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Paracetamol (acetaminophen, APAP) is the most frequently used analgesic drug worldwide. However, patients in several specific populations can have an increased exposure to toxic APAP metabolites. Therefore, APAP-protein adducts have been proposed as an alternative marker for the assessment of APAP intoxications and as an effective tool to study and steer APAP treatment in patients with an increased risk of APAP-induced liver damage. These adducts have been determined in plasma or serum as a matrix. Blood microsampling allows the determination of a variety of analytes, including protein adducts, in a drop of blood, facilitating convenient followup of patients in a home-sampling context, as well as repeated sampling of pediatric patients. We therefore evaluated the use of blood-based volumetric microsamples for the quantification of APAP-protein adducts. Quantitative methods for the determination of APAP-protein adducts in dried blood and dried plasma volumetric absorptive microsamples were developed and validated. Also a preliminary evaluation of pediatric patient dried blood microsamples was conducted. Method validation encompassed the evaluation of selectivity, carry over, calibration model, accuracy and precision, matrix effect, recovery and the effect of the hematocrit on the recovery, dilution integrity, and stability. All pre-set acceptance criteria were met, except for stability. Spiking of blank blood with APAP revealed a concentration-dependent ex vivo formation of APAP-protein adducts, resulting in a response for the measurand APAP-Cys, with an apparent role for the red blood cell fraction. Analysis of authentic samples, following intake of APAP at therapeutic dosing, revealed much higher APAP-Cys concentrations in dried blood vs. dried plasma samples, making interpretation of the results in the context of published intervals difficult. In addition, in contrast to what was observed during method validation, the data obtained for the patient samples showed a high and unacceptable variation. We conclude that, for a combination of reasons, dried blood is not a suitable matrix for the quantification of APAP-protein adducts via the measurement of the APAP-Cys digestion product. The collection of plasma or serum, either in the form of a liquid sample or a dried microsample for this purpose is advised.

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.

Liquid chromatography (LC) hyphenated to mass spectrometry is currently the most widely used means of determining metabolic phenotypes via both untargeted and targeted analysis. At present a range of analytical separations, including reversed-phase, hydrophilic interaction and ion-pair LC are employed to maximise metabolome coverage with ultra (high) performance liquid chromatography (UHPLC) increasingly displacing conventional high performance liquid chromatography because of the need for short analysis times and high peak capacity in such applications. However, it is widely recognized that these methodologies do not entirely solve the problems facing researchers trying to perform comprehensive metabolic phenotyping and in addition to these "routine" approaches there are continuing investigations of alternative separation methods including 2-dimensional/multi column approaches. These involve either new stationary phases or multidimensional combinations of the more conventional materials currently used, as well as application of miniaturization or "new" approaches such as supercritical HP and UHP- chromatographic separations. There is also a considerable amount of interest in the combination of chromatographic and ion mobility separations, with the latter providing both an increase in resolution and the potential to provide additional structural information via the determination of molecular collision cross section data. However, key problems remain to be solved including ensuring quality, comparability across different laboratories and the ever present difficulty of identifying unknowns.

Animal studies remain an essential part of drug discovery since in vitro models are not capable of describing the complete living organism. We developed and qualified a microchip electrophoresis-electrochemical detection (MCE-EC) method for rapid analysis of morphine in mouse plasma using a commercial MCE-EC device. Following liquid-liquid extraction (LLE), we achieved within-run precision of 3.7 and 4.5% (coefficient of variation, CV, n = 6) and accuracy of 106.9% and 100.7% at biologically relevant morphine concentrations of 5 and 20 µM in plasma, respectively. The same method was further challenged by morphine detection in mouse brain homogenates with equally good within-run precision (7.8% CV, n = 5) at 1 µM concentration. The qualified method was applied to analyze a set of plasma and brain homogenate samples derived from a behavioral animal study. After intraperitoneal administration of 20 mg/kg morphine hydrochloride, the detected morphine concentrations in plasma were between 6.7 and 17 µM. As expected, the morphine concentrations in the brain were significantly lower, ca. 80–125 nM (280–410 pg morphine/mg dissected brain), and could only be detected after preconcentration achieved during LLE. In all, the microchip-based separation system is proven feasible for rapid analysis of morphine to provide supplementary chemical information to behavioral animal studies.

