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Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.

Background Weaning is known to result in barrier dysfunction and villus atrophy in the immediate post-weaning phase, and the magnitude of these responses is hypothesized to correlate with changes in the glutathione (GSH) redox system. Therefore, these parameters were simultaneously measured throughout the weaning phase, in piglets differing in birth weight category and weaning age, as these pre-weaning factors are important determinants for the weaning transition. Low birth weight (LBW) and normal birth weight (NBW) littermates were assigned to one of three weaning treatments; i.e. weaning at 3 weeks of age (3w), weaning at 4 weeks of age (4w) and removal from the sow at 3 d of age and fed a milk replacer until weaning at 3 weeks of age (3d3w). For each of these treatments, six LBW and six NBW piglets were euthanized at 0, 2, 5, 12 or 28 d post-weaning piglets, adding up 180 piglets. Results Weaning increased the glutathione peroxidase activity on d 5 post-weaning in plasma, and duodenal and jejunal mucosa. Small intestinal glutathione-S-transferase activity gradually increased until d 12 post-weaning, and this was combined with a progressive rise of mucosal GSH up till d 12 post-weaning. Oxidation of the GSH redox status (GSH/GSSG Eh) was only observed in the small intestinal mucosa of 3d3w weaned piglets at d 5 post-weaning. These piglets also demonstrated increased fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase fluxes in the duodenum and distal jejunum during the experiment, and specifically demonstrated increased FD4 fluxes at d 2 to d 5 post-weaning. On the other hand, profound villus atrophy was observed during the weaning transition for all weaning treatments. Finally, LBW and NBW piglets did not demonstrate notable differences in GSH redox status, small intestinal barrier function and histo-morphology throughout the experiment. Conclusion Although moderate changes in the GSH redox system were observed upon weaning, the GSH redox status remained at a steady state level in 3w and 4w weaned piglets and was therefore not associated with weaning induced villus atrophy. Conversely, 3d3w weaned piglets demonstrated GSH redox imbalance in the small intestinal mucosa, and this co-occurred with a temporal malfunction of their intestinal barrier function.

Being traditionally utilized mainly as appetizers and herbal teas as well as used to ease abdominal pains, colds and gastrointestinal issues, the genusSalviaL. has gained significant consideration owing to its remarkable phytochemicals and industrial importance. The present study aimed to develop and validate an LC–MS/MS method for the qualitative and quantitative investigation of 19 fingerprint phytochemicals in six endemicSalviaspecies. The validation parameters of the developed LC–MS/MS method were repeatability (intermediate precision), recovery (accuracy), limits of detection and quantification, linearity and uncertainty (U% at 95% confidence level (k = 2)). Reversed-phase HPLC separation and mass spectrometry parameters were optimized for each analyte. Ethanol extracts of the studiedSalviaspecies collected in three consecutive years were screened for their fingerprint phytochemicals by using the developed and validated LC–MS/MS method. Moreover, studiedSalviaspecies were subjected to multivariate analysis such as principal component analysis techniques to demonstrate the variabilities in phytochemical contents by years and parts of the samples. Roots, flowers, leaves, branches and whole plant of theSalviaspecies collected in 2015, 2016 and 2017 were used for the analyses. It was observed that the roots and branches ofSalviaspecies were similar in terms of their salvianolic acid A, caffeic acid, and 6,7-dehydroroyleanone components. Accordingly, apigenin, rosmarinic acid, luteolin 7-O-glucoside, caffeic acid, salvianolic acid B, and 6,7-dehydroroyleanone were notable phytochemicals that were present in the studiedSalviaspecies.

Graphical abstract, The recent approval of messenger RNA (mRNA)-based vaccines to combat the SARS-CoV-2 pandemic highlights the potential of both conventional mRNA and self-amplifying mRNA (saRNA) as a flexible immunotherapy platform to treat infectious diseases. Besides the antigen it encodes, mRNA itself has an immune-stimulating activity that can contribute to vaccine efficacy. This self-adjuvant effect, however, will interfere with mRNA translation and may influence the desired therapeutic outcome. To further exploit its potential as a versatile therapeutic platform, it will be crucial to control mRNA’s innate immune-stimulating properties. In this regard, we describe the mechanisms behind the innate immune recognition of mRNA and provide an extensive overview of strategies to control its innate immune-stimulating activity. These strategies range from modifications to the mRNA backbone itself, optimization of production and purification processes to the combination with innate immune inhibitors. Furthermore, we discuss the delicate balance of the self-adjuvant effect in mRNA vaccination strategies, which can be both beneficial and detrimental to the therapeutic outcome.

Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function., Carminic acid is a widely applied red colorant that is still harvested from insects because its biosynthesis is not fully understood. Here, the authors identify and characterize a membrane-bound C-glucosyltransferase catalyzing the final step during carminic acid biosynthesis.

Membrane lipid composition varies greatly within submembrane compartments, different organelle membranes, and also between cells of different cell stage, cell and tissue types, and organisms. Environmental factors (such as diet) also influence membrane composition. The membrane lipid composition is tightly regulated by the cell, maintaining a homeostasis that, if disrupted, can impair cell function and lead to disease. This is especially pronounced in the brain, where defects in lipid regulation are linked to various neurological diseases. The tightly regulated diversity raises questions on how complex changes in composition affect overall bilayer properties, dynamics, and lipid organization of cellular membranes. Here, we utilize recent advances in computational power and molecular dynamics force fields to develop and test a realistically complex human brain plasma membrane (PM) lipid model and extend previous work on an idealized, "average" mammalian PM. The PMs showed both striking similarities, despite significantly different lipid composition, and interesting differences. The main differences in composition (higher cholesterol concentration and increased tail unsaturation in brain PM) appear to have opposite, yet complementary, influences on many bilayer properties. Both mixtures exhibit a range of dynamic lipid lateral inhomogeneities ("domains"). The domains can be small and transient or larger and more persistent and can correlate between the leaflets depending on lipid mixture, Brain or Average, as well as on the extent of bilayer undulations.

