When subcellular chemical imaging enlightens our understanding on intestinal absorption, intracellular fate and toxicity of PFOA in vitro.

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant that accumulates in the human body, leading to major health issues. Upon oral uptake, the gastrointestinal tract is the first biological barrier against PFOA. However, the localization of PFOA and its impact on the intestinal wall are largely unknown. Here we achieve a breakthrough in the knowledge of intestinal absorption, intracellular fate and toxicity of PFOA using in vitro assays combined with novel analytical imaging techniques. For the first time, we localized PFOA in the cytosol of Caco-2 cells after acute exposure using high spatial resolution mass spectrometry imaging, and we estimated the PFOA cytosolic concentration. Knowing that PFOA enters and accumulates in the intestinal cells, we also performed common toxicity assays assessing cell metabolic activity, membrane integrity, oxidative stress response, and cell respiration. This study integrating powerful analytical techniques with widely used toxicology assays provides insightful information to better understand potential negative impacts of PFOA and opens new opportunities in toxicology and life science in general.

Exposure to bisphenol A in European women from 2007 to 2014 using human biomonitoring data - The European Joint Programme HBM4EU.

BACKGROUND: Bisphenol A (BPA; or 4,4'-isopropylidenediphenol) is an endocrine disrupting chemical. It was widely used in a variety of plastic-based manufactured products for several years. The European Food Safety Authority (EFSA) recently reduced the Tolerable Daily Intake (TDI) for BPA by 20,000 times due to concerns about immune-toxicity. OBJECTIVE: We used human biomonitoring (HBM) data to investigate the general level of BPA exposure from 2007 to 2014 of European women aged 18-73 years (n = 4,226) and its determinants. METHODS: Fifteen studies from 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Israel, Luxembourg, Slovenia, Spain, Sweden, and the United Kingdom) were included in the BPA Study protocol developed within the European Joint Programme HBM4EU. Seventy variables related to the BPA exposure were collected through a rigorous post-harmonization process. Linear mixed regression models were used to investigate the determinants of total urine BPA in the combined population. RESULTS: Total BPA was quantified in 85-100 % of women in 14 out of 15 contributing studies. Only the Austrian PBAT study (Western Europe), which had a limit of quantification 2.5 to 25-fold higher than the other studies (LOQ=2.5 µg/L), found total BPA in less than 5 % of the urine samples analyzed. The geometric mean (GM) of total urine BPA ranged from 0.77 to 2.47 µg/L among the contributing studies. The lowest GM of total BPA was observed in France (Western Europe) from the ELFE subset (GM=0.77 µg/L (0.98 µg/g creatinine), n = 1741), and the highest levels were found in Belgium (Western Europe) and Greece (Southern Europe), from DEMOCOPHES (GM=2.47 µg/L (2.26 µg/g creatinine), n = 129) and HELIX-RHEA (GM=2.47 µg/L (2.44 µg/g creatinine), n = 194) subsets, respectively. One hundred percent of women in 14 out of 15 data collections in this study exceeded the health-based human biomonitoring guidance value for the general population (HBM-GVGenPop) of 0.0115 µg total BPA/L urine derived from the updated EFSA's BPA TDI. Variables related to the measurement of total urine BPA and those related to the main socio-demographic characteristics (age, height, weight, education, smoking status) were collected in almost all studies, while several variables related to BPA exposure factors were not gathered in most of the original studies (consumption of beverages contained in plastic bottles, consumption of canned food or beverages, consumption of food in contact with plastic packaging, use of plastic film or plastic containers for food, having a plastic floor covering in the house, use of thermal paper…). No clear determinants of total urine BPA concentrations among European women were found. A broader range of data planned for collection in the original questionnaires of the contributing studies would have resulted in a more thorough investigation of the determinants of BPA exposure in European women. CONCLUSION: This study highlights the urgent need for action to further reduce exposure to BPA to protect the population, as is already the case in the European Union. The study also underscores the importance of pre-harmonizing HBM design and data for producing comparable data and interpretable results at a European-wide level, and to increase HBM uptake by regulatory agencies.