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.

Due to its multifunctional and complex role in the honey bee colony functioning and health (construction material allowing food storage, brood rearing, thermoregulation, mediation in chemical and mechanical communication, substrate for pathogens, toxins and waste), Apis mellifera beeswax has been widely studied over the last five decades. This is supported by a comprehensive set of scientific reports covering different aspects of beeswax research. In this article, we present an overview of the methods for studying chemical, biological, constructional, and quality aspects of beeswax. We provide a detailed description of the methods for investigating wax scales, comb construction and growth pattern, cell properties, chemical composition of beeswax using different analytical tools, as well as the analytical procedures for provenancing beeswax and beeswax-derived compounds based on the hydrogen isotope ratio (IRMS). Along with classical physico-chemical and sensory analysis, we describe more precise and accurate methods for detection of adulterants in beeswax (GC-MS and FTIR-ATR). Moreover, we present methods for studying the influence of beeswax (comb foundation) adulteration on comb construction. Analytical protocols for determining the pesticide residues using different chromatographic and spectroscopic techniques are also described. As beeswax is an agent of high risk for the transmission of bee diseases, we present methods for detection of pathogens in beeswax. To ensure the reproducibility of experiments and results, we present best practice approaches and detailed protocols for all methods described, as well as their advantages and disadvantages. COST ActionEuropean Cooperation in Science and Technology (COST) [FA0803]; Ricola Foundation - Nature Culture We would like to acknowledge the Ricola Foundation - Nature & Culture, and COST Action FA0803 for funding the COLOSS (Prevention of honey bee COlony LOSSes) network and making the BEEBOOK possible. We would also like to express our gratitude to Editor Vincent Dietemann for his kind support all the way, and for providing helpful and constructive suggestions.

Introduction As large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC–MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening. Objectives To develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system. Methods A rapid untargeted HILIC LC–MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation. Results The rapid HILIC–MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC–IMS–MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS information compared against the Metlin MS/MS database and, in addition, both calculated and measured CCS values from an experimentally derived CCS database. Conclusion A rapid metabolic profiling method for the analysis of polar metabolites has been developed. The method has the advantages of speed and both reducing sample and solvent consumption compared to conventional profiling methods. The addition of IMS added an additional dimension for feature detection and the identification of metabolites. Electronic supplementary material The online version of this article (10.1007/s11306-019-1474-9) contains supplementary material, which is available to authorized users.

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

A systematic approach was developed to investigate the stability of gentamicin sulfate (GS) and GS/poly (lactic-co-glycolic acid) (PLGA) coatings on hydroxyapatite surfaces. The influence of environmental factors (light, humidity, oxidation and heat) upon degradation of the drug in the coatings was investigated using liquid chromatography with evaporative light scattering detection and mass spectrometry. GS coated rods were found to be stable across the range of environments assessed, with only an oxidizing atmosphere resulting in significant changes to the gentamicin composition. In contrast, rods coated with GS/PLGA were more sensitive to storage conditions with compositional changes being detected after storage at 60 °C, 75% relative humidity or exposure to light. The effect of γ-irradiation on the coated rods was also investigated and found to have no significant effect. Finally, liquid chromatography–mass spectrometry analysis revealed that known gentamines C1, C1a and C2 were the major degradants formed. Forced degradation of gentamicin coatings did not produce any unexpected degradants or impurities., Graphical abstract fx1, Highlights • Method developed for extraction and analysis of an antimicrobial coated orthopedic device. • Evaluated the effect of light, humidity, oxygen and heat on the storage stability of drug coatings. • Also evaluated the influence of γ-ray sterilization on the chemical composition of drug coatings.