Terminalia laxiflora , Terminalia brownii and Anogeissus leiocarpus are used as decoctions, macerations, infusions and fumigations in East and West African traditional medicine for treatment of infectious diseases and their symptoms. Using this ethnopharmacological information as a guideline for our research and owing to the fact that these species have not been subjected to in depth antibacterial and phytochemical studies, thirty-nine extracts of various polarities of the stem bark, stem wood and roots were studied for growth inhibitory effects against the human pathogenic bacteria Staphylococcus epidermidis , Staphylococcus aureus , Micrococcus luteus and Pseudomonas aeruginosa . Our results indicate that the studied species contain antibacterial compounds of a wide range of polarities. All polar root extracts of T. laxiflora and various polar extracts of T. brownii roots, including hot water decoctions, gave broad-spectrum antibacterial effects and low MIC values of 39 μg/ml. The main ellagitannins in an ethyl acetate extract of the root of T. laxiflora were found to be corilagin and its derivative and punicalagin. A methanol extract of the roots of T. brownii contained methyl-(S)-flavogallonate and its derivative as the main identified ellagitannins. Moreover, both Terminalia species were found to contain ellagic acid xylopyranoside and methyl ellagic acid xyloside and pure ellagic acid was present in T. brownii . Pure punicalagin did not give as low MIC as an ethyl acetate extract of the roots of T. laxiflora , containing punicalagin as one of its main compounds, although this ellagitannin totally inhibited the growth of S. aureus at 125 μg/ml and P. aeruginosa at 500 μg/ml. Similarly, pure ellagic and gallic acid gave higher MIC values than the methanolic root extract of T. brownii against S. aureus and P. aeruginosa . Moreover, a Sephadex LH-20 fraction of the methanolic extract of the roots of T. brownii , enriched with methyl-(S)-flavogallonate and its isomer, gave higher MIC values than the crude methanolic extract. These results suggest that the polyphenols in the extracts might act synergistically with each other. A methanolic soxhlet extract of the roots of A. leiocarpus , containing ampelopsin, aromadendrin, taxifolin, pinosylvin and 4′-methylpinosylvin gave a low MIC value of 39 μg/ml against all bacterial strains used in this investigation. Our results demonstrate that the roots, stem bark and stem wood of T. brownii , T. laxiflora and A. leiocarpus are rich sources of (new) antimicrobial compounds and justify the uses of these plants for treatment of infections in African traditional medicine.

Human milk oligosaccharides have been recognized as an important, functional biomolecule in mothers’ milk. Moreover, these oligosaccharides have been recognized as the third most abundant component of human milk, ranging from 10−15 g/L in mature milk and up to and over 20 g/L reported in colostrum. Initially, health benefits of human milk oligosaccharides were assigned via observational studies on the differences between breastfed and bottle fed infants. Later, pools of milk oligosaccharides were isolated and used in functional studies and in recent years more specific studies into structure−function relationships have identified some advanced roles for milk oligosaccharides in the healthy development of infants. In other research, the levels, diversity, and complexity of human milk oligosaccharides have been studied, showing a wide variation in results. This review gives a critical overview of challenges in the analysis of human milk oligosaccharides. In view of the myriad functions that can be assigned, often to specific structures or classes of structures, it is very relevant to assess the levels of these structures in the human milk correctly, as well as in other biological sample materials. Ultimately, the review makes a case for a comparative, inter-laboratory study on quantitative human milk oligosaccharide analysis in all relevant biological samples.

Quercetin has been shown to alleviate mucosal damage and modulate the glutathione (GSH) redox system in the colon of rodents. In the current study, we assessed whether quercetin was able to mitigate small intestinal dysfunction in weaned pigs. Here, 224 weaned piglets were fed a diet containing quercetin at either 0, 100, 300, or 900 mg/kg diet until d14 post-weaning, followed by a common basal diet until d42. Eight animals per treatment were sampled at d5 and d14 post-weaning. In these animals, the small intestinal histomorphology, barrier function, and protein abundance of occludin, caspase-3, and proliferating cell nuclear antigen were assessed. None of these parameters were affected, and neither did quercetin improve performance up to d42 post-weaning. The GSH redox system was evaluated in blood, small intestinal mucosa, and liver. Quercetin did not affect the glutathione peroxidase, glutathione reductase, and glutamate&ndash, cysteine ligase activity in these tissues. In contrast, the hepatic glutathione transferase (GST) activity was significantly increased by quercetin supplementation at d5 post-weaning of 100, 300, and 900 mg/kg. Importantly, d5 was characterized by a more oxidized GSH redox status. To conclude, dietary quercetin had little effect on the small intestine, but did upregulate hepatic GST in the occurrence of redox disturbance.

Fat is an important component of human milk and infant formula (IF), delivering half of the energy a baby needs. Nowadays, mostly vegetable fats are used in IFs; however, the use of bovine milk fat in formulas is currently increasing. Bovine milk fat contains a composition of fatty acids and lipid components different from those of vegetable fats. We have compared the lipid profile of human and bovine milk with infant formulas with different fat sources. Furthermore, current knowledge of how infant digestion, absorption, metabolic responses, gut immunity, microbiota and/or cognition is affected by dietary fat is reviewed. The possible opportunities and drawbacks of the application of bovine milk fat in infant nutrition are described. Future perspectives for the development of IF containing bovine milk fat and future research directions are highlighted.

Sphingolipids (SPs) have both a structural role in the cell membranes and a signaling function that regulates many cellular processes. The enormous structural diversity and low abundance of many SPs pose a challenge for their identification and quantification. Recent advances in lipidomics, in particular liquid chromatography (LC) coupled with mass spectrometry (MS), provide methods to detect and quantify many low-abundant SP species reliably. Here we use LC-MS to compile a "murine sphingolipid atlas," containing the qualitative and quantitative distribution of 114 SPs in 21 tissues of a widely utilized wild-type laboratory mouse strain (C57BL/6). We report tissue-specific SP fingerprints, as well as sex-specific differences in the same tissue. This is a comprehensive, quantitative sphingolipidomic map of mammalian tissues collected in a systematic fashion. It will complement other tissue compendia for interrogation into the role of SP in mammalian health and disease. ispartof: CELL REPORTS vol:35 issue:11 ispartof: location:United States status: published

Bovine whey is an important ingredient in human nutrition and contains many biofunctional, glycosylated proteins. Knowledge on the glycoprotein composition of whey and whey products is valuable for the dairy industry. This paper describes a method for the characterisation of whey, or whey powders, by N-linked glycoprofile analysis. Application of the method for analysis of whey protein products showed clear differences in glycoprotein composition between concentrate, isolate and demineralised whey powders. The quantitative potential was explored by screening 100 pooled farm milk samples. IgG and lactoferrin protein concentrations determined by N-glycoprofile analysis matched well with ELISA results. The protein concentration of GlyCAM-1 was determined to be ≥1 mg mL−1. The approaches presented in this work allow simultaneous concentration estimation of the three major whey glycoproteins, lactoferrin, IgG and GlyCAM-1 on the basis of their N-linked glycoprofiles, also in highly processed samples where conventional methods of detection (ELISA) are less suitable.