Exposure to Phthalates in European Children, Adolescents and Adults since 2005: A Harmonized Approach Based on Existing HBM Data in the HBM4EU Initiative.

Phthalates are mainly used as plasticizers and are associated inter alia with adverse effects on reproductive functions. While more and more national programs in Europe have started monitoring internal exposure to phthalates and its substitute 1,2-Cyclohexanedicarboxylic acid (DINCH), the comparability of results from such existing human biomonitoring (HBM) studies across Europe is challenging. They differ widely in time periods, study samples, degree of geographical coverage, design, analytical methodology, biomarker selection, and analytical quality assurance level. The HBM4EU initiative has gathered existing HBM data of 29 studies from participating countries, covering all European regions and Israel. The data were prepared and aggregated by a harmonized procedure with the aim to describe-as comparably as possible-the EU-wide general population's internal exposure to phthalates from the years 2005 to 2019. Most data were available from Northern (up to 6 studies and up to 13 time points), Western (11; 19), and Eastern Europe (9; 12), e.g., allowing for the investigation of time patterns. While the bandwidth of exposure was generally similar, we still observed regional differences for Butyl benzyl phthalate (BBzP), Di(2-ethylhexyl) phthalate (DEHP), Di-isononyl phthalate (DiNP), and Di-isobutyl phthalate (DiBP) with pronounced decreases over time in Northern and Western Europe, and to a lesser degree in Eastern Europe. Differences between age groups were visible for Di-n-butyl phthalate (DnBP), where children (3 to 5-year olds and 6 to 11-year olds) had lower urinary concentrations than adolescents (12 to 19-year-olds), who in turn had lower urinary concentrations than adults (20 to 39-year-olds). This study is a step towards making internal exposures to phthalates comparable across countries, although standardized data were not available, targeting European data sets harmonized with respect to data formatting and calculation of aggregated data (such as developed within HBM4EU), and highlights further suggestions for improved harmonization in future studies.

Operation of a pilot-scale lipid accumulation technology employing parameters to select Microthrix parvicella for biodiesel production from wastewater.

Wastewater treatment plants (WWTPs) may play a crucial role in shifting to a zero-emission future by becoming more sustainable and contributing to the circular economy (CE). Recovered lipids from urban sewage can serve as a raw material for biofuel production contributing to a waste reduction, mitigation of natural resources depletion and reinforcing security and energy independence. A novel, pilot-scale lipid accumulation technology (LAT) employing parameters to select M. parvicella for the biofuel/biodiesel production was implemented on a side stream of an urban WWTP. The LAT proved its concept as the average amount of extracted lipids accumulated in the bioreactors was three-fold higher when compared to the lipids existing in activated sludge. The average transesterification of extracted lipids to biodiesel resulted in a 1.6 % yield, meaning that from 1 kg of dried sludge, 16 g of biodiesel could be formed. The biodiesel produced complies with European standard specifications (EN14214).

From science to policy: How European HBM indicators help to answer policy questions related to phthalates and DINCH exposure.