The anodic voltammetric behavior of an antibiotic drug, lincomycin hydrochloride (LIN) at gold electrode (GE) has been investigated using cyclic and linear sweep voltammetry. The dependence of the current on pH, concentration and scan rate were investigated to optimize the experimental conditions for the determination of lincomycin. The anodic peak was characterized and the process was adsorption-controlled. The number of electrons transferred in the oxidation process was calculated. In the range of 8.0×10−6 to 1.0×10−4 M, the current measured by linear sweep voltammetry presents a good linear property as a function of the concentration of lincomycin with a detection limit of 1.7×10−7 M with good selectivity and sensitivity. The proposed method was successfully applied to lincomycin determination in pharmaceutical dosage form and in urine as a real sample. This method can be employed in clinical analysis, quality control and routine determination of drugs in pharmaceutical formulations.

We report on an experimental approach to measure the pressure stability and mechanical strength of monolithic silica capillary columns with different diameters (50 and 100 mu m i.d.) and considering two different domain sizes, typical for the second generation monoliths or smaller. The approach consists of exposing the capillaries to ultra-high pressures (gradually stepwise increased from 20 to 80 MPa), with intermediate measurements of the column efficiency, permeability and retention factors to check the mechanical stability of the bed. It was observed that all tested columns withstood the imposed pressure stress, i.e., all the tested parameters remained unaffected up till the maximal test pressure of 80 MPa. The applied pressure gradient corresponded to 320 MPa/m. The two 100 mu m i.d.-capillary columns were also exposed to pressures between 80 and 90 MPa for a prolonged time (8 h), and this did not cause any damage either. (C) 2016 Elsevier B.V. All rights reserved.

Monitoring the levels of the ceramides (Cer) d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1 and ratios thereof in human plasma empowers the prediction of fatal outcome of coronary artery disease (CAD). We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for clinical-scaled measurement of the four distinct ceramides. Rapid plasma precipitation was accomplished in 96-well format. Excellent extraction recoveries in the range of 98-109 % were achieved for each ceramide. Addition of corresponding D-7-labeled ceramide standards facilitated precise quantification of each plasma ceramide species utilizing a novel short 5-min LC-MS/MS method. Neither matrix interference nor carryover was observed. Robust intra- and inter-assay accuracy and precision 0.99, were achieved for all analytes. Short-term bench top, long-term plasma, and extract stability demonstrated that the distinct ceramides were stable in the conditions evaluated. The validity of the methodology was demonstrated by determining the precise ceramide concentrations in a small CAD case-control study. Thus, our LC-MS/MS methodology features simple sample preparation and short analysis time for accurate quantification of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1, designed for routine analysis.

Effluents from wastewater treatment plants (WWTPs) are a major source of estrogenic compounds to the aquatic environment. In the present work, estrogenic activities of effluents from eight municipal WWTPs in Finland were studied. The main objectives of the study were to quantify the concentrations of selected estrogenic compounds, to evaluate their contribution to estrogenic potency and to test the feasibility of the commercial bioassays for wastewater analysis. The effluent samples were analyzed by two in vitro tests, i.e. ERα-CALUX(®) and ELISA-E2, and by liquid chromatography mass spectrometry for six estrogenic compounds: estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), 17α-estradiol and bisphenol A (BPA). Estrogenic effects were found in all of the effluent samples with both of the bioassays. The concentrations measured with ELISA-E2 (8.6-61.6 ng/L) were clearly higher but exhibited a similar pattern than those with chemical analysis (E2

The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6.This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at −20 °C and one set was analyzed after lyophilization.In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants.The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.

In this work, poly(ethylene glycol dimethacrylate-N-methacryloyl-L-tryptophan methyl ester) [poly(EGDMA-MATrp)] microbeads (average diameter = 106–300 µm), were synthesized by suspension polymerization technique. The microbeads showed strong affinity and allowed for solid phase extraction (SPE) of two common pesticides; pirimicarb and propoxur in environmental water samples. Some SPE process parameters such as the desorption solvent type, solvent volume, pH and salt concentration were optimized. When it was coupled with high pressure liquid chromatography (HPLC-PDA) for the determination of analytes, the linear ranges were about 10–800 ng mL−1 and the detection limits were 0.95–2.85 ng mL−1. Also the correlation coefficients (0.9949–0.9991) and precisions (2.15–2.52% RSD, n = 3) were determined. The technique was also applied to the enrichment of these pesticides in various environmental water samples. However, no target analyte was detected in the samples. Therefore, samples were spiked and recovery values were obtained in the range of 95.8–104.2% with RSDs of 2.6–6.6%.