International audience; The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.

International audience; 3-Carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) is a metabolite of furan fatty acid and a marker of fish oil intake. CMPF is described as a protein-bound uremic toxin and interacts with free oxygen radicals, which can induce cell damages. However, the clinical consequences of CMPF accumulation in haemodialysis patients remain poorly documented. The aims of this study are to investigate potential association between CMPF levels and (i) biochemical and nutritional parameters; (ii) cardiovascular events and (iii) mortality. Two hundred and fifty-two patients undergoing maintenance haemodialysis were included. Routine clinical biochemistry tests and assay for CMPF by HPLC technique were performed at the inclusion. Body composition parameters were measured using a bioimpedance spectroscopy method. The enrolled patients were prospectively monitored for cardiovascular events and mortality. CMPF level was positively correlated with nutritional parameters and lean mass and is significantly higher in patients without protein-energy wasting. However, the multivariate linear regression analysis indicated that CMPF level was not independently associated with albumin, prealbumin, creatinemia and body mass index. Elevated serum CMPF was not associated with mortality and cardiovascular morbidity. Our results indicate that CMPF is not a relevant uremic toxin in haemodialysis and in contrast could be a marker of healthy diet and omega 3 intakes.

Background: Given the growing interest in the health benefits of vitamin K, there is great need for development of new high-throughput methods for quantitative determination of vitamin K in plasma. We describe a simple and rapid method for measurement of plasma vitamin K1 (phylloquinone [PK]) and K2 (menaquinones [MK]-4 and -7). Furthermore, we investigated the association of fasting plasma vitamin K with functional vitamin K insufficiency in renal transplant recipients (RTR). Methods: We used HPLC-tandem mass spectrometry with atmospheric pressure chemical ionization for measurement of plasma PK, MK-4, and MK-7. Solid-phase extraction was used for sample clean-up. Mass spectrometric detection was performed in multiple reaction monitoring mode. Functional vitamin K insufficiency was defined as plasma desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) >500 pmol/L. Results: Lower limits of quantitation were 0.14 nmol/L for PK and MK-4 and 4.40 nmol/L for MK-7. Linearity up to 15 nmol/L was excellent. Mean recoveries were >92%. Fasting plasma PK concentration was associated with recent PK intake (ρ=0.41, p=0.002) and with plasma MK-4 (ρ=0.49, p Conclusions: The developed rapid and easy-to-use LC-MS/MS method for quantitative determination of PK, MK-4, and MK-7 in human plasma may be a good alternative for the labor-intensive and time-consuming LC-MS/MS methods and enables a higher sample throughput.

This study aimed to assess the lipophilic and phenolic extractives composition of cork of Quercus suber L. grown in Algeria by gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatograhy-mass spectrometry (UHPLC-MS), respectively. The phenolic cork oak extract was also evaluated for its scavenging activity towards DPPH radical, ABTS radical scavenging, reducing power and antibacterial activity against Staphylococcus aureus ATCC, Listeria innocua, Escherichia coil and Pseudomonas aeruginosa. The lipophilic fraction is mainly composed of thirty three compounds, with cerine, friedeline and betulinic acid as the major components. Furthermore, eighteen phenolic compouds were detected in the phenolic extract, with protocatechuic, ellagic and gallic acids as the major compounds. The methanol:water cork extract showed high DPPH scavenging ability with an ICso value of 5.69 mu g mL(-1) and an ABTS enhanced scavenging capacity when compared to BHT and trolox. A reducing power higher than BHA was also found. In addition, the phenolic fraction of cork was shown to inhibit the growth of Staphelococcus aureus (12.1 mm) and Pseudomonas aeroginisa (10.07 mm). These results indicate that algerian cork oak constitutes a good source of natutal bioactive compounds. Morever, this study is a relevant contribution to the valorization of algerian cork industrial residues, for which novel applications could be considered, namely the use of these extracts in food or nutraceutical industry. (C) 2015 Elsevier B.V. All rights reserved.

Lichens and free-living terrestrial algae are widespread across many habitats and develop successfully in ecosystems where a cold winter limits survival. With the goal of comparing photoprotective responses in free-living and lichenized algae, the physiological responses to chilling and photochilling conditions were studied in three lichens and their isolated algal photobionts together as well as in a fourth free-living algal species. We specifically addressed the following questions: (i) Are there general patterns of acclimation in green algae under chilling and photochilling stresses? (ii) Do free-living algae exhibit a similar pattern of responses as their lichenized counterparts? (iii) Are these responses influenced by the selection pressure of environmental conditions or by the phylogenetic position of each species? To answer these questions, photosynthetic fluorescence measurements as well as pigment and low molecular weight carbohydrate pool analyses were performed under controlled laboratory conditions. In general, photochemical efficiency in all free-living algae decreased with increasing duration of the stress, while the majority of lichens maintained an unchanged photochemical activity. Nevertheless, these patterns cannot be generalized because the alga Trebouxia arboricola and the lichen Ramalina pollinaria (associated with Trebouxia photobionts) both showed a similar decrease in photochemical efficiency. In contrast, in the couple Elliptochloris bilobata-Baeomyces rufus, only the algal partner exhibited a broad physiological performance under stress. This study also highlights the importance of the xanthophyll cycle in response to the studied lichens and algae to photochilling stress, while the accumulation of sugars was not related to cold acclimation, except in the alga E. bilobata. The differences in response patterns detected among species can be mainly explained by their geographic origin, although the phylogenetic position should also be considered, especially in some species. FM received a fellowship for PhD students (2012-2015) from the Government of the Basque Country as well as a mobility grant which allowed the collaboration between University of Basque Country (UPV/EHU) and University of Rostock. LG thanks the Deutsche Forschungsgemeinschaft for financial support (DFG GU 1278/1-1). The work was also supported by the project BFU 2010-15021 and CTM2014-53902-C2-2-P from the MINECO and the European Regional Development Fund ERDF (FEDER), and by the Basque Government (UPV/EHU-GV IT-624-13 and UPV/EHU-GV IT-1018-16).

