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Endothelial cells (ECs) secrete different paracrine signals that modulate the function of adjacent cells; two examples of these paracrine signals are nitric oxide (NO) and neuregulin-1 (NRG1), a cardioprotective growth factor. Currently, it is undetermined whether one paracrine factor can compensate for the loss of another. Herein, we hypothesized that NRG1 can compensate for endothelial NO synthase (eNOS) deficiency. We characterized eNOS null and wild-type (WT) mice by cardiac ultrasound and histology and we determined circulating NRG1 levels. In a separate experiment, eight groups of mice were divided into four groups of eNOS null mice and WT mice; half of the mice received angiotensin II (ANG II) to induce a more severe phenotype. Mice were randomized to daily injections with NRG1 or vehicle for 28 days. eNOS deficiency increased NRG1 plasma levels, indicating that ECs increase their NRG1 expression when NO production is deleted. eNOS deficiency also increased blood pressure, lowered heart rate, induced cardiac fibrosis, and affected diastolic function. In eNOS null mice, ANG II administration not only increased cardiac fibrosis but also induced cardiac hypertrophy and renal fibrosis. NRG1 administration prevented cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. Moreover, Nrg1 expression in the myocardium is shown to be regulated by miR-134. This study indicates that administration of endothelium-derived NRG1 can compensate for eNOS deficiency in the heart and kidneys. NEW & NOTEWORTHY ECs compensate for eNOS deficiency by increasing the secretion of NRG1. NRG1 administration prevents cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. NRG1 expression is regulated by miR-134.

Lignin is a renewable and abundant source for production of bio-based chemicals and is a valuable alternative to crude oil to obtain aromatic building blocks. It is built from aromatic units with strong chemical linkages, which need to be cleaved to enable the use of the aromatic compounds in industrial applications. In addition to depolymerizing lignin, efficient fractionation and conversion of the resulting complex mixtures is an essential step in the valorization of lignin derivatives for different applications. In this work, we studied the separation of a lignin oil obtained by catalytic cleavage in supercritical ethanol (scEtOH) of technical lignin produced by means of soda pulping of wheat straw. The use of six commercial polymeric nanofiltration (NF) membranes and one in-house developed Grignard-functionalized ceramic membrane was investigated for the fractionation of a mixture of lignin derivatives. Separation by molecular weight (MW) was observed with the polymeric NP030 membrane but not with the other membranes tested. The development of a protocol using this membrane in the diafiltration mode for fractionation of crude lignin oils (CLOs) is discussed.

An interlaboratory comparison was done for the analysis of carotenoids in freeze-dried mango. The study was performed from July to September 2018. Mango fruit was freeze-dried, homogenized, and packaged under vacuum conditions in portions of 6 g (test sample). Two test samples were sent to the participating laboratories for analysis. Laboratory results were rated using Z-scores in accordance with ISO 13528 and ISO 17043. The standard deviation for proficiency assessment (also called target standard deviation) was determined using a modified Horwitz function and varied between 10% and 25%, depending on the analyte. Out of 14 laboratories from 10 different countries, 9 laboratories (64%) obtained a satisfactory performance (Z ≤ 2) for the analysis of β-carotene. While for 7 laboratories that analyzed α-carotene, (9Z)-β-carotene, β-cryptoxanthin, and zeaxanthin, 4 laboratories (57%) obtained a satisfactory performance. However, only 2 laboratories out of 7 (29%) obtained a satisfactory performance for lutein. Based on the comparability of the analytical results, this study concludes that freeze-dried mango pulp can be used as a reference material for the analysis of α and β-carotene, (9Z)-β-carotene, β-cryptoxanthin, and zeaxanthin by applying different analytical procedures for their extraction and quantification., This work was performed within the frame of the TEAM EC2017TEA442A103 VLIR-UOS project “Improving Ecuadorian child nutrition by using mango by-products as potential sources of bioactive compounds”. JV-Ch wants to acknowledge the quality technical support of Samara Fernández de Souza from VITO. VM-P acknowledges Mayra Anaguano from EPN. AZM acknowledges Fabiane C. Petry for the carotenoid analysis, FAPESP (grant 2018/23752-1) and CNPq (grant 309182/2018-2).