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.

International audience; Haemochromatosis is defined as systemic iron overload of genetic origin, caused by a reduction in the concentration of the iron regulatory hormone hepcidin, or a reduction in hepcidin-ferroportin binding. Hepcidin regulates the activity of ferroportin, which is the only identified cellular iron exporter. The most common form of haemochromatosis is due to homozygous mutations (specifically, the C282Y mutation) in HFE, which encodes hereditary haemochromatosis protein. Non-HFE forms of haemochromatosis due to mutations in HAMP, HJV or TFR2 are much rarer. Mutations in SLC40A1 (also known as FPN1; encoding ferroportin) that prevent hepcidin-ferroportin binding also cause haemochromatosis. Cellular iron excess in HFE and non-HFE forms of haemochromatosis is caused by increased concentrations of plasma iron, which can lead to the accumulation of iron in parenchymal cells, particularly hepatocytes, pancreatic cells and cardiomyocytes. Diagnosis is noninvasive and includes clinical examination, assessment of plasma iron parameters, imaging and genetic testing. The mainstay therapy is phlebotomy, although iron chelation can be used in some patients. Hepcidin supplementation might be an innovative future approach.

Vitamin K₂ (menaquinone) concentrations were measured in a wide range of cheeses and the effects of fat content, ripening and origin of the cheeses were investigated. Moreover, the menaquinone content of cheese was compared with that of other foods known to contain vitamin K₂. It was found that cheese and curd are the most important sources of long-chain menaquinones in the Western diet and, in general, hard cheeses are richer in menaquinones than soft cheeses. However, the actual menaquinone content varies substantially and is dependent on the type of cheese, the time of ripening, the fat content and the geographic area where the cheeses are produced. Given the fact that poor vitamin K status has been mentioned as a risk factor for cardiovascular disease and mortality, while there is no clear evidence for adverse cardiovascular effects of dairy fats, cheese should be considered as a recommendable component in a heart-healthy diet.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a tetrahydroisoquinoline derivative whose presence in humans was first detected in the urine of Parkinsonian patients on l-DOPA (l-dihydroxyphenylalanine) medication. Thus far, multiple hypotheses regarding its physiological/pathophysiological roles have been proposed, especially related to Parkinson’s disease or alcohol addiction. The aim of this review was to outline studies related to salsolinol, with special focus on in vivo and in vitro experimental models. To begin with, the chemical structure of salsolinol together with its biochemical implications and the role in neurotransmission are discussed. Numerous experimental studies are summarized in tables and the most relevant ones are stressed. Finally, the ability of salsolinol to cross the blood–brain barrier and its possible double-faced neurobiological potential are reviewed.

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C-18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%).

A sensitive and specific method for the quantitative determination of Fumonisin B1 (FB1), its partially hydrolysed metabolites pHFB1a+b and hydrolysed metabolite HFB1, and Fumonisin B2 (FB2) in broiler chicken plasma using ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis((R)) Ostro(TM) 96-well plate. Chromatography was performed on an Acquity HSS-T3 column, using 0.3% formic acid and 10 mM ammonium formate in water, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the two multiple reaction monitoring transitions were monitored for each component for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity (r 0.99) was achieved over the concentration ranges tested (1-500 ng/mL for FB1 and FB2; 0.86-860 ng/mL for pHFB1a; 0.72-1430 ng/mL for pHFB1b and 2.5-2500 ng/mL for HFB1). Limits of quantification (LOQ) and detection (LOD) in plasma ranged between 0.72 to 2.5 ng/mL and 0.03 to 0.17 ng/mL, respectively. The results for the within-day and between-day precision and accuracy fell within the specified ranges. Moreover, the method was transferred to an UPLC high-resolution mass spectrometry (HR-MS) instrument in order to determine potential metabolites of HFB1, such as N-acyl-HFB1s and phase II metabolites. The method has been successfully applied to investigate the toxicokinetics and biotransformation of HFB1 in broiler chickens.