Within the European Human Biomonitoring (HBM) Initiative HBM4EU we derived HBM indicators that were designed to help answering key policy questions and support chemical policies. The result indicators convey information on chemicals exposure of different age groups, sexes, geographical regions and time points by comparing median exposure values. If differences are observed for one group or the other, policy measures or risk management options can be implemented. Impact indicators support health risk assessment by comparing exposure values with health-based guidance values, such as human biomonitoring guidance values (HBM-GVs). In general, the indicators should be designed to translate complex scientific information into short and clear messages and make it accessible to policy makers but also to a broader audience such as stakeholders (e.g. NGO's), other scientists and the general public. Based on harmonized data from the HBM4EU Aligned Studies (2014-2021), the usefulness of our indicators was demonstrated for the age group children (6-11 years), using two case examples: one phthalate (Diisobutyl phthalate: DiBP) and one non-phthalate substitute (Di-isononyl cyclohexane-1,2- dicarboxylate: DINCH). For the comparison of age groups, these were compared to data for teenagers (12-18 years), and time periods were compared using data from the DEMOCOPHES project (2011-2012). Our result indicators proved to be suitable for demonstrating the effectiveness of policy measures for DiBP and the need of continuous monitoring for DINCH. They showed similar exposure for boys and girls, indicating that there is no need for gender focused interventions and/or no indication of sex-specific exposure patterns. They created a basis for a targeted approach by highlighting relevant geographical differences in internal exposure. An adequate data basis is essential for revealing differences for all indicators. This was particularly evident in our studies on the indicators on age differences. The impact indicator revealed that health risks based on exposure to DiBP cannot be excluded. This is an indication or flag for risk managers and policy makers that exposure to DiBP still is a relevant health issue. HBM indicators derived within HBM4EU are a valuable and important complement to existing indicator lists in the context of environment and health. Their applicability, current shortcomings and solution strategies are outlined.

MdMYB52 regulates lignin biosynthesis upon the suberization process in apple.

Our previous studies, comparing russeted vs. waxy apple skin, highlighted a MYeloBlastosys (Myb) transcription factor (MdMYB52), which displayed a correlation with genes associated to the suberization process. The present article aims to assess its role and function in the suberization process. Phylogenetic analyses and research against Arabidopsis thaliana MYBs database were first performed and the tissue specific expression of MdMYB52 was investigated using RT-qPCR. The function of MdMYB52 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight differentially regulated genes in response MdMYB52. Transcriptomic data were supported by analytical chemistry and microscopy. A massive decreased expression of photosynthetic and primary metabolism pathways was observed with a concomitant increased expression of genes associated with phenylpropanoid and lignin biosynthesis, cell wall modification and senescence. Interestingly key genes involved in the synthesis of suberin phenolic components were observed. The analytical chemistry displayed a strong increase in the lignin content in the cell walls during MdMYB52 expression. More specifically, an enrichment in G-Unit lignin residues was observed, supporting transcriptomic data as well as previous work describing the suberin phenolic domain as a G-unit enriched lignin-like polymer. The time-course qPCR analysis revealed that the observed stress response, might be explain by this lignin biosynthesis and by a possible programmed senescence triggered by MdMYB52. The present work supports a crucial regulatory role for MdMYB52 in the biosynthesis of the suberin phenolic domain and possibly in the fate of suberized cells in russeted apple skins.

Whey- and Soy Protein Isolates Added to a Carrot-Tomato Juice Alter Carotenoid Bioavailability in Healthy Adults.

Recent findings suggested that proteins can differentially affect carotenoid bioaccessibility during gastro-intestinal digestion. In this crossover, randomized human trial, we aimed to confirm that proteins, specifically whey- and soy-protein isolates (WPI/SPI) impact postprandial carotenoid bioavailability. Healthy adults (n = 12 males, n = 12 females) were recruited. After 2-week washout periods, 350 g of a tomato-carrot juice mixture was served in the absence/presence of WPI or SPI (50% of the recommended dietary allowance, RDA ≈ 60 g/d). Absorption kinetics of carotenoids and triacylglycerols (TAGs) were evaluated via the triacylglycerol-rich lipoprotein (TRL) fraction response, at timed intervals up to 10 h after test meal intake, on three occasions separated by 1 week. Maximum TRL-carotenoid concentration (Cmax) and corresponding time (Tmax) were also determined. Considering both genders and carotenoids/TAGs combined, the estimated area under the curve (AUC) for WPI increased by 45% vs. the control (p = 0.018), to 92.0 ± 1.7 nmol × h/L and by 57% vs. SPI (p = 0.006). Test meal effect was significant in males (p = 0.036), but not in females (p = 0.189). In males, significant differences were found for phytoene (p = 0.026), phytofluene (p = 0.004), α-carotene (p = 0.034), and ß-carotene (p = 0.031). Cmax for total carotenoids (nmol/L ± SD) was positively influenced by WPI (135.4 ± 38.0), while significantly lowered by SPI (89.6 ± 17.3 nmol/L) vs. the control (119.6 ± 30.9, p < 0.001). Tmax did not change. The results suggest that a well-digestible protein could enhance carotenoid bioavailability, whereas the less digestible SPI results in negative effects. This is, to our knowledge, the first study finding effects of proteins on carotenoid absorption in humans.