Cardiac arrhythmias are associated with cardiovascular morbidity and mortality. Cardiac electrophysiology studies (EPS) use intra-cardiac catheter recording and stimulation for profound evaluation of the heart's electrical properties. The main clinical applica-tion is investigation and treatment of rhythm disorders. These techniques have been translated to the murine setting to open opportunities for detailed evaluation of the impact of different characteristics (including genetics) and interventions on cardiac electrophysiology and-pathology. Currently, a detailed description of the technique of murine transjugular EPS (which is the standard route of catheter introduction) is lacking. This article provides detailed information on EPS in mice via the transjugular route. This includes catheter placement, stimulation protocols, intracardiac tracing interpretation, artifact reduction, and surface ECG recording. In addition, reference values as obtained in C57BL/6N mice are presented for common electrophysiological pa-rameters. This detailed methodological description aims to increase accessibility and standardization of EPS in mice. Ultimately, also human research and patient care may benefit from translation of the knowledge obtained in preclinical models using this technique. NEW & NOTEWORTHY Electrophysiology studies (EPS) allow in-depth evaluation of cardiac electrophysiology and-pathology. These techniques have been adapted to the murine setting for (translational) studies, mainly focusing on arrhythmogenesis. Despite the frequent application of EPS via the transjugular route, a thorough description of the technique is currently lacking. This article aims to function as a comprehensive guide, also elaborating (for the first time) on nonsurgical aspects such as cathe-ter positioning, tracing artifacts, stimulation protocols, and reference values.

Aims Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. Methods and results In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4−/− mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4−/− mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. Conclusion The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

Non-target analysis (NTA) employing high-resolution mass spectrometry is a commonly applied approach for the detection of novel chemicals of emerging concern in complex environmental samples. NTA typically results in large and information-rich datasets that require computer aided (ideally automated) strategies for their processing and interpretation. Such strategies do however raise the challenge of reproducibility between and within different processing workflows. An effective strategy to mitigate such problems is the implementation of inter-laboratory studies (ILS) with the aim to evaluate different workflows and agree on harmonized/standardized quality control procedures. Here we present the data generated during such an ILS. This study was organized through the Norman Network and included 21 participants from 11 countries. A set of samples based on the passive sampling of drinking water pre and post treatment was shipped to all the participating laboratories for analysis, using one pre-defined method and one locally (i.e. in-house) developed method. The data generated represents a valuable resource (i.e. benchmark) for future developments of algorithms and workflows for NTA experiments., Measurement(s)chemical • drinking waterTechnology Type(s)high resolution mass spectrometry • non-target analysis • InterlaboratoryFactor Type(s)methodSample Characteristic - Environmentlaboratory environment Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.15028665

Development of analytical methods for the characterization (particle size determination, chemical identification, and quantification) of the low µm-range microplastic (MPs; 1-10 µm) and nanoscale plastic (NPs; 1-1000 nm) debris in environmental matrices is a quickly emerging scientific field and has gained considerable attention, not only within the scientific community, but also on the part of policy makers and the general public. However, due to the limited sensitivity of the current state of the art monitoring techniques, detection of MPs and NPs in water is one of the biggest challenges for their monitoring, source identification and, ultimately, risk assessment.As it is evident that no single method will provide all the information required for a complete characterization of MPs and NPs in water, the present work is aimed to give an overview of different complementary analytical methodologies showing considerable promise for the particle size determination, chemical identification, and quantification of MPs and NPs [1]. In addition, results of three case studies will be included to adequately address the smallest fractions in plastic debris size determination, making such approaches worthwhile to be further explored.The first case study offers a novel method based on the use of inductively coupled plasma-mass spectrometry operated in single-event mode and relies on our previous work where for the first time ever single particle inductively coupled plasma-mass spectrometry based on carbon monitoring was successfully used for the detection, particle size characterization and particle number concentration of polystyrene MPs [2]. The second case study further explore light scattering methods, including nanoparticle tracking analysis or dynamic light scattering, for MPs and NPs particle size distribution and particle number in water. Finally, the capabilities of size exclusion chromatography in combination with online detection techniques such as UV-visible absorption spectrometry will be presented for the particle size determination of smallest fraction of NPs (1-100 nm). M.V. is a senior postdoctoral fellow of the Research Foundation – Flanders (FWO 12ZD120N). References[1] Velimirovic M., Tirez K., Voorspoels S., Vanhaecke F. (2020) Recent developments in mass spectrometry for the characterization of micro- and nanoscale plastic debris in the environment, Analytical and Bioanalytical Chemistry, 1-9.[2] Bolea-Fernandez E., Rua-Ibarz A., Velimirovic M., Tirez K., and Vanhaecke F. (2020) Detection of microplastics using inductively coupled plasma-mass spectrometry (ICP-MS) operated in single-event mode. Journal of Analytical Atomic Spectrometry 35, 455-460.