In this study, an ID-LC-MS method was developed and validated for the determination of 14 sulfonamides in milk samples. Recovery results were found to be in the range of 91%-114% for milk samples. The relative measurement uncertainty was between 7.5%-12.7%. Validated method was performed on milk samples obtained from market and street vendors. The amount of sulfonamides in the analyzed samples was found to be below the legal limits (Sulfamethazine: 6.46 +/- 0.76 ng/g and sulfisoxazole: 7.3 +/- 0.71 ng/g).

International audience; Individual molecular components of soil organic matter (SOM) have been studied since the early 19th century. Their characterization is essential, because knowledge of the molecular structures constituting SOM allows for the detailed understanding of its origin and the processes implicated in soil C sequestration. This provides the basis for target-oriented development of management practices to optimize ecosystem services provided by soil. The aim of this review is to give an overview about the major analytical developments and the information gain that could be achieved by application of molecular methods in SOM research. Up to the 1980s, studies concentrated on the characterization of the chemical nature of SOM. Analyses were mainly based on soluble OM compounds, which were fractionated chemically into humic and fulvic acids. During the 1990s, the focus changed and scientists were more interested in biological processes controlling SOM formation and its dynamics. Introduction of physical fractionation and combination of molecular and isotopic techniques allowed assessment of composition, origin, and turnover of SOM within specific localizations in the mineral soil matrix. Analyses of the dynamics of single molecules led to a massive change of paradigms. Long residence times of SOM are no longer explained by chemical recalcitrance but by microbial products being stabilized by the interaction with soil minerals and microbial inaccessibility. In recent years, techniques yielding results with high molecular and spatial resolution were introduced, which will allow the acquisition of much more detailed information, moving a step further toward elucidating the nature and properties of SOM.

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.

A method for the quantitative determination of folates in rodent diets is very important for correct interpretation of folate intake during feeding trials, given the possible discrepancy between the actual folate concentration in the diet and that mentioned on the product sheet. Liquid chromatography tandem-mass spectrometry is the method of choice to differentiate and quantify the individual folate species present. This discrepancy may be accounted for by, e.g., inaccurate folic acid supplementation and/or the presence of endogenous reduced and substituted folates. We developed a method, validated based on FDA guidelines, that allows the measurement of added and endogenous folates by quantitative determination of 5 folate monoglutamates with linear ranges from 8 mu g to 2 mg/kg feed. This information, combined with feed intake data, allows insight into the actual folate intake in animal feeding studies. The relevance of this method was illustrated by the analysis of several feed samples of varying composition, by the investigation of the effect of casein incorporation, and by evaluating the variability of the folate content between pellets and production batches.

International audience; A composite 30-cm capillary was prepared. The head of the capillary was a 1.5-cm original and miniaturized aptamer-based monolithic affinity support that was in-line coupled to the end of the capillary used for capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The device was used for the preconcentration, separation and quantification of ochratoxin A (OTA) as a test solute. The 1.5-cm preconcentration unit consists of a fritless affinity monolithic bonded with 5'-SH-modified oligonucleotide aptamers. A vinyl spacer was used for thiol-ene photoclick chemistry with a 5 min irradiation at 365 nm. Photografting allowed to confine the binding reaction to the desired silica monolithic segment, upstream the empty section of the CE capillary using an UV mask. The photografting procedure was optimized preparing 10-cm capillary monoliths for nano-LC. The retention factors of cationic solutes in ion-exchange nano-LC allowed to follow the aptamer binding on the monolith. The reproducibility of the photografting process was satisfactory with inter-capillary variation lower than 10%. The aptamer bonding density can be increased by successive graftings of 100 mu M aptamer concentration solution (5 pmol/cm/grafting). The optimal conditions to successfully perform the in-line coupling (preconcentration, elution and separation of OTA) with the composite capillary were adjusted depending on individual requirements of each step but also insuring compatibility. Under optimized conditions, OTA was successfully preconcentrated and quantified down to 0.1 pg (percolation of 2.65 mu L of a 40 ng/L OTA solution). A quantitative recovery of OTA (93 +/- 2%) was achieved in a single elution of 30 pg percolated OTA amount. The reproducibility of the overall process was satisfactory with a relative standard deviation lower than 10% with negligible non-specific adsorption. This device was applied for the preconcentration and analysis of OTA in beer and wine at the ppb level within a total analysis time of 30 min.