Background: In this study the physiological implications of artificial rearing were investigated. Low (LBW) and normal birth weight (NBW) piglets were compared as they might react differently to stressors caused by artificial rearing. In total, 42 pairs of LBW and NBW piglets from 16 litters suckled the sow until d19 of age or were artificially reared starting at d3 until d19 of age. Blood and tissue samples that were collected after euthanasia at 0, 3, 5, 8 and 19 d of age. Histology, ELISA, and Ussing chamber analysis were used to study proximal and distal small intestine histo-morphology, proliferation, apoptosis, tight junction protein expression, and permeability. Furthermore, small intestine, liver and systemic redox parameters (GSH, GSSG, GSH-Px and MDA) were investigated using HPLC. Results: LBW and NBW artificially reared piglets weighed respectively 40 and 33% more than LBW and NBW sow-reared piglets at d19 (P < 0.01). Transferring piglets to a nursery at d3 resulted in villus atrophy, increased intestinal FD-4 and HRP permeability and elevated GSSG/GSH ratio in the distal small intestine at d5 (P < 0.05). GSH concentrations in the proximal small intestine remained stable, while they decreased in the liver (P < 0.05). From d5 until d19, villus width and crypt depth increased, whereas PCNA, caspase-3, occludin and claudin-3 protein expressions were reduced. GSH, GSSG and permeability recovered in artificially reared piglets (P < 0.05). Conclusion: The results suggest that artificial rearing altered the morphology, permeability and redox state without compromising piglet performance. The observed effects were not depending on birth weight.

International audience; Folates, also known as B9 vitamins, serve as donors and acceptors in one-carbon (C1) transfer reactions. The latter are involved in synthesis of many important biomolecules, such as amino acids, nucleic acids and vitamin B5. Folates also play a central role in the methyl cycle that provides one-carbon groups for methylation reactions. The important functions fulfilled by folates make them essential in all living organisms. Plants, being able to synthesize folates de novo, serve as an excellent dietary source of folates for animals that lack the respective biosynthetic pathway. Unfortunately, the most important staple crops such as rice, potato and maize are rather poor sources of folates. Insufficient folate consumption is known to cause severe developmental disorders in humans. Two approaches are employed to fight folate deficiency: pharmacological supplementation in the form of folate pills and biofortification of staple crops. As the former approach is considered rather costly for the major part of the world population, biofortification of staple crops is viewed as a decent alternative in the struggle against folate deficiency. Therefore, strategies, challenges and recent progress of folate enhancement in plants will be addressed in this review. Apart from the ever-growing need for the enhancement of nutritional quality of crops, the world population faces climate change catastrophes or environmental stresses, such as elevated temperatures, drought, salinity that severely affect growth and productivity of crops. Due to immense diversity of their biochemical functions, folates take part in virtually every aspect of plant physiology. Any disturbance to the plant folate metabolism leads to severe growth inhibition and, as a consequence, to a lower productivity. Whereas today's knowledge of folate biochemistry can be considered very profound, evidence on the physiological roles of folates in plants only starts to emerge. In the current review we will discuss the implication of folates in various aspects of plant physiology and development.

This study has been aimed at providing a qualitative and quantitative evaluation of selected phytochemicals such as phenolic acids, flavonoids, oleuropein, fatty acids profile, and volatile oil compounds, present in wild olive leaves harvested in Portugal, as well as at determining their antioxidant and cytotoxic potential against human melanoma HTB-140 and WM793, prostate cancer DU-145 and PC-3, hepatocellular carcinoma Hep G2 cell lines, as well as normal human skin fibroblasts BJ and prostate epithelial cells PNT2. Gallic, protocatechuic, p-hydroxybenzoic, vanillic acids, apigenin 7-O-glucoside, luteolin 7-O-glucoside, and rutin were identified in olive leaves. The amount of oleuropein was equal to 22.64 g/kg dry weight. (E)-Anethole (32.35%), fenchone (11.89%), and (Z)-3-nonen-1-ol (8%) were found to be the main constituents of the oil volatile fraction, whereas palmitic, oleic, and alpha-linolenic acid were determined to be dominating fatty acids. Olive leaves methanol extract was observed to exerted a significant, selective cytotoxic effect on DU-145 and PC-3 cell lines. Except the essential oil composition, evaluated wild olive leaves, with regard to their quantitative and qualitative composition, do not substantially differ from the leaves of other cultivars grown for industrial purposes and they reveal considerable antioxidant and cytotoxic properties. Thus, the wild species may prove to be suitable for use in traditional medicine as cancer chemoprevention. Polish Ministry of Science and Higher Education [K/ZDS/004122, K/ZDS/006239, K/ZDS/006228] info:eu-repo/semantics/publishedVersion

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Haptoglobin (Hp) is an acute phase protein that has recently been linked to components of the metabolic syndrome (MetS). We aimed to evaluate Hp as marker of MetS, and to assess its association with long-term outcome in renal transplant recipients (RTR). We measured plasma Hp in a prospective cohort of 699 stable RTR and 149 healthy controls. Median plasma Hp concentration in RTR was 1.4 [interquartile range (IQR), 1.0–1.8] g/L, which was higher compared to 1.1 [0.9–1.4] g/L in controls (P

CHEMTAX was used to assess the relative contribution of the main phytoplankton classes to the total concentration of Chlorophyll a (Chl a) from the waters off SW coast of Portugal. Sampling campaigns were carried out during all seasons from 2008 to 2012, at three stations located 2, 10 and 18 km from the coast. Samples were taken from the surface, mid-Secchi and Secchi depth, for the determination of Chl a and other phytoplanktonic pigments by HPLC. Supporting data were also obtained including dissolved inorganic nutrients, salinity, transparency, temperature and upwelling indices. The CHEMTAX results were also related to microscopy counts and also spectral analysis of absorption of other samples from the same sampling campaigns. The pigment results showed that diatoms dominated from early spring to summer, coinciding with upwelling conditions, while cryptophytes, prymnesiophytes and prasinophytes dominated in autumn and winter, coinciding with seasonal stratification. Although the contribution of cyanobacteria to total Chl a was generally low, there were occasional sampling campaigns where it was exceptionally high, but these appeared not to be related to upwelling. Dinoflagellates and chrysophytes were minority groups although the pigment marker peridinin that was used to distinguish dinoflagellates was not adequate for distinguishing all the members of this group. CHEMTAX was particularly useful for discriminating between the smaller (0–20 μm) classes of the microplankton that could not be easily identified by microscopy., This research was funded by European Space Agency as part of the project “Technical assistance for the validation of MERIS marine products at Portuguese oceanic and coastal sites” (contract n° 21464/08/I-OL). P.C. Goela and S. Cristina are funded by PhD grants from FCT (SFRH/BD/78356/2011 and SFRH/BD/78354/2011, respectively); S. Danchenko is funded by Erasmus Mundus EMJD MACOMA; A. Newton is funded by EU FP7 project DEVOTES (grant agreement no. 308392), www.devotes-project.eu. J. Icely is funded by EU FP7 AQUA_USER (grant agreement no. 607325) www.aqua-users.eu.

