochemical configurations of an aryltetralin-type lignan other than podophyllotoxin (PTOX).

International audience; Due to their pronounced cytotoxic activity, a number of aryltetralin lignans (ATLs), such as podophyllotoxin (PTOX), are used as antitumor compounds. The production of such molecules from entire plants or plant cell-tissue-organ cultures is thus of interest to the pharmaceutical industry. Hairy root cultures constitute a good tool not only for phytochemical production but also for investigating plant secondary metabolism. This work reports on the growth and ATL biosynthesis in two hairy root cultures of Linum album Kotschy ex Boiss. and Linum flavum. The kinetics of accumulation of the intermediates of MPTOX biosynthesis and of their glucosylated forms are described over a 21-day period of growth. An accumulation of non-glucosylated forms of the ATLs during the exponential phase of the cultures is followed by an accumulation of the glucosylated forms during the stationary phase. Our results show a strong coordination of the biosynthetic paths derived from deoxypodophyllotoxin via deoxypodophyllotoxin 6-hydroxylase and deoxypodophyllotoxin 7-hydroxylase, and a coordinated glucosylation of podophyllotoxin, methoxypodophyllotoxin, and 5'-demethoxymethoxypodophyllotoxin. Furthermore, our results suggest an important role of β-peltatin-6-glucoside formation in the control of ATL accumulation in Linum hairy root cultures.

Studies on plant secondary metabolites have been increasing over the last 50 years. These molecules are known to play a major role in the adaptation of plants to their environment, but also represent an important source of active pharmaceuticals. Plant cell culture technologies were introduced at the end of the 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using in vitro systems, have been extensively studied with the objective of improving the production of secondary plant compounds. Undifferentiated cell cultures have been mainly studied, but a large interest has also been shown in hairy roots and other organ cultures. Specific processes have been designed to meet the requirements of plant cell and organ cultures in bioreactors. Despite all of these efforts of the last 30 years, plant biotechnologies have led to very few commercial successes for the production of valuable secondary compounds. Compared to other biotechnological fields such as microorganisms or mammalian cell cultures, this can be explained by a lack of basic knowledge about biosynthetic pathways, or insufficiently adapted reactor facilities. More recently, the emergence of recombinant DNA technology has opened a new field with the possibility of directly modifying the expression of genes related to biosyntheses. It is now possible to manipulate the pathways that lead to secondary plant compounds. Many research projects are now currently being carried out and should give a promising future for plant metabolic engineering.

Background The combination of paclitaxel and bevacizumab was EMA-approved as first-line therapy in metastatic breast cancer. Moreover, in vitro studies showed a potential antiangiogenic synergistic effect of paclitaxel and bevacizumab. Methods Between November 2008 and March 2010, this case series study included 15 patients with metastatic non squamous-cell lung carcinoma (NSCLC). Those were bevacizumab eligible and received the same regimen used in metastatic breast cancer with weekly paclitaxel (80 mg/m 2 , days 1, 8 and 15) and bevacizumab (10 mg/kg at days 1 and 15) after at least one prior line of chemotherapy. Efficacy was evaluated by CT-scan and PET-FDG every two months. Circulating endothelial progenitor cells (CEP) and circulating endothelial cells (CEC) levels were explored in a subset of patients. Results Median age 56 (36-75), female: 47%, never smokers: 27%, adenocarcinoma: 100%, PS 0-1: 87% and PS 3: 13%. All patients were treated with a first-line platinum-based doublet with or without bevacizumab and 70% of them with erlotinib in the second-line. No major toxicity was observed. Partial response (PR) rate was 44% (31-63%) using RECIST criteria on CT-scan, and 65% (29-88%) with PET FDG. PS improved in 33% of the cases. Median progression free survival was 4.6 months. An increase of CEC and CEP was observed in patients with NSCLC treated with paclitaxel and bevacizumab. Conclusion In this retrospective series, our results suggest efficacy signal in pre-treated metastatic NSCLC and warrant further assessment in a randomized clinical trial.

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l−1 of NaH2PO4), nitrate (2527 mg l−1 of KNO3), carbon source (10 g l−1 of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (tD equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems.

Kaposi sarcoma (KS) is a tumor caused by human herpesvirus 8, also known as KS-associated herpesvirus. Epidemiological varieties are represented by an epidemic form, strongly associated with AIDS, classic form, endemic form, and transplant-related form in association with severely immunosuppressive drug regimens, pointing to a role for immunodeficiency in the development of KS. We report the case of a woman who had preexisting immune system damage because of recurrent Hodgkin's lymphoma and presented with KS of the pelvis with high FDG uptake on PET/CT mimicking nodal relapse. Clinicians and nuclear medicine physicians should be aware that KS can present as intense FDG uptake. Moreover, our case emphasizes the importance of obtaining a biopsy.