Long-term sustainability and high resource use efficiency are major goals for high quality baking wheat production throughout the world. Present strategies are low input systems such as organic agriculture or improved conventional systems (integrated). The fertilisation level and strategy, crop protection as well as preceding crop effects may modulate system performance with respect to wheat grain yield, quality and environmental performance of the systems. Our aim was to evaluate data of winter wheat (Triticum aestivum L.) performance from the DOK long-term systems experiment in Switzerland comparing two mixed organic (biodynamic and bioorganic: BIODYN and BIOORG) and a mixed conventional cropping system (CONFYM) using mineral fertilisers and farmyard manure at two fertilisation intensities (level 1: 50% of standard fertilisation, level 2: standard fertilisation) since 1978. A conventional system was fertilised exclusively minerally at level 2 (CONMIN) and a control remained unfertilised (NOFERT). We compared crop yields, baking quality parameters, the nitrogen use efficiency and the effect of maize and potatoes as preceding crops obtained between 2003 and 2010 along with long-term soil sustainability parameters. The mean grain yields across both fertiliser levels of the organic cropping systems (BIODYN and BIOORG) were 64% of CONFYM, whereas crude protein contents were 79% of CONFYM at fertilisation level 2 and achieved 90% at level 1. The main driving factor of lower yields was a reduction of the numbers of ears per m(2) and the thousand kernel weight. The apparent nitrogen use efficiency decreased with increasing N fertilisation. Doubling the organic fertilisation in the organic systems only slightly improved wheat grain yields but was not able to improve grain baking quality, due to low mineral N additions via slurry and farmyard manure. In contrast the effects of the preceding crop potatoes in comparison with preceding silage maize outperformed the organic fertilisation effects, resulting in 33% higher yields and 11% higher crude protein contents. The yield components recorded in the case of preceding potatoes demonstrated a more synchronised nutrient supply throughout the wheat development. Over all low input systems and both fertilisation levels in the conventional mixed farm system at half standard fertilisation (level 1) performed best with distinctly higher grain yields and crude protein contents than in the organic systems with standard fertilisation. However, all systems, organic and conventional, with the low or zero organic fertiliser inputs performed poorly considering the long-term soil quality parameters, indicating a degradation of soil quality. The DOK long-term experiment allows an integrated view on the performance of baking wheat production and long-term sustainability. The results emphasise the importance of a sufficient supply of soils with organic fertilisers as well as the need to improve the availability of organic nitrogen and synchrony between nutrient supply and demand in organic baking wheat production, beside the selection of a suitable preceding crop.

In agriforensics, time of administration is often debated when illegal drug residues, such as clenbuterol, are found in frequently traded cattle. In this proof-of-concept work, the feasibility of obtaining retrospective timeline information from segmented calf tail hair analyses has been studied. First, an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) hair analysis method was adapted to accommodate smaller sample sizes and in-house validated. Then, longitudinal 1 cm segments of calf tail hair were analyzed to obtain clenbuterol concentration profiles. The profiles found were in good agreement with calculated, theoretical positions of the clenbuterol residues along the hair. Following assessment of the average growth rate of calf tail hair, time of clenbuterol administration could be retrospectively determined from segmented hair analysis data. The data from the initial animal treatment study (n = 2) suggest that time of treatment can be retrospectively estimated with an error of 3-17 days.