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of 1H NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in &ldquo, hairy root&rdquo, organ cultures of R. strica.

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2011 and mid- 2012. It covers the major mycotoxins aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. A section on mycotoxins in botanicals and spices is also included. Methods for mycotoxin determination continue to be developed using a wide range of analytical systems ranging from rapid immunochemical-based methods to the latest advances in mass spectrometry. This review follows the format of previous reviews in this series (i.e. sections on individual mycotoxins), but due to the rapid spread and developments in the field of multimycotoxin methods by liquid chromatography-tandem mass spectrometry, a separate section has been devoted to advances in this area of research.

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on EstroG-100 (TM) as a novel food (NF) submitted pursuant to Regulation (EC) No 258/97 of the European Parliament and of the Council. The NF is EstroG-100 (TM), a hot-water extract of a mixture of three herbal roots (Cynanchum wilfordii Hemsley, Phlomis umbrosa Turcz. and Angelica gigas Nakai), which is concentrated and spray-dried. The information provided on the composition, specifications and stability of the NF is sufficient, and does not raise safety concerns. The applicant intends to use EstroG-100 (TM) in food supplements, with a proposed maximum intake level of 514 mg/day. The target population is post-menopausal women. The Panel considers that the information provided does not raise safety concerns as regards the genotoxicity of the NF. The Panel considers that the no-observed-adverse effect level (NOAEL) derived from the subchronic 90-day oral toxicity study with EstroG-100 (TM), which was supported by observations in other studies, is 500 mg/kg body weight (bw) per day. Taking into account the NOAEL and the proposed maximum intake level, the Panel considers that the margin of safety of 68 is not sufficient. Based on the absence of chronic toxicity data, increase in effects with exposure duration in toxicity studies, and the absence of investigations of liver parameters and haematology in human studies, the Panel applies the uncertainty factor of 200 to derive the maximum safe intake level for the NF. Thus, the Panel concludes that the NF, EstroG-100 (TM), is safe for the use in food supplements at the maximum intake level of 175 mg/day for an adult of 70 kg bw. (C) 2016 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

Bovine whey protein products are used as a base ingredient in infant formulas to optimize the amino acid pattern to a more human-like composition. Although the protein composition of bovine milk has been studied in detail, glycosylation details of commercial whey protein products are missing. To this end, both the N- and O-glycans of such a protein concentrate were sequentially released, the N-glycans enzymatically and the O-glycans chemically (reducing and nonreducing conditions). For the structural analysis of the N- and O-glycans a combination of MALDI-TOF-MS, one-dimensional H-1 NMR spectroscopy, Wisteria floribunda agglutinin affinity chromatography, HPAEC-PAD profiling, and HPLC-FD profiling (2-aminobenzamide derivatives), together with exoglycosidase treatments, were used. A mixture of over 60 N-glycans and 10 O-glycans was characterized, giving a detailed insight into the glycosylation of a bovine whey protein product, Deminal 90, which is applied as an ingredient for infant formulas.

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography-high-resolution mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5-7.7 % and within-lab reproducibility (RSD(WLR)) of 17 %. The method was used for analysis of100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops.

International audience; Sphingolipids are emerging as important mediators of cellular and developmental processes in plants, and advances in lipidomics have yielded a wealth of information on the composition of plant sphingolipidomes. Studies using Arabidopsis thaliana showed that the dihydroxy long-chain base (LCB) is desaturated at carbon position 8 (d18:1?8). This raised important questions on the role(s) of sphingosine (d18:1?4) and sphingosine-1-phosphate (d18:1?4-P) in plants, as these LCBs appear to be absent in A.similar to thaliana. Here, we surveyed 21 species from various phylogenetic groups to ascertain the position of desaturation of the d18:1 LCB, in order to gain further insights into the prevalence of d18:1?4 and d18:1?8 in plants. Our results showed that d18:1?8 is common in gymnosperms, whereas d18:1?4 is widespread within nonseed land plants and the Poales, suggesting that d18:1?4 is evolutionarily more ancient than d18:1?8 in Viridiplantae. Additionally, phylogenetic analysis indicated that the sphingolipid ?4-desaturases from Viridiplantae form a monophyletic group, with Angiosperm sequences falling into two distinct clades, the Eudicots and the Poales. We propose that efforts to elucidate the role(s) of d18:1?4 and d18:1?4-P should focus on genetically tractable Viridiplantae species where the d18:1 LCB is desaturated at carbon position 4.

Microorganisms depend on their ability to modulate their metabolic composition according to specific circumstances, such as different phases of the growth cycle and circadian rhythms, fluctuations in environmental conditions, as well as experimental perturbations. A thorough understanding of these metabolic adaptations requires the ability to comprehensively identify and quantify the metabolome of bacterial cells in different states. In this review, we present an overview of the diverse metabolomics approaches recently adopted to explore the metabolism of a wide variety of microorganisms. Focusing on a selection of illustrative case studies, we assess the different experimental designs used and explore the major achievements and remaining challenges in the field. We conclude by discussing the important complementary information provided by computational methods such as genome-scale metabolic modeling, which enable an integrated analysis of metabolic state changes in the context of overall cellular physiology.