Impact of Protein-Enriched Plant Food Items on the Bioaccessibility and Cellular Uptake of Carotenoids.

Carotenoids are lipophilic pigments which have been associated with a number of health benefits, partly related to antioxidant effects. However, due to their poor solubility during digestion, carotenoid bioavailability is low and variable. In this study, we investigated the effect of frequently consumed proteins on carotenoid bioaccessibility and cellular uptake. Whey protein isolate (WPI), soy protein isolate (SPI), sodium caseinate (SC), gelatin (GEL), turkey and cod, equivalent to 0/10/25/50% of the recommended dietary allowance (RDA, approx. 60g/d), were co-digested gastro-intestinally with carotenoid-rich food matrices (tomato and carrot juice, spinach), and digesta further studied in Caco-2 cell models. Lipid digestion, surface tension and microscopic visualization were also carried out. Co-digested proteins positively influenced the micellization of carotenes (up to 3-fold, depending on type and concentration), especially in the presence of SPI (p < 0.001). An increased cellular uptake was observed for xanthophylls/carotenes (up to 12/33%, p < 0.001), which was stronger for matrices with an initially poor carotenoid micellization (i.e., tomato juice, p < 0.001), similar to what was encountered for bioaccessibility. Turkey and cod had a weaker impact. Significant interactions between carotenoids, lipids and proteins were observed during digestion. Co-digested proteins generally improved lipid digestion in all matrices (p < 0.001), especially for carrot juice, though slight decreases were observed for GEL. Protein impact on the surface tension was limited. In conclusion, proteins generally improved both carotenoid bioaccessibility and cellular uptake, depending on the matrices and carotenoid-type (i.e., carotene vs. xanthophylls), which may be relevant under specific circumstances, such as intake of carotenoid-rich food items low in lipids.

Handling wine pomace: The importance of drying to preserve phenolic profile and antioxidant capacity for product valorization.

Four different wine grape pomaces (GP) (Vitis vinifera) varieties, Auxerrois, Pinot Blanc, Gamay and Pinot Noir, and obtained from white, rosé or red wine vinification, were considered for possible valorization in food supplement industry. Stabilization of GP by drying is paramount prior to further processing in the valorization chain, as GP might suffer spoilage over time. The objectives of this work were therefore to: evaluate the effect of microbiological spoilage and drying on the polyphenol profile and antioxidant capacity of GP; define a drying procedure by comparing kinetics of freeze-drying (FD) and vacuum oven (VO) (at 60 °C and 40 °C). Microbiological spoilage led to significant losses (P < 0.01) of antioxidant capacity (40% to 87%) and total phenolic content (70% to 90%), while drying had no significant effect. FD and VO at 60 °C drying kinetics exhibited similar drying curves, and a dry weight (DW) plateau was reached by 48 hr. In contrast VO at 40 °C required 170 hr to reach similar DW values, pointing out the importance of temperature when opting for VO technology. Antioxidant capacity of GP extracts did not differ between drying methods. Interestingly, GPs from white and rosé wines (AUX, PB, and GAM) had up to 3.5 times higher content (P < 0.001) of total polyphenols compared to PN, obtained from red wine. These results reinforce the importance of drying of GP as a pretreatment, which otherwise could result in significant product degradation. Additionally, we propose white and rosé GP as more interesting sources for valorization, with higher phenolic content, compared to red wine GP.