Background: Therapeutic drug monitoring is useful in the treatment of tuberculosis to assure adequate exposure, minimize antibiotic resistance, and reduce toxicity. Salivary therapeutic drug monitoring could reduce the risks, burden, and costs of blood-based therapeutic drug monitoring. This systematic review compared human pharmacokinetics of antituberculosis drugs in saliva and blood to determine if salivary therapeutic drug monitoring could be a promising alternative. Methods: On December 2, 2016, PubMed and the Institute for Scientific Information Web of Knowledge were searched for pharmacokinetic studies reporting human salivary and blood concentrations of antituberculosis drugs. Data on study population, study design, analytical method, salivary Cmax, salivary area under the time-concentration curve, plasma/serum Cmax, plasma/serum area under the time-concentration curve, and saliva-plasma or saliva-serum ratio were extracted. All included articles were assessed for risk of bias. Results: In total, 42 studies were included in this systematic review. For the majority of antituberculosis drugs, including the first-line drugs ethambutol and pyrazinamide, no pharmacokinetic studies in saliva were found. For amikacin, pharmacokinetic studies without saliva-plasma or saliva-serum ratios were found. Conclusions: For gatifloxacin and linezolid, salivary therapeutic drug monitoring is likely possible due to a narrow range of saliva-plasma and saliva-serum ratios. For isoniazid, rifampicin, moxifloxacin, ofloxacin, and clarithromycin, salivary therapeutic drug monitoring might be possible; however, a large variability in saliva-plasma and saliva-serum ratios was observed. Unfortunately, sali-vary therapeutic drug monitoring is probably not possible for doripenem and amoxicillin/clavulanate, as a result of very low salivary drug concentrations.

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).

After being used for decades in clinical screening, dried blood spots (DBS) have recently received considerable attention for their application in pharmacokinetic and toxicokinetic studies in rodents. The goal of this study was to develop and apply a DBS-based assay for a pharmacokinetic study of paclitaxel (PTX) and its metabolites in SCID/Beige mice. A fast and sensitive UHPLC-MS/MS method has been developed for the simultaneous determination of PTX, its three metabolites (6 alpha-hydroxy-paclitaxel, 3'-p-hydroxypacli taxel, and 6a,3'-p-dihydroxy-paclitaxel) and its stereoisomer 7-epi-p aclitaxel. The 10 mu L DBS sample was extracted with methanol for 20 min at 37 degrees C. After dilution of the extracts with water in a ratio of 1:1, the analytes were separated on a reversed-phase 2.1 mm I.D. column using gradient elution. The total run time was 2.5 min. The analytes were detected by use of multiple reaction monitoring mass spectrometry. The extraction recoveries of the compounds were all greater than 60%, resulting in a quantification limit of 1 ng/ml. The calibration curves ranged from 1 to 1000 ng/ml. The intra-day and inter-day imprecision (%CV) across three validation runs over four quality control levels were less than or equal to 14.6%. The accuracy was within +/-11.9% in terms of relative error. The described method is advantageous in terms of its ease-of-use and speed compared to other published PTX assays. The method's usefulness was demonstrated by applying it to a preclinical pharmacokinetic investigation of PTX and its metabolites in SCID/Beige mice with an intraperitoneal administration of 50 mg/kg Abraxane (R).

International audience; RATIONALE: The first step in the diagnosis of oligosaccharidoses is to evidence abnormal oligosaccharides excreted in urine, usually performed by the poorly sensitive but efficient thin layer chromatography (TLC) method. Developing a tandem mass spectrometry (MS/MS) technique could be of great interest to replace TLC. METHODS: Abnormal underivatized oligosaccharides have been recently studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, allowing the unambiguous identification of oligosaccharidoses. Based on this previous work, we developed an advantageous and efficient liquid chromatography (LC)/MS/MS method using a more common triple quadrupole tandem mass spectrometer for oligosaccharides analysis. RESULTS: Oligosaccharidoses (n = 97) and control (n = 240) urine samples were analysed. A specific pattern was obtained for each oligosaccharidosis using this method. In urine, it allows not only the identification of all the oligosaccharidoses previously identified by TLC (fucosidosis, alphamannosidosis, aspartylglucosaminuria, GM1 gangliosidosis, sialidosis, galactosialidosis and Schindler disease), but also extends the field of diagnosis to mucolipidosis type II, Sandhoff disease, and beta-mannosidosis. The same technique was applied to 16 amniotic fluid supernatants from oligosaccharidosis-affected foetuses (n = 16) compared with 37 unaffected. All the affected foetuses could be clearly identified: sialidosis (n = 3), galactosialidosis (n = 4), aspartylglucosaminuria (n = 1), mucolipidosis type II (n = 4) or GM1 gangliosidosis (n = 4). This technique can be applied to early prenatal diagnosis as well as to the oligosaccharidosis screening in the case of non-immune hydrops fetalis. CONCLUSIONS: The method is quick and easy to run, with an LC analysis time of 13 min per sample. The quantitative validation could not be obtained in the absence of a specific standard and of a labelled internal standard for each compound. Even if this LC/MS/MS method is only qualitative, it is very specific and much more sensitive than TLC. It allows the urinary screening of oligosaccharidoses, even mild or late-onset forms, and the screening of antenatal forms in amniotic fluid. Copyright (C) 2017 John Wiley & Sons, Ltd.