International audience; Food supplements can contain polycyclic aromatic hydrocarbons (PAH). The European Food Safety Authority (EFSA) has defined 16 priority PAH that are both genotoxic and carcinogenic and identified 8 priority PAH (PAH8) or 4 of these (PAH4) as good indicators of the toxicity and occurrence of PAH in food. The current study aims to determine benzo[a]pyrene and other EFSA priority PAH in different categories of food supplements containing botanicals and other ingredients. In 2003 up to 2008, benzo[a]pyrene exceeded the limit of quantification (LOQ) in 553 (44%) of 1258 supplements with a lower bound mean of 3.37 µg kg-1. In 2008 and 2009, benzo[a]pyrene and 12 other EFSA priority PAH were determined in 333 food supplements. Benzo[a]pyrene exceeded the LOQ in 210 (63%) food supplements with a lower bound mean of 5.26 µg kg-1. Lower bound mean levels for PAH4 and PAH8 (-indeno[1,2,3-cd]pyrene) were 33.5 µg kg-1 and 40.5 µg kg-1, respectively. Supplements containing resveratrol, Ginkgo biloba, St. John's wort and propolis showed relatively high PAH4 levels in 2008 and 2009. Before 2008, supplements with these ingredients and also dong quai, green tea or valerian contained relatively high benzo[a]pyrene levels. On average, PAH4 intake resulting from food supplement use will be at the lower end of the range of contributions of main food groups to PAH4 exposure, although individual food supplements can contribute significantly to PAH4 exposure. Regular control of EFSA indicator PAH levels in food supplements may prove a way forward to further reduce the intake of PAH from food.

In the past decades, there has been a renewed interest in the use of natural dye plants for textile dyeing, e.g. Reseda luteola (weld). Its main yellow dye constituents are the flavones luteolin-7,3'-O-diglucoside, luteolin-7-O-glucoside and luteolin. The aim of this work was to develop a simple validated industrially usable quantitative method to assess the flavone content of R. luteola samples. The flavones were overnight extracted from the dried and ground aerial parts of the plant at room temperature via maceration with methanol-water 8:2. Afterwards, they were quantified through internal standardisation against chrysin by RP-HPLC-UV at 345 nm. The efficiency of the one-step extraction was 95%. The limits of detection (LOD) and quantitation (LOQ) were ≤ 1 ng and ≤ 3 ng, respectively, providing ample sensitivity for the purpose. The precision expressed as relative standard deviation of the entire method was

Recently, it was found that applying curvilinear shear flow in a cone-cone shearing device to wheat flour dough induces separation, resulting in a gluten-enriched fraction in the apex of the cone and gluten-depleted fraction at the outer part. This article describes whether fractionation of the various proteineous components occurs during and after separation of Soissons wheat flour. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high performance liquid chromatography (SE-HPLC) were found to be suitable techniques for this. It is concluded that all protein fractions migrate to the center of the cone as a result of which the composition of the gluten-enriched fraction remains rather similar to that in the original flour. However, the larger glutenin polymer fraction migrated faster, as a result of which the concentration of large polymers was increased with a factor 2.4 compared to that of Soissons flour. The concentration of monomers in the gluten-enriched fraction was decreased to 70% of the original concentration in the original wheat flour.

This review highlights developments in mycotoxin analysis and sampling over a period between mid-2008 and mid-2009. It covers the major mycotoxins: aflatoxins, alternaria toxins, cyclopiazonic acid, fumonisins, ochratoxin, patulin, trichothecenes and zearalenone. Developments in mycotoxin analysis continue, with emphasis on novel immunological methods and further description of LC-MS and LC-MS/MS, particularly as multimycotoxin applications for different ranges of mycotoxins. Although falling outside the main emphasis of the review, some aspects of natural occurrence have been mentioned, especially if linked to novel method developments.

PURPOSE. The vitreous body of the human eye undergoes progressive morphologic changes with aging. Since the enzymatic collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are known to be important for the integrity of the collagen matrix, the presence in the vitreous on aging was studied.METHODS. Vitreous bodies (VBs; n = 143) from 119 donors (age 4-80 years; mean +/- SD, 54.3 +/- 17.0 years) were carefully dissected. After weighing and freeze-drying, all samples were analyzed by high performance liquid chromatography. Left and right eyes of 24 donors were compared and, for age-related phenomena, 119 single eyes were used.RESULTS. Within one donor, no significant differences were found between left and right eyes. On aging, VB wet weight (4.42 +/- 0.84 g) accumulates until 35 years and decreases thereafter. Collagen content (0.30 +/- 0.14 mg), HP per triple helix (TH; 0.55 +/- 0.18), and (HP plus LP)/TH (0.61 +/- 0.19) increase until 50 years followed by a decrease, whereas LP/TH (0.057 +/- 0.018) accumulates until 50 years and remains constant thereafter. The ratio between HP and LP (range, 0.42 - 31.0; median, 10.0) is constant over time.CONCLUSIONS. The accumulation of enzymatic collagen cross-links until 50 years is consistent with collagen maturation and possible collagen synthesis in the human vitreous body. The decline of collagen cross-links after 50 years is consistent with collagen breakdown. (Invest Ophthalmol Vis Sci. 2009; 50: 1041-1046) DOI: 10.1167/iovs.08-1714

A thermophilic spore-forming bacterium (strain AMP) was isolated from a thermophilic methanogenic bio- reactor that was fed with cobalt-deprived synthetic medium containing methanol as substrate. 16S rRNA gene analysis revealed that strain AMP was closely related to the aceto- genic bacterium Moorella thermoacetica DSM 521 T (98.3% sequence similarity). DNA-DNA hybridization showed 75.2 § 4.7% similarity to M. thermoacetica DSM 521 T , suggesting that strain AMP is a M. thermoacetica strain. Strain AMP has a unique one-carbon metabolism compared to other Moorella species. In media without cobalt growth of strain AMP on methanol was only sus- tained in coculture with a hydrogen-consuming methano- gen, while in media with cobalt it grew acetogenically in the absence of the methanogen. Addition of thiosulfate led to sulWde formation and less acetate formation. Growth of strain AMP with CO resulted in the formation of hydrogen as the main product, while other CO-utilizing Moorella strains produce acetate as product. Formate supported growth only in the presence of thiosulfate or in coculture with the methanogen. Strain AMP did not grow with H2/ CO 2 , unlike M. thermoacetica (DSM 521 T ). The lack of growth with H2/CO2 likely is due to the absence of cyto- chrome b in strain AMP.