Arterial stiffness is an important predictor of cardiovascular risk. Clinical studies have demonstrated that arterial stiffness increases in cancer patients treated with the chemotherapeutic doxorubicin (DOX). However, the mechanisms of DOX-induced arterial stiffness remain largely unknown. This study aimed to evaluate artery stiffening in DOX-treated mice using in vivo and ex vivo techniques. Male C57BL/6J mice were treated for 2 weeks with 2 mg/kg (low dose) or 4 mg/kg (high dose) of DOX weekly. Arterial stiffness was assessed in vivo with ultrasound imaging (abdominal aorta pulse wave velocity (aaPWV)) and applanation tonometry (carotid-femoral PWV) combined with ex vivo vascular stiffness and reactivity evaluation. The high dose increased aaPWV, while cfPWV did not reach statistical significance. Phenylephrine (PE)-contracted aortic segments showed a higher Peterson’s modulus (Ep) in the high dose group, while Ep did not differ when vascular smooth muscle cells (VSMCs) were relaxed by a NO donor (DEANO). In addition, aortic rings of DOX-treated mice showed increased PE contraction, decreased basal nitric oxide (NO) index and impaired acetylcholine-induced endothelium-dependent relaxation. DOX treatment contributed to endothelial cell loss and reduced endothelial nitric oxide synthase (eNOS) expression in the aorta. In conclusion, we have replicated DOX-induced arterial stiffness in a murine model and this aortic stiffness is driven by impaired endothelial function, contributing to increased vascular tone.

In Europe, 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) and 4-methylimidazole (4-MEI) are – to a certain level – allowed to be present in the food colours ammonia caramel (E 150c) and sulphite ammonia caramel (E 150d). Besides their presence in food colours, exposure to these contaminants may also include other dietary sources. This study describes the occurrence of THI and 4-MEI in a wide variety of food products (n = 522) purchased from the Belgian market and their dietary intake in Belgian consumers from 15 years old onwards. THI was found to be present in 22.4% of the investigated foods at a level up to 551 µg/kg. For 4-MEI (57.7% quantifiable), concentrations up to 2,835 µg/kg were observed. The average dietary intake amounted to 0.02–0.36 µg kg−1 bw−1 day for THI and 0.4–3.7 µg kg−1 bw−1 day for 4-MEI. Coffee, cola and beer were contributing most to the dietary THI and 4-MEI intake in Belgium.

Traffic-related air pollution (TRAP) exposure is associated with negative health outcomes. Changes in DNA methylation level may be an important mechanism through which air pollution can induce its effects. The objective of this study was to evaluate the association between DNA methylation in blood and personally measured TRAP exposure. Global DNA methylation in whole blood was analyzed with HPLC in a population of 55 healthy adults (average age 41 years). TRAP was assessed for each participant with a portable aethalometer measuring black carbon (BC). Exposure measurements were collected during one typical working week. These data were used in combination with ambient levels measured at a reference site to derive subchronic BC exposure. Urinary trans,trans-muconic acid (t,t-MA), a metabolite of benzene, was used as an internal proxy of traffic exposure. DNA methylation levels were associated with short- and subchronic BC exposure. An IQR increase in BC exposure on lag 24 h (477 ng/m3), lag 48 h (491 ng/m3), lag 1 week (314 ng/m3) and subchronic exposure (618 ng/m3) were associated with a decrease in DNA methylation levels of respectively 0.0020% (− 0.0040 to − 0.0001, p = 0.047), 0.0028% (− 0.0054 to − 0.0001, p = 0.043), 0.0024% (− 0.0043 to − 0.0005, p = 0.019), and 0.025% (− 0.048 to − 0.0015, p = 0.044). In addition, an IQR increase in t,t-MA (0.135 mg/l) was associated with a 0.0021% (− 0.0033 to − 0.0008, p = 0.0019) decrease in global DNA methylation levels. Analysis of a panel of cytokines in blood samples failed to demonstrate an association between inflammatory and oxidative stress biomarkers and TRAP or DNA methylation. In a panel of healthy adults, we found negative associations between total DNA methylation and markers of TRAP exposure. Considering that change in DNA methylation concentration is a biological marker connecting environmental and lifestyle exposures and disease development trajectories, our results warrant further study.

The human TRP protein family comprises a family of 27 cation channels with diverse permeation and gating properties. The common theme is that they are very important regulators of intracellular Ca2+ signaling in diverse cell types, either by providing a Ca2+ influx pathway, or by depolarising the membrane potential, which on one hand triggers the activation of voltage-gated Ca2+ channels, and on the other limits the driving force for Ca2+ entry. Here we focus on the role of these TRP channels in vascular smooth muscle and cardiac striated muscle. We give an overview of highlights from the recent literature, and highlight the important and diverse roles of TRP channels in the pathophysiology of the cardiovascular system. The discovery of the superfamily of Transient Receptor Potential (TRP) channels has significantly enhanced our knowledge of multiple signal transduction mechanisms in cardiac muscle and vascular smooth muscle cells (VSMC). In recent years, multiple studies have provided evidence for the involvement of these channels, not only in the regulation of contraction, but also in cell proliferation and remodeling in pathological conditions. The mammalian family of TRP cation channels is composed by 28 genes which can be divided into 6 subfamilies groups based on sequence similarity: TRPC (Canonical), TRPM (Melastatin), TRPML (Mucolipins), TRPV (Vanilloid), TRPP (Policystin) and TRPA (Ankyrin-rich protein). Functional TRP channels are believed to form four-unit complexes in the plasma, each of them expressed with six transmembrane domain and intracellular N and C termini. Here we review the current knowledge on the expression of TRP channels in both muscle types, and discuss their functional properties and role in physiological and pathophysiological processes.