A proportion of the plant's L-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolitesmay act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-dilcetogulonate (DKG), preparations of which can non-enzymically generate H2O2 and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H2O2 and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with lambda(max) 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major H2O2-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pK(a) approximate to 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula C6H6O5, i.e. it is a reduction product of DKG (C6H8O7). In conclusion, compound (1) is the major H2O2-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate -> dehydroascorbic acid -> DKG -> (1). We hypothesise that (1) generates apoplastic H2O2 (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence. (C) 2017 The Authors. Published by Elsevier Inc.

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.

The beneficial effects of honey on human health have long been recognized. Today, many of those positive effects have been studied to elucidate its mode of action. This review briefly summarizes the best studied features of honey, highlighting it as an appealing alternative medicine. In these reports, the health benefits of honey range from antioxidant, immunomodulatory, and anti-inflammatory activity to anticancer action, metabolic and cardiovascular benefits, prebiotic properties, human pathogen control, and antiviral activity. These studies also support that the honey's biological activity is mainly dependent on its floral or geographic origin. In addition, some promising synergies between honey and antibiotics have been found, as well as some antiviral properties that require further investigation. Altogether, these studies show that honey is effectively a nutraceutical foodstuff. info:eu-repo/semantics/publishedVersion

In this study, the stability constants of the copper(II) and aluminium(III) complexes of quercetin were determined potentiometrically by using Calvin-Bjerrum and Irwing Rossotti methods. The protonation constants of quercetin were found: logK(1) = 11.15 +/- 0.118, logK(2) = 10.42 +/- 0.144, logK(3) = 9.44 +/- 0.162, logK(4) = 8.28 +/- 0.151. For copper complex formation constant was found by Irwing-Rossotti method: logK(1) = 19.92 +/- 0.367 and for aluminium complex complex: logK(1) = 23.02 +/- 0.459. From the results, the components of quercetin / metal complexes are given as 1/1 both copper(II) and aluminium(III). A reversed phase high pressure liquid chromatographic method was developed for the metal complexes which were prepared according to potentiometric results with the ratio quercetin / metal = 1/1. Mobile phase was 0.01 M HClO4/8.33 x 10(-5) M quercetin in Methanol (40/60), column was XTerra RP18, 5 mu m, 4.6 x 150 mm, detector was Diode Array Detector at lambda = 373 ve 421 nm (band width 4nm).

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.

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.

A targeted reversed-phase gradient UPLC-MS/MS assay has been developed for the quantification/monitoring of amino acids and amino-containing compounds in human plasma and serum using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQTag UltraTM). Derivatization of the target amino-containing compounds reagent required minimal sample preparation and resulted in analytes with excellent chromatographic and mass spectrometric properties. The resulting method, which requires only 10 µl of sample, provides the reproducible and robust separation of 66 analytes in 7.5 minutes, including baseline resolution of isomers such as e.g. leucine and isoleucine. The assay has been validated for the quantification of 33 amino compounds (predominantly amino acids) over a concentration range from 2-20 and 800µM. Intra- and inter-day accuracy of between 0.05-15.6 and 0.78 -13.7 % and precision between 0.91-16.9 % and 2.12-15.9 % were obtained. A further 33 biogenic amines can be monitored in samples for relative changes in concentration rather than quantification. Application of the assay to samples derived from healthy controls and patients suffering from acetaminophen (APAP, paracetamol) induced acute liver failure (ALF) showed significant differences in the amounts of aromatic and branched chain amino acids between the groups as well as a number of other analytes, including the novel observation of increased concentrations of sarcosine in ALF patients. The properties of the developed assay, including short analysis time, make it suitable for high throughput targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and epidemiological environments.

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.