Capillary zone electrophoresis (CZE) was tested for reproducibility and was shown to be a suitable method for estimating the relative concentration of ¿-lactalbumin, ß-lactoglobulin, ¿S2-casein (¿S2-CN), ¿S1-CN, ¿-CN, and ß-CN in milk. This study showed how the method can also be used to determine the relative concentration of two different phosphorylation states of ¿S1-CN (¿S1-CN-8P and ¿S1-CN-9P) and three of ¿S2-CN (¿S2-CN-10P, ¿S2-CN-11P and ¿S2-CN-12P). Furthermore, with CZE it was possible to determine most common genetic variants including the ¿-CN E variant that has not been identified by CZE before. Analyses of milk samples of 1948 Dutch Holstein-Friesian cows showed large variation in both the relative protein concentration and the relative concentration of different phosphorylation states for all milk proteins studied. Correlations between different phosphorylation states of ¿S1- and ¿S2-CN indicated that the relative concentration of specific phosphorylation states of ¿S1- and ¿S2-CN were related to each other.

Burned greasy deposits were found inside shells of the large Nile bivalve Chambardia rubens, excavated in an eight- to tenth- century AD church of the Coptic monastery of Bawit, Egypt, and supposedly used as oil lamps. The residues were subjected to a combination of chromatographic residue analysis techniques. The rather high concentrations of unsaturated fatty acids, as analysed by gas chromatography (GC) in the methylated extract, suggest the presence of a vegetal oil. Analysis of the stable carbon isotopes (delta C-13 values) of the methyl esters also favoured plants over animals as the lipid source. In the search for biomarkers by GC coupled to mass spectrometry on a silylated extract, a range of diacids together with high concentrations of 13,14-dihydroxydocosanoate and 11,12-dihydroxyeicosanoate were found. These compounds are oxidation products of erucic acid and gondoic acid, which are abundantly present in seeds of Brassicaceae plants. Liquid chromatography coupled to mass spectrometry analysis showed low concentrations of unaltered triglycerides, but revealed sizeable amounts of triglycerides with at least one dihydroxylated acyl chain. The unusual preservation of dihydroxylated triglycerides and alpha,omega-dicarboxylic acids can be related to the dry preservation conditions. Analysis of the stereoisomers of the dihydroxylated fatty acids allows one to determine whether oxidation took place during burning of the fuel or afterwards. The results prove that the oil of rapeseed (Brassica napus L.) or radish (Raphanus sativus L.) was used as illuminant in early Islamic Egypt, and that not only ceramic lamps but also mollusk shells were used as fuel containers. ispartof: Analytical and Bioanalytical Chemistry vol:390 issue:2 pages:783-793 ispartof: location:Germany status: published

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopep-tides have a major role in the antagonism of B. subtilis toward P. fusca.

Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655 and effects on levels of 5-HT, precursors and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

International audience; The end-use value of durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.) is mainly governed by its grain protein concentration and composition. Adjustment of variables to compensate for inter-annual and location variations in semolina quality leads to high cost for processors in the wheat industry. A better understanding of the mechanisms governing environmental variations of grain protein composition is thus required. Here, a field experiment was setup in a Mediterranean environment with the aim to analyze the effect of sowing date and nitrogen (N) fertilization on the dynamics of grain dry mass, water, protein composition and glutenin polymer size distribution for durum wheat cv. Creso. The results indicated that (1) grain dry mass accumulation was related to grain water concentration and stopped at 44.9% independently of the growing conditions and N supply; (2) during the grain filling period as well as at ripeness maturity, the quantity of the different protein fractions scaled with the quantity of N per grain; (3) SDS-extractable glutenin polymers were produced continuously until the same grain water concentration as the dry mass deposition was reached; (4) SDS-unextractable polymeric proteins were found as early as 7 days after anthesis and their rate of accumulation increased sharply when grain dry mass was 60% of its final value and proceeded at a constant rate until ripeness maturity, thus suggesting that the insolubilization of glutenin polymers is not directly related to the rapid loss of water after physiological maturity, but rather to the continuous dehydration of the grain. (C) 2014 Elsevier B.V. All rights reserved.

A recent development in algal pigment analysis by high-performance liquid chromatography (HPLC) is the application of automation. An optimization of a complete sampling and analysis protocol applied specifically in automation has not yet been performed. In this paper we show that automation can only be successful if the various methodological aspects of the sampling and analysis protocol are considered in coherence. We introduce an optimized protocol that involves freeze-drying of the sample, subsequent extraction in 90% acetone and the application of water-packing during analysis. The method was evaluated on both natural plankton populations and a broad spectrum of microalgal cultures: Thalassiosira weisflogii (Bacillariophyceae), Emiliania huxleyi (Prymnesiophyceae), Phaeocystis globosa and Phaeocystis antarctica (Pryrymesiophyceac) and Pyramimonas sp. (Prasinophyceae). Whereas pigment extracts were unstable in methanol, with recorded chlorophyll a losses from 10% to 60% per day, pigment degradation rates in acetone were generally less than 1% over 18 h storage in the autosampler (4 degrees C). In addition, it was found that the extraction efficiency of acetone significantly increased upon freeze-drying prior to extraction. Increases as high as 50-60% were measured in P. antarctica. The application of water-packing of the sample during injection resulted in improved peak shape and peak separation, without diluting the pigment concentrations. Automation is especially beneficial for application in the field, when mixed algal assemblages and low biomass put a high demand on the sensitivity as well as reproducibility of the method. (c) 2006 Elsevier B.V. All rights reserved.

An original approach based on successive size-exclusion and hydrophilic interaction HPLC (HILIC) was developed to purify traces of Ni species from a plant aqueous extract. The degree of purity achieved was for the first time sufficient for the identification, in a natural sample, of a number of non-covalent metal complexes by electrospray Q-TOFMS/MS. Nickel complexes with malate, citrate, histidine, EDTA and nicotianamine (NA) were identified in the roots, xylem, shoots and their protoplasts of a metal hyperaccumulator plant Thlaspi caerulescens. The quantitative recovery of the most stable of these complexes (with EDTA and NA) allowed their quantitative determination by SEC-ICP-MS.