Insights into Lignan Composition and Biosynthesis in Stinging Nettle (Urtica dioica L.).

Stinging nettle (Urtica dioica L.) has been used as herbal medicine to treat various ailments since ancient times. The biological activity of nettle is chiefly attributed to a large group of phenylpropanoid dimers, namely lignans. Despite the pharmacological importance of nettle lignans, there are no studies addressing lignan biosynthesis in this plant. We herein identified 14 genes encoding dirigent proteins (UdDIRs) and 3 pinoresinol-lariciresinol reductase genes (UdPLRs) in nettle, which are two gene families known to be associated with lignan biosynthesis. Expression profiling of these genes on different organs/tissues revealed a specific expression pattern. Particularly, UdDIR7, 12 and 13 displayed a remarkable high expression in the top internode, fibre tissues of bottom internodes and roots, respectively. The relatively high expression of UdPLR1 and UdPLR2 in the young internodes, core tissue of bottom internode and roots is consistent with the high accumulation of lariciresinol and secoisolariciresinol in these tissues. Lignan quantification showed a high abundance of pinoresinol in roots and pinoresinol diglucosides in young internodes and leaves. This study sheds light on lignan composition and biosynthesis in nettle, providing a good basis for further functional analysis of DIRs and PLRs and, ultimately, engineering lignan metabolism in planta and in cell cultures.

Impact of jasmonic acid on lignification in the hemp hypocotyl.

Phytohormones are crucial molecules regulating plant development and responses to environmental challenges, including abiotic stresses, microbial and insect attacks. Most notably, phytohormones play important roles in the biosynthesis of lignocellulosics. Jasmonates are involved in secondary growth and secondary metabolism, such as phenylpropanoids and lignin biosyntheses. At the physiological and molecular levels, the actions of phytohormones depend on subtle concentration changes, as well as antagonistic equilibria between two or more of these molecules. In this article, we investigate the consequences of jasmonic acid (JA) spraying on young hemp hypocotyls. First, we show that JA application results in changes in the monomeric composition of lignin. Second, we highlight that, five days after application, JA leads to an increase in salicylic acid (SA) content in hemp hypocotyls. These results are discussed in the light of the known antagonism between JA and SA at both the physiological and molecular levels.

Pesticide residue profiles in bee bread and pollen samples and the survival of honeybee colonies-a case study from Luxembourg.

Pesticide residues (112 compounds) were quantified by GC-MS/MS or LC-MS/MS in 85 bee bread samples and 154 pollen samples obtained from five apiaries each with three or four colonies (genotype Buckfast) in Luxembourg over the period 2011-2013. Thiacloprid, chlorfenvinphos, tebuconazole, and methiocarb were found most frequently in bee bread while thiacloprid, permethrin-cis, and permethrin-trans were detected most frequently in the pollen samples. Three neonicotinoid insecticides (clothianidin, imidacloprid, and thiamethoxam) that were restricted by an EU regulation in 2013 after our sampling campaign was finished were each found in less than 8% of the pollen or bee bread samples. The maximum concentrations of thiacloprid, metazachlor, and methiocarb measured in the pollen collected by a group of honeybee colonies (n = 5) without survivors within the 3-year period of observation were 86.20 ± 10.74 ng/g, 2.80 ± 1.26 ng/g, and below the limit of quantification, respectively. The maximum concentrations of the same compounds measured in the pollen collected by a group of honeybee colonies with significantly (P = 0.02) more survivors (7 out of 9) than expected, if the survivors had been distributed randomly among the groups of colonies, were 11.98 ± 2.28 ng/g, 0.44 ± 0.29 ng/g, and 8.49 ± 4.13 ng/g, respectively. No honeybee colony that gathered pollen containing more than 23 ng/g thiacloprid survived the 3-year project period. There was no statistically significant association between pesticide residues in the bee bread and the survival of the colonies. Actions already taken or planned and potential further actions to protect bees from exposure to pesticides are discussed.