The conditions for tricaprylin synthesis using Lipozyme (immobilized 1,3-specific Mucor miehei lipase) in a medium solely composed of glycerol and caprylic acid (octanoic acid) in stoichiometric amounts are 60°C with air ventilation in an open batch. The kinetics of mono-, di-, and triotanoylglycerol synthesis and caprylic acid esterification are correlated with those corresponding to water production and water activity along the esterification reaction. The technical feasibility of studying the time course of the reaction and water loss monitoring only by weight and Karl-Fisher measurements is also described.

Enzymatic synthesis of tricaprylin from caprylic acid (octanoic acid) and glycerol was carried out under solvent-free conditions. Kinetics of mono-, di- and tricaprylin synthesis and caprylic acid esterification in standard conditions (free glycerol) were slightly different from those obtained in the case of silica gel adsorbed glycerol. The critical role of the silica gel which behaves as a "reservoir" is proposed and it was shown that the use of silica gel facilitates reactions and improves the conversion yields at high glycerol concentrations.

Les métabolites secondaires végétaux sont des substances naturelles qui interviennent dans l’adaptation des plantes à leur environnement. Certains de ces métabolites secondaires végétaux sont dotés de propriétés pharmacologiques remarquables, qui en font des médicaments de premier ordre (paclitaxel de l’if, alcaloïdes indoliques de la pervenche de Madagascar etc.). Une caractéristique générale de ces métabolites secondaires végétaux tient à leur très faible concentration chez les plantes. Cette faible production végétale pose le problème de l’approvisionnement durable en ces composés naturels pour l’industrie pharmaceutique. Depuis les années 70, la biotechnologie végétale s’est saisie de la question. Diverses technologies ont été développées, comme les cultures de cellules végétales indifférenciées en bioréacteurs ou la culture de « hairy roots ». Les exemples de succès technologiques, s’ils existent, restent toutefois en nombre restreint. Le Laboratoire Agronomie et Environnement (UMR 1121, Université de Lorraine-INRA) a développé depuis 1996 une technologie alternative, reposant sur la culture de plantes médicinales en serres, pour la production de métabolites secondaires à partir de racines. Cette technologie intitulée « plantes à traire » consiste à récupérer à partir des racines, et de manière non destructive, les composés naturels recherchés. La même plante peut donc être utilisée indéfiniment afin de récolter les molécules d’intérêt. Cette technologie est aujourd’hui utilisée par la société Plant Advanced Technologie SA, spin-off du laboratoire, qui l’exploite à des fins de production de principes actifs tels que des alcaloïdes, terpènes ou composés polyphénoliques., Plant secondary metabolites display important functions related to plant stress adaptation. Some secondary compounds also exhibit remarkable pharmaceutical properties such as Paclitaxel or monoterpene alkaloids, respectively extracted from yew tree and Madagascar periwinkle. A general problem related to these secondary compounds is due to their low abundance in plant tissues. This is an issue for the pharmaceutical industry, constantly looking for reliable and sustainable sources of natural compounds. Since the ‘70s, plant biotechnologies have been developed to specifically address this question, with examples such as plant cell cultures or hairy roots in bioreactors. However, success stories, if they exist, are quite limited in this technological domain. Laboratoire Agronomie et Environnement (UMR 1121, Université de Lorraine-INRA) has developed since 1996 an alternative technology, based on greenhouse grown plants, and specifically designed to recover plant root metabolites in a non-destructive way for plants. With this so-called “plant milking technology”, the same plant can be used again so that to harvest the high-added value molecules. This technology is now exploited by the company Plant Advanced Technologies SA, a spin-off from the lab, for the production of alkaloids, terpenes or polyphenolic compounds from plants.

The production of secondary metabolites, and more specifically alkaloids, from medicinal plants is still an important objective for many research programs. When natural lead compounds have been discovered and when chemical synthesis cannot be easily performed, the extraction and purification of biomolecules from entire plants is generally the preferred solution. However, it is now established that plant cells and tissue cultures in bioreactors can constitute an alternative solution to this agronomical approach. Our research programs devoted to the production of tropane alkaloids from Datura innoxia and furanocoumarins from Ruta graveolens have shown that hydroponics and aeroponics, techniques situated in-between field and fermentor scales, enable the entire plants to be used as efficient bioreactors. Revisiting scientific advances made in the past decades, the ethical, legal, biological and technological aspects are discussed in the light of the most recent literature, in order to establish a roadmap for further developments of plant secondary metabolite production systems.

The effects of Tween 20 as permeabilizing agent on tropane alkaloids fromDatura innoxia Mill. hairy root cultures have been studied. The kinetics of the alkaloid release is detailed and shows three different stages: an initial rapid increase of the alkaloid level within the roots and in the culture medium, followed by a slower but higher increase of the alkaloid concentration in the medium. During this phase, the alkaloid concentration within the roots returned to a lower value. Finally, after a longer time, the quantity of hyoscyamine in the medium decreased significantly with a variable rate. According to the total alkaloid content per flask determinations under different conditions, it is clearly demonstrated that Tween treatment permeabilized the roots, but also acted as an inducer.