A reliable extrapolation of neurochemical alterations from a mouse model to human metabolic brain disease requires knowledge of neurotransmitter levels and related compounds in control mouse brain. C57BL/6 is a widely used background strain for knockout and transgenic mouse models. A prerequisite for reliable extrapolation from mouse brain to the human condition is the existence of analogous distribution patterns of neurotransmitters and related compounds in control mouse and human brain. We analysed regional distribution patterns of biogenic amines, neurotransmitter and non-neurotransmitter amino acids, and cholinergic markers. Distribution patterns were compared with known neurotransmitter pathways in human brain. The present study provides a reference work for future analyses of neurotransmitters and related compounds in mouse models bred in a C57BL/6 background strain.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400?mg?kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

The aim of this study was to investigate the chemopreventive effects of widely consumed cruciferous vegetables, namely Brussels sprouts and red cabbage towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced preneoplastic lesions [liver glutathione-S-transferase placental positive (GST-P(+)) foci and colonic aberrant crypt foci (ACF)]. Male F344 rats were treated with IQ (100 mg/kg bw/g) on 10 alternating days and received drinking water supplemented with Brussels sprouts and red cabbage juices (5% v/v) before and during the carcinogen treatment. From each vegetable two different cultivars were tested. Brussels sprouts reduced the frequency of IQ-induced aberrant foci in both organs (41-52% in the colon and 27-67% in the liver). Also, Brussels sprouts drastically diminished (85-91%) the size of liver GST-P(+) foci, but no such effect was seen in the colon. With red cabbage, the size of liver GST-P(+) foci was markedly reduced (41-83%) whereas the foci frequency was only moderately decreased (19-50%). No protection was seen in the colon after treatment with red cabbage. Cooking (10 min, 100 degrees C) of the vegetables had no influence on their protective effects. The stronger chemoprotective effects of Brussels sprouts may be due to the fact that the overall glucosinolate contents were substantially (2-3-fold) higher than those of the cabbage cultivars, but it was not possible to attribute the reduction of preneoplastic lesions to specific glucosinolates. The activities of hepatic UDP-glucuronosyltransferase form 2 (UDPGT-2) and cytochrome P4501A2 were increased by both vegetables. The induction effect of Brussels sprouts on the activity of UDPGT-2 was more marked than that of the red cabbage cultivars, suggesting that increased glucuronidation of IQ may account for the reduction of the preneoplastic lesions. Our findings support the assumption that Brassica vegetables protect against the carcinogenic effects of heterocyclic amines.

Two edible plants in Southeast Asia, Gynura bicolor and G.divaricata, are not only known to be nutritive but also useful as medicinal herbs. Previous phytochemical investigation of Gynura species showed the presence of hepatotoxic pyrrolizidine alkaloids (PAs), indicating the toxic risk of using these two plants. The present study was designed to analyze the distribution of PA components and tried to evaluate the preliminary toxicity of these two Gynura species. Eight samples of G.bicolor and G.divaricata from five different Chinese locations were collected and their specific PAs were qualitatively characterized by applying an UPLC/MS/MS spectrometry method. Using a pre-column derivatization HPLC method, the total retronecine ester-type PAs in their alkaloids extracts were quantitatively estimated as well. Finally, their genotoxicity was investigated with an effective high-throughput screening method referred to as Vitotox test and their potential cytotoxicity was tested on HepG2 cells. It was found that different types of PAs were widely present in Gynura species collected from south of China. Among them, no significant genotoxic effects were detected with serial concentrations through the present invitro assay. However, the cytotoxicity assay of Gynura plants collected from Jiangsu displayed weak activity at the concentration of 100mg/ml. It is important to note that this research validates in part the indication that the use of Gynura species requires caution.

Oxidant-mediated tissue injury is key to the pathogenesis of acute lung injury. Glutathione- S-transferases (GSTs) are important detoxifying enzymes that catalyze the conjugation of glutathione with toxic oxidant compounds and are associated with acute and chronic inflammatory lung diseases. We hypothesized that attenuation of cellular GST enzymes would augment intracellular oxidative and metabolic stress and induce lung cell injury. Treatment of murine lung epithelial cells with GST inhibitors, ethacrynic acid (EA), and caffeic acid compromised lung epithelial cell viability in a concentration-dependent manner. These inhibitors also potentiated cell injury induced by hydrogen peroxide (H2O2), tert-butyl-hydroperoxide, and hypoxia and reoxygenation (HR). SiRNA-mediated attenuation of GST-π but not GST-μ expression reduced cell viability and significantly enhanced stress (H2O2/HR)-induced injury. GST inhibitors also induced intracellular oxidative stress (measured by dihydrorhodamine 123 and dichlorofluorescein fluorescence), caused alterations in overall intracellular redox status (as evidenced by NAD+/NADH ratios), and increased protein carbonyl formation. Furthermore, the antioxidant N-acetylcysteine completely prevented EA-induced oxidative stress and cytotoxicity. Whereas EA had no effect on mitochondrial energetics, it significantly altered cellular metabolic profile. To explore the physiological impact of these cellular events, we used an ex vivo mouse-isolated perfused lung model. Supplementation of perfusate with EA markedly affected lung mechanics and significantly increased lung permeability. The results of our combined genetic, pharmacological, and metabolic studies on multiple platforms suggest the importance of GST enzymes, specifically GST-π, in the cellular and whole lung response to acute oxidative and metabolic stress. These may have important clinical implications.

Most secreted and cell membrane proteins in mammals are glycosylated. Many of these glycoproteins are also prevalent in milk and play key roles in the biomodulatory properties of milk and ultimately in determining milk's nutritional quality. Although a significant amount of information exists on the types and roles of free oligosaccharides in milk, very little is known about the glycans associated with milk glycoproteins, in particular, the biological properties that are linked to their presence. The main glycoproteins found in bovine milk are lactoferrin, the immunoglobulins, glycomacropeptide, a glycopeptide derived from kappa-casein, and the glycoproteins of the milk fat globule membrane. Here, we review the glycoproteins present in bovine milk, the information currently available on their glycosylation and the biological significance of their oligosaccharide chains.

The neurotoxin β-N-methylamino-l-alanine (BMAA) is suspected to play a role in the neurological diseases amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. BMAA production by cyanobacteria has been reported and contact with cyanobacteria infested waters or consumption of aquatic organisms are possible pathways to human exposure. However, there is little consensus regarding whether BMAA is present in cyanobacteria or not, and if so, at what concentrations. The aim of this review is to indicate the current state of knowledge on the presence of BMAA in aquatic ecosystems. Some studies have convincingly shown that BMAA can be present in aquatic samples at the µg/g dry weight level, which is around the detection limit of some equally credible studies in which no BMAA was detected. However, for the majority of the reviewed articles, it was unclear whether BMAA was correctly identified, either because inadequate analytical methods were used, or because poor reporting of analyses made it impossible to verify the results. Poor analysis, reporting and prolific errors have shaken the foundations of BMAA research. First steps towards estimation of human BMAA exposure are to develop and use selective, inter-laboratory validated methods and to correctly report the analytical work.