Iron-impregnated zeolite catalyst for efficient removal of micropollutants at very low concentration from Meurthe river.

In this paper, for the first time, faujasite Y zeolite impregnated with iron (III) was employed as a catalyst to remove a real cocktail of micropollutants inside real water samples from the Meurthe river by the means of the heterogeneous photo-Fenton process. The catalyst was prepared by the wet impregnation method using iron (III) nitrate nonahydrate as iron precursor. First, an optimization of the process parameters was conducted using phenol as model macro-pollutant. The hydrogen peroxide concentration, the light wavelength (UV and visible) and intensity, the iron loading immobilized, as well as the pH of the solution were investigated. Complete photo-Fenton degradation of the contaminant was achieved using faujasite containing 20 wt.% of iron, under UV light, and in the presence of 0.007 mol/L of H2O2 at pH 5.5. In a second step, the optimized process was used with real water samples from the Meurthe river. Twenty-one micropollutants (endocrine disruptors, pharmaceuticals, personal care products, and perfluorinated compounds) including 17 pharmaceutical compounds were specifically targeted, detected, and quantified. All the initial concentrations remained in the range of nanogram per liter (0.8-88 ng/L). The majority of the micropollutants had a large affinity for the surface of the iron-impregnated faujasite. Our results emphasized the very good efficiency of the photo-Fenton process with a cocktail of a minimum of 21 micropollutants. Except for sulfamethoxazole and PFOA, the concentrations of all the other microcontaminants (bisphenol A, carbamazepine, carbamazepine-10,11-epoxide, clarithromycin, diclofenac, estrone, ibuprofen, ketoprofen, lidocaine, naproxen, PFOS, triclosan, etc.) became lower than the limit of quantification of the LC-MS/MS after 30 min or 6 h of photo-Fenton treatment depending on their initial concentrations. The photo-Fenton degradation of PFOA can be neglected. The photo-Fenton degradation of sulfamethoxazole obeys first-order kinetics in the presence of the cocktail of the other micropollutants.

Insights into the molecular regulation of monolignol-derived product biosynthesis in the growing hemp hypocotyl.

BACKGROUND: Lignin and lignans are both derived from the monolignol pathway. Despite the similarity of their building blocks, they fulfil different functions in planta. Lignin strengthens the tissues of the plant, while lignans are involved in plant defence and growth regulation. Their biosyntheses are tuned both spatially and temporally to suit the development of the plant (water conduction, reaction to stresses). We propose to study the general molecular events related to monolignol-derived product biosynthesis, especially lignin. It was previously shown that the growing hemp hypocotyl (between 6 and 20 days after sowing) is a valid system to study secondary growth and the molecular events accompanying lignification. The present work confirms the validity of this system, by using it to study the regulation of lignin and lignan biosynthesis. Microscopic observations, lignin analysis, proteomics, together with in situ laccase and peroxidase activity assays were carried out to understand the dynamics of lignin synthesis during the development of the hemp hypocotyl. RESULTS: Based on phylogenetic analysis and targeted gene expression, we suggest a role for the hemp dirigent and dirigent-like proteins in lignan biosynthesis. The transdisciplinary approach adopted resulted in the gene- and protein-level quantification of the main enzymes involved in the biosynthesis of monolignols and their oxidative coupling (laccases and class III peroxidases), in lignin deposition (dirigent-like proteins) and in the determination of the stereoconformation of lignans (dirigent proteins). CONCLUSIONS: Our work sheds light on how, in the growing hemp hypocotyl, the provision of the precursors needed to synthesize the aromatic biomolecules lignin and lignans is regulated at the transcriptional and proteomic level.

Fate and effects of silver nanoparticles on early life-stage development of zebrafish (Danio rerio) in comparison to silver nitrate.