Microalgae are promising sources of lipids for applications in food, fuel, pharmaceutics and cosmetics. However, these lipids are also subject to lipolysis reactions during processing and storage. Especially during wet biomass storage, substantial amounts of free fatty acids (FFA) are formed, which has negative effects on flavor and nutritional value and can cause downstream processing problems in biodiesel production. This study contributes to a better understanding of the underlying processes leading to lipolysis by focusing on cell wall differences between species. Therefore, in a first phase, a comparison was made between the lipolytic stability of Nannochloropsis oculata and T-Isochrysis lutea , two species reported to have substantial differences in cell wall structure and strength. Nannochloropsis appeared to be stable during the first days of wet storage, while in T-Isochrysis , lipolysis started immediately after harvest. In a second experiment, the influence of cell disruption by high pressure homogenization (HPH) on lipolytic stability during wet storage of Nannochloropsis was investigated. It was observed that the HPH treatment induced lipolysis, as the FFA content was rapidly increasing immediately after the treatment, in contrast to the control sample, in which the FFA content was constant during the first days of storage. The integrity of the microalgal cell was thus found to play a crucial role in lipid stability during short-term post-harvest wet storage.

There is a lack of data for individual oligomeric procyanidins in apples and apple extracts. Our aim was to develop, validate and evaluate an analytical method for the separation, identification and quantification of monomeric and oligomeric flavanols in apple extracts. To achieve this, we prepared two types of flavanol extracts from freeze-dried apples; one was an epicatechin-rich extract containing ∼30% (w/w) monomeric (−)-epicatechin which also contained oligomeric procyanidins (Extract A), the second was an oligomeric procyanidin-rich extract depleted of epicatechin (Extract B). The parameters considered for method optimisation were HPLC columns and conditions, sample heating, mass of extract and dilution volumes. The performance characteristics considered for method validation included standard linearity, method sensitivity, precision and trueness. Eight laboratories participated in the method evaluation. Chromatographic separation of the analytes was best achieved utilizing a Hilic column with a binary mobile phase consisting of acidic acetonitrile and acidic aqueous methanol. The final method showed linearity for epicatechin in the range 5–100 μg/mL with a correlation co-efficient >0.999. Intra-day and inter-day precision of the analytes ranged from 2 to 6% and 2 to 13% respectively. Up to dp3, trueness of the method was >95% but decreased with increasing dp. Within laboratory precision showed RSD values, This research has received funding from the European Community’s Seventh Framework Programme (FP7 under agreement no. 312090, project BACCHUS) and the Biotechnology and Biological Sciences Research Council (UK) through an Institute Strategic Programme Grant (‘Food and Health’; Grant No: BB/J004545/1) to the Institute of Food Research.

Background We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. Material and methods Six hundred 14–15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid as a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. Results Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel and t,t-muconic acid were inversely associated with the alkaline comet assay. Conclusion This cross-sectional study found associations between current environmental exposure to (potential) human carcinogens in 14–15-year-old Flemish adolescents and short-term (oxidative) damage to DNA. Prospective follow-up will be required to investigate whether long-term effects may occur due to complex environmental exposures.

Background Decreased eNOS activity is the hallmark of endothelial dysfunction and is associated with cardiovascular and renal disorders. Besides NO, endothelial cells produce numerous other small molecules, peptides, and proteins, which modulate the function of adjacent cells. For instance, neuregulin-1 (NRG-1) is an endothelium-derived growth factor, which plays crucial roles in cardiovascular development, has cardioprotective properties, and induces growth and differentiation of cell types in different organs, including the kidney. Purpose Although the cardioprotective effects of endothelium-derived NO and NRG-1 are well established, their interaction is not clear. Therefore, we studied the interaction between the NO/eNOS and NRG-1/ErbB signalling pathways in a transgenic eNOS knock-out mouse model (eNOS−/−) treated with subpressor doses of angiotensin II (AngII). Methods eNOS−/− mice and their wild type (WT) littermates (n=64, 15 weeks old) were randomized for implantation of osmotic minipumps with AngII (400 ng/kg.min) for 28 days or sham surgery. Mice were randomized to receive either daily NRG-1 injections (20 μg/kg, intraperitoneal) or vehicle for 4 weeks (n=8/group). Hypertrophy and fibrosis were measured in left ventricle (LV) and kidneys using echography and immunohistochemical staining. Results Although blood pressure was higher in eNOS−/− mice compared to their WT littermates, it was unaffected by a subpressor dose of AngII. Masson's trichrome staining showed that AngII significantly increased LV (interstitial and perivascular) and renal fibrosis in eNOS−/− mice, but not in WT controls (see figure for LV data). NRG-1 reversed this AngII-induced LV and renal fibrosis caused by eNOS deficiency. There was also significant hypertrophy of LV and kidneys in eNOS−/− mice treated with AngII, which was again normalized by NRG-1 treatment. Moreover, NRG-1 significantly attenuated albuminuria induced by eNOS deficiency under AngII treatment. Conclusions This study demonstrates that the anti-fibrotic and anti-hypertrophic effects of NRG-1 are independent from the NO/eNOS pathway in both heart and kidney. Strikingly, NRG-1 is able to compensate for some of the negative effects of eNOS deficiency, at least in conditions of AngII stimulation. Acknowledgement/Funding supported by university of Antwerp

Rationale: TRPM4 is a Ca 2+ -activated non-selective cation channel that is abundantly expressed in the heart. Mutations in the Trpm4 gene are associated with human cardiac conduction disorders, including Progressive Familial Heart Block type I (PFHBI) and Brugada Syndrome. However, the mechanistic role and in vivo significance of TRPM4 in the triggering of cardiac arrhythmias is still completely unclear. Objective: To investigate the role of TRPM4 during pathological Ca 2+ handling in the heart. Methods and results: Using three in vivo pro-arrhythmic assays, we found that Trpm4 -/- mice show a reduced arrhythmic burden compared to control mice. First, aconitine intoxication resulted in severe cardiac arrhythmias, both in WT and Trpm4 -/- animals, but Trpm4 -/- mice developed significantly less ventricular ectopic beats (VEBs) and showed a lower arrhythmic score. Second, during ischemia-reperfusion, induced by 30 min of LAD ligation, significantly more WT animals developed arrhythmias compared to Trpm4 -/- mice. Third, catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, carrying mutations in RyR2, were subjected to a stress test. Significantly more RyR +/R2474S -Trpm4 +/+ animals developed arrhythmias compared to RyR +/R2474S -Trpm4 -/- mice. Conclusion: Our data establish that TRPM4 represents a novel target in the prevention and treatment of cardiac arrhythmias.

Previously, it has been observed that lipolysis occurring during (even short term) wet storage of microalgal biomass causes high free fatty acid (FFA) concentrations in the biomass. These FFA have a negative impact for different applications of microalgal lipids, e.g. downstream processing problems in biodiesel production, off-flavors and loss of nutritional value for food applications. However, it is not clear which factors influence lipolysis in microalgal biomass and which lipid class are more susceptible to lipolysis. In this study, wet biomass of T-Isochrysis lutea was stored at 20°C, 4°C and -20°C during 3 weeks. The extent of lipolysis was followed by analyzing the lipid classes distribution by ultrahigh-performance liquid chromatography−accurate mass mass spectrometry (UHPLC-amMS) and the FFA content. It was observed that FFA were formed very rapidly during post-harvest storage of wet biomass at 20°C and 4°C, the rate of this process being faster at 20°C than at 4°C, while almost no lipolysis was observed at -20°C. However, the FFA content levelled off after several days of storage because FFA reacted with alcohols to form fatty acyl esters. publisher: Elsevier articletitle: Lipolysis in T-Isochrysis lutea during wet storage at different temperatures journaltitle: Algal Research articlelink: http://dx.doi.org/10.1016/j.algal.2016.07.003 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved. ispartof: Algal Research vol:18 pages:281-287 status: published

Algal lipids have gained wide interest in various applications ranging from biofuels to nutraceuticals. Given their complex nature composed of different lipid classes, a deep knowledge between extraction conditions and lipid characteristics is essential. In this paper, we investigated the influence of different pretreatments on lipid extraction with supercritical CO2 by a lipidomic approach. Pretreatment was found to double the total extraction yield, thereby reaching 23.1 wt.% comparable to the 26.9 wt.% obtained with chloroform/methanol. An increase in acylglycerides was concurrently observed, together with a nearly doubling of free fatty acids indicative of partial hydrolysis. Moreover, an alteration in the distribution of glyco- and phospholipids was noted, especially promoting digalactosyldiglycerides and phosphatidylcholine as compared to monogalactosyldiglycerides and phosphatidylglycerol. At optimized conditions, supercritical CO2 extraction provided a lipid extract richer in neutral lipids and poorer in phospholipids as compared to chloroform/methanol, though with a very similar fatty acid distribution within each lipid class.

A dietary exposure assessment of food emulsifiers E481 (sodium stearoyl-2-lactylate) and E482 (calcium stearoyl-2-lactylate) in the Belgian population was performed. Nationally representative food consumption data from the Belgian National Food Consumption Surveys 2004 (BNFCS2004) and 2014 (BNFCS2014) were used for calculations. A conservative approach (combining individual food consumption data with the maximum permitted level (MPL) of foods (tier 2), was compared with more refined estimates (combining individual food consumption data with actual concentrations measured in food products available on the Belgian market (tier 3)). Estimated daily intakes were compared to the acceptable daily intake (ADI) of the stearoyl-2-lactylates. The results of tier 2 demonstrated that 92% of the children (3-9 years), 53% of the adolescents (10-17 years), 15% of the adults (18-64 years) and 26% of the elderly (64-98 years) had a potential intake higher than the ADI. When replacing the MPL with maximum analysed concentration levels in foods, daily intake estimates decreased dramatically. The estimated daily intake of the food emulsifiers was below the ADI for all age groups, except for a small percentage of children (1.9%) for which the intake exceeded the ADI. The main contributors to the exposure of E481 and E482 were bread, rolls and fine bakery wares.