The use of silver nanomaterials in everyday products, such as cosmetics, textiles, certain types of packaging, etc. is increasing, leading to their release into the environment, including aquatic ecosystems. This last point initiated this investigation on the toxicological effects of Ag nanoparticles (Ag NPs) in the aquatic model organism Danio rerio. For this purpose, zebrafish larvae were exposed to 20nm bare Ag NPs at different concentrations and AgNO3, used as a positive control for Ag+ ions toxicity, at the beginning of their foraging behaviour to determine adverse effects on fitness parameters. We used secondary ion mass spectrometry (SIMS) to determine the localization of Ag and transcriptomics (microarray) to determine the toxicity at the level of gene expression in fish larvae. Exposure to Ag NPs did not result in adverse effects on survival and growth of the fish. However, SIMS analysis showed that Ag NPs mainly concentrate around liver blood vessels and in the interstitial tissue between the intestine and the liver. Gene expression profiles revealed that AgNO3 and Ag NPs impacted common pathways, suggesting similar targets, such as the phototransduction system. However, the Ag NPs showed a broader set of genes impacted following the exposure, including the circadian clock regulation and the photoreception, suggesting specific particle-related effects in addition to those induced by ions.

Differential Lipid Composition and Gene Expression in the Semi-Russeted "Cox Orange Pippin" Apple Variety.

Russeting is characterized by a particular rough and brown phenotype, which is mainly due to the accumulation of suberin in the inner part of the epidermal cell walls. In our previous bulk transcriptomic analysis, comparing fully russeted, and waxy apple varieties, showed, in apple fruit skin, a massive decreased expression of cutin, wax and some pentacyclic triterpene biosynthesis genes in the russeted varieties, with an expected concomitant enhanced expression of the suberin biosynthetic genes. In the present work, we performed a deep investigation of the aliphatic composition of the cutin, suberin, waxes, and triterpenes in the waxy and russeted patches of the semi-russeted apple variety "Cox Orange Pippin." A targeted gene expression profiling was performed to validate candidate genes which were identified in our previous work and might be involved in the respective metabolic pathways. Our results showed that a decrease of cuticular waxes, ursolic acid and oleanolic acid, accompanied by an accumulation of alkyl-hydroxycinamates and betulinic acid, occurs in the russeted patches. The suberin monomer composition is characterized by specific occurrence of 20, 22, and 24 carbon aliphatic chains, whereas cutin is mainly represented by common C16 and C18 aliphatic chains. This work depicts, for the first time in apple, the complex composition of suberin, cutin, waxes and triterpenes, and confirms the strong interplay between these epidermal polymers in apple fruit skin.

No influence of supplemental dietary calcium intake on the bioavailability of spinach carotenoids in humans.

Dietary carotenoid intake, especially from fruits and vegetables, has been associated with a reduced incidence of several chronic diseases. However, its bioavailability can vary, depending on the food matrix and host factors. Recently, it has been suggested that divalent minerals negatively impinge on carotenoid bioavailability by reducing bile-salt and non-esterified fatty-acid levels in the gut, which normally aid in emulsifying carotenoids. The aim of the present study was to investigate whether supplemental Ca would negatively influence carotenoid absorption in humans. A total of twenty-five healthy, non-obese men (age: 20-46 years, BMI<30 kg/m2) were recruited for this postprandial, randomised, crossover, double-blinded trial. Following a randomised block design, each participant received (after 2-week washout periods), on three occasions separated by 1 week, 270 g of spinach-based meals (8·61 (sd 1·08) mg carotenoids/100 g fresh weight), supplemented with 0, 500 or 1000 mg of Ca (as calcium carbonate), with each participant acting as his or her own control. Blood samples were collected at regular postprandial intervals for up to 10 h following test meal intake, and standardised lunches were served. TAG-rich lipoprotein fractions were separated and carotenoid concentrations determined. AUC for meals without supplemented Ca were 22·72 (sem 2·78) nmol×h/l (lutein), 0·19 (sem 3·90) nmol×h/l (ß-carotene) and 2·80 (sem 1·75) nmol×h/l (ß-cryptoxanthin). No significant influence of supplementation with either 500 or 1000 mg of supplemental Ca was found. In conclusion, Ca - the most abundant divalent mineral in the diet - given at high but physiological concentrations, does not appear to have repercussions on the bioavailability of carotenoids from a spinach-based meal.

MdMyb93 is a regulator of suberin deposition in russeted apple fruit skins.

A comparison of the transcriptomes of russeted vs nonrusseted apple skins previously highlighted a tight relationship between a gene encoding an MYB-type transcription factor, MdMYB93, and some key suberin biosynthetic genes. The present work assesses the role of this transcription factor in the suberization process. A phylogenetic analysis of MdMYB93 and Arabidopsis thaliana MYBs was performed and the function of MdMYB93 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight the MdMYB93-regulated genes. Ultraperformance liquid chromatography-triple time-of-flight (UPLC-TripleTOF) and GC-MS were used to investigate alterations in phenylpropanoid, soluble-free lipid and lipid polyester contents. A massive accumulation of suberin and its biosynthetic precursors in MdMYB93 agroinfiltrated leaves was accompanied by a remobilization of phenylpropanoids and an increased amount of lignin precursors. Gene expression profiling displayed a concomitant alteration of lipid and phenylpropanoid metabolism, cell wall development, and extracellular transport, with a large number of induced transcripts predicted to be involved in suberin deposition. The present work supports a major role of MdMYB93 in the regulation of suberin deposition in russeted apple skins, from the synthesis of monomeric precursors, their transport, polymerization, and final deposition as suberin in primary cell wall.

Inhibition of multixenobiotic resistance transporters (MXR) by silver nanoparticles and ions in vitro and in Daphnia magna.

The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATP-binding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MXR). In this study we first evaluated, in vitro, the interaction of silver nanoparticles (Ag NPs, 20, 23 and 27nm), Ag 200nm particles and Ag ions (AgNO3) with MXR efflux transporters using MDCKII and the P-gp over-expressing MDCKII-MDR1 cells and calcein-AM as a substrate of the transporters. Next the in vivo modulation of MXR activity was studied in Daphnia magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. The common environmental contaminants perfluorooctane sulfonate and bisphenol A, previously observed to interfere with the P-gp in vitro, also inhibited the efflux of calcein in vivo. Small-sized Ag NPs (with biomolecules present on the surface) and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1 cells, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides evidence of the interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in D. magna, and should be taken into account when Ag NP toxicity is assessed.

Origin and Dynamics of Rare Earth Elements during Flood Events in Contaminated River Basins: Sr-Nd-Pb Isotopic Evidence.

In order to precisely quantify the contribution of anthropogenic activities and geogenic sources to the dissolved and suspended loads of rivers we have combined for the first time Rare Earth Element (REE) concentrations with Sr-Nd-Pb isotope ratios. We observed enrichments in Anthropogenic Rare Earth Elements (AREE) for dissolved (Gd) and suspended (Ce and Nd) loads of river water. During flood events, AREE anomalies progressively disappeared and gave way to the geogenic chemical signature of the basin in both dissolved and suspended loads. The isotopic data confirm these observations and shed new light on the trace elements sources. On the one hand, dissolved loads have peculiar isotopic characteristics and carry mainly limestone-derived and anthropogenic Sr and Nd as well as significant amounts of anthropogenic Pb. On the other hand, the results clearly indicate that anthropogenic contributions impact the suspended loads in all hydrological conditions. This study demonstrates that anthropogenic contributions to the river may change not only Pb but also Sr and Nd isotopic compositions in both dissolved and suspended loads. This is of importance for future provenance studies.