Pomegranates are one of the main highly valuable sources of ellagitannins. Despite the potential health benefits of these compounds, reliable data on their content in pomegranates and derived extracts and food products is lacking, as it is usually underestimated due to their complexity, diversity, and lack of commercially available standards. This study describes a new method for the analysis of the extractable and nonextractable ellagitannins based on the quantification of the acid hydrolysis products that include ellagic acid, gallic acid, sanguisorbic acid dilactone, valoneic acid dilactone, and gallagic acid dilactone in pomegranate samples. The study also shows the occurrence of ellagitannin C-glycosides in pomegranates. The method was optimized using a pomegranate peel extract. To quantify nonextractable ellagitannins, freeze-dried pomegranate fruit samples were directly hydrolyzed with 4 M HCl in water at 90 °C for 24 h followed by extraction of the pellet with dimethyl sulfoxide/methanol (50:50, v/v). The method was validated and reproducibility was assessed by means of an interlaboratory trial, showing high reproducibility across six laboratories with relative standard deviations below 15%. Their applicability was demonstrated in several pomegranate extracts, different parts of pomegranate fruit (husk, peels, and mesocarp), and commercial juices. A large variability has been found in the ellagitannin content (150-750 mg of hydrolysis products/g) and type (gallagic acid/ellagic acid ratios between 4 and 0.15) of the 11 pomegranate extracts studied.

Scope: Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. Method and results: We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. Conclusions: In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability.

Caramel colours are used by the food industry in a wide range of foods and beverages. During their manufacturing, low molecular weight compounds such as 4-methylimidazole (4-MEI), the structural isomer of 4-MEI, 2-methylimidazole (2-MEI) and 2-acetyl-4-tetrahydroxy-butylimidazole (THI) are generated. The presence of these inevitable by-products of caramel manufacturing can be hazardous to human health. This publication describes an isotope dilution Ultra-High-performance Liquid Chromatography tandem mass spectrometric method (UHPLC-MS/MS) that was developed and validated for the simultaneous quantification of these impurities in both beverages/liquids and foods. A limit of quantification of 5 μg/kg was obtained for 4-MEI and THI. The expanded measurement uncertainty (U; k = 2) for these compounds was below 51% in beverages/liquids and below 56% in foods. As higher measurement uncertainties were obtained for 2-MEI, the developed analytical procedure can only be used in a semi-quantitative way for this compound.

Alternative plasticizers and flame retardants (FRs) have been introduced as replacements for banned or restricted chemicals, but much is still unknown about their metabolism and occurrence in humans. We identified the metabolites formed in vitro for four alternative plasticizers (acetyltributyl citrate (ATBC), bis(2-propylheptyl) phthalate (DPHP), bis(2-ethylhexyl) terephthalate (DEHTP), bis(2ethylhexyl) adipate (DEHA)), and one FR (2,2-bis (chloromethyl)-propane-1,3-diyltetrakis(2-chloroethyl) bisphosphate (V6)). Further, these compounds and their metabolites were investigated by LC/ESI-Orbitrap-MS in urine and finger nails collected from a Norwegian cohort. Primary and secondary ATBC metabolites had detection frequencies (% DF) in finger nails ranging from 46 to 95%. V6 was identified for the first time in finger nails, suggesting that this matrix may also indicate past exposure to FRs as well as alternative plasticizers. Two isomeric forms of DEHTP primary metabolite were highly detected in urine (97% DF) and identified in finger nails, while no DPHP metabolites were detected in vivo. Primary and secondary DEHA metabolites were identified in both matrices, and the relative proportion of the secondary metabolites was higher in urine than in finger nails; the opposite was observed for the primary metabolites. As many of the metabolites present in in vitro extracts were further identified in vivo in urine and finger nail samples, this suggests that in vitro assays can reliably mimic the in vivo processes. Finger nails may be a useful noninvasive matrix for human biomonitoring of specific organic contaminants, but further validation is needed.

The influents of plants treating complex industrial wastewaters from third parties may contain a large variety of often unknown or unidentified potentially harmful substances. The conventional approach of assessing and regulating the effluents of these plants is to set emission limit values for a limited set of physicochemical parameters, such as heavy metals, biological oxygen demand, chemical oxygen demand and adsorbable organic halogen compounds. The objective of this study was to evaluate the relevance of physicochemical parameters for setting emission limit values for such plants based on a comparison of effluent analyses by physicochemical and biological assessment tools. The results show that physicochemical parameters alone are not sufficient to evaluate the effectiveness of the water treatment plants for removing hazardous compounds and to protect the environment. The introduction of toxicity limits and limits for the total bioaccumulation potential should be considered to supplement generic parameters such as chemical oxygen demand and adsorbable organic halogens. A recommendation is made to include toxicity screening as a technique to consider in the determination of best available techniques (BAT) during the upcoming revision of the BAT reference document for the waste treatment industries to provide a more rational basis in decisions on additional treatment steps.

TRPM4 is a Ca2+-activated non-selective cation channel that belongs to the family of the Transient Receptor Potential (TRP) ion channels. Importantly, TRPM4 is impermeable for Ca2+ and is involved in different Ca2+-dependent cell functions, such as exocytosis, contraction and cell death. Trpm4 is known to be expressed in atrial and ventricular cardiomyocytes. The interest in the functional role of TRPM4 in the heart has risen further by the discovery of Trpm4 mutations that are linked to cardiac conduction disorders, including Progressive Familial Heart Block type I (PFHBI) and Brugada Syndrome. Both gain-of-function and loss-of-function mutation were described in patients with cardiac conduction diseases. Recently, our group showed that TRPM4 plays a role during the late repolarization phase of the action potential in murine ventricular cardiomyocytes and that deletion of the Trpm4 gene leads to shorter ventricular action potentials. To characterize if deletion of Trpm4 has an effect on the conduction properties of the heart, an in depth electrophysiological study was performed in living mice. An octapolar catheter was inserted into the right atrium and ventricle of the heart to measure intracardial electrograms. The atrial-His (AH) and His-ventricular (HV) intervals were calculated and no differences were found between WT and Trpm4-deficient mice. Additionally, more detailed conduction parameters of the heart were determined by use of programmed electrical stimulation (PES) protocols. Sinus node recovery time (SNRT) was not different between WT and Trpm4-deficient mice. Effective refractory period of atrium (AERP), AV node (AVNERP) and ventricle (VERP) were in the same range in WT and Trpm4-deficient mice. Wenckebach periodicity, the parameter for AV nodal conduction, was also not different between WT and Trpm4-deficient mice. These results suggest that deletion of Trpm4 has no effects on the conduction properties of the murine heart.

Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin–NFAT pathway and the development of pathological hypertrophy. Electronic supplementary material The online version of this article (doi:10.1007/s00395-015-0501-x) contains supplementary material, which is available to authorized users.

In the recent years hair has been increasingly used as alternative matrix in human biomonitoring (HBM) of environmental pollutants. Sampling advantages and time integration of exposure assessment seems the most attractive features of hair matrix. In the current study, a novel miniaturized method was developed and validated for measuring 15 perfluoroalkyl substances (PFAS), including perfluoro n-butanoic acid (PFBA), perfluoro n-pentanoic acid (PFPeA), perfluoro n-hexanoic acid (PFHxA), perfluoro n-heptanoic acid (PFHpA), perfluor n-octanoic acid (PFOA), perfluoro n-nonanoic acid (PFNA), perfluoro tetradecanoic acid (PFreDA), perfluorobutane sulfonic acid (PFBS), perfluoro pentane sulfonic acid (PFPeS), perfluorohexane sulfonic acid (PFHxS), perfluoroheptane sulfonic acid (PFHpS), perfluorooctane sulfonic acid (PFOS), perfluorononane sulfonic acid (PFNS), perfluorodecane sulfonic acid (PFDS) and perfluorododecane sulfonic acid (PFDoS) in human hair by liquid chromatography tandem mass spectrometry (LC-MS/MS). After extraction using ethyl acetate, dispersive ENVI-Carb was used for clean-up. Good intra- and inter-day precision for low (LQ 5 ng/g hair) and high spike (HQ 15 ng/g) levels were achieved (in general RSD < 10%). The accuracy was assessed using recoveries (%), which ranged between 68-118% (LQ) and 70-121% (HQ). The instrumental limit of detection (LODi) and limit of quantification (LOQ(i)) were between 1-4 pg/g hair and 3-13 pg/g hair, respectively. The method limit of quantification (LOQ(m)) ranged between 6 and 301 pg/g hair. The PFAS levels were measured in 30 human hair samples indicating that the levels are low (14-1534 pg/g hair). Some PFAS were not present in any hair sample (e.g. PFHpA, PFTeDA, PFNA, PFPeS, PFHpS, PFOS and PFNS), while other PFAS were frequently detected (PFBA, PFPeA, PFHxA, PFOA, PFBS, PFHxS, PFOS, PFDS and PFDoS) in human hair. Although levels in general were low, there is evidence of higher human exposure to some analytes, such as PFBA, PFPeA, PFHxA, PFOA, PFBS, PFHxS, and PFDoS. The current study shows that hair is a suitable alternative non-invasive matrix for exposure assessment of PFAS. (C) 2015 Elsevier B.V. All rights reserved.

TRPM4 is a Ca(2+)-activated nonselective cation channel. The channel is activated by an increase of intracellular Ca(2+) and is regulated by several factors including temperature and Pi(4,5)P2. TRPM4 allows Na(+) entry into the cell upon activation, but is completely impermeable to Ca(2+). Unlike TRPM5, its closest relative in the transient receptor potential family, TRPM4 proteins are widely expressed in the body. Currents with properties that are reminiscent of TRPM4 have been described in a variety of tissues since the advent of the patch clamp technology, but their physiological role is only beginning to be clarified with the increasing characterization of knockout mouse models for TRPM4. Furthermore, mutations in the TRPM4 gene have been associated with cardiac conduction disorders in human patients. This review aims to overview the currently available data on the functional properties of TRPM4 and the current understanding of its physiological role in healthy and diseased tissue.

RATIONALE: The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases and Brugada syndrome. Transient receptor potential member 4 (TRPM4) proteins con...

Rationale : The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases and Brugada syndrome. Transient receptor potential member 4 (TRPM4) proteins constitute Ca 2+ -activated, but Ca 2+ -impermeable, nonselective cation channels and are expressed both in atrial and in ventricular cardiomyocytes. The physiological function of TRPM4 in the heart remains, however, incompletely understood. Objective : To establish the role of TRPM4 in cardiac muscle function. Methods and Results : We used TRPM4 knockout mice and performed patch-clamp experiments, membrane potential measurements, microfluorometry, contractility measurements, and in vivo pressure–volume loop analysis. We demonstrate that TRPM4 proteins are functionally present in mouse ventricular myocytes and are activated on Ca 2+ -induced Ca 2+ release. In Trpm4 −/− mice, cardiac muscle displays an increased β-adrenergic inotropic response both in vitro and in vivo. Measurements of action potential duration show a significantly decreased time for 50% and 90% repolarization in Trpm4 −/− ventricular myocytes. We provide evidence that this change in action potential shape leads to an increased driving force for the L-type Ca 2+ current during the action potential, which explains the altered contractility of the heart muscle. Conclusions : Our results show that functional TRPM4 proteins are novel determinants of the inotropic effect of β-adrenergic stimulation on the ventricular heart muscle.

We recently proposed that the transient receptor potential melastatin 5 (TRPM5) cation channel contributes to glucose-induced electrical activity of the β cell and positively influences glucose-induced insulin release and glucose homeostasis. In this study, we investigated Trpm5 expression and function in pancreatic islets from mouse models of type II diabetes. Gene expression analysis revealed a strong reduction of Trpm5 mRNA levels in pancreatic islets of db/db and ob/ob mice. The glucose-induced Ca(2+) oscillation pattern in db/db and ob/ob islets mimicked those of Trpm5 (-/-) islets. Leptin treatment of ob/ob mice not only reversed the diabetic phenotype seen in these mice but also upregulated Trpm5 expression. Leptin treatment had no additional effect on Trpm5 expression levels when plasma insulin levels were comparable to those of the vehicle-injected control group. In murine β cell line, MIN6, insulin downregulated TRPM5 expression in a dose-dependent manner, unlike glucose or leptin. In conclusion, our data show that increased plasma insulin levels downregulate TRPM5 expression in pancreatic islets from leptin-deficient mouse models of type 2 diabetes.

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard'' of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

Background: DNA methylation changes are potential pathways of environmentally induced health effects. We investigated whether exposure to ambient concentrations of NO2, PM10, PM2.5 and O3 and traffic parameters were associated with global DNA methylation in blood of healthy adults. Methods: 48 non-smoking adults (25 males) with a median age of 39 years were sampled in winter and summer. Global DNA methylation in whole blood (% 5-methyl-2′-deoxycytidine, %5mdC) was analyzed with HPLC. Exposure to air pollutants at the home address was assessed using interpolated NO2, PM10, PM2.5 and O3 concentrations for various exposure windows (60- to 1-day moving average exposures and yearly averages) and GIS-based traffic parameters. Associations between pollutants and %5mdC were tested with multiple mixed effects regression models. Results: Average %5mdC (SD) was 4.30 (0.08) in winter and 4.29 (0.08) in summer. Men had higher %5mdC compared to women both in winter (4.32 vs. 4.26) and summer (4.31 vs. 4.27). When winter and summer data were analyzed together, various NO2, PM10 and PM2.5 moving average exposures were associated with changes in %5mdC (95% CI) ranging from −0.04 (−0.09 to 0.00) to −0.14 (−0.28 to 0.00) per IQR increase in pollutant. NO2, PM10, PM2.5 and O3 moving average exposures were associated with decreased %5mdC (95% CI) varying between −0.01 (−0.03 to 0.00) and −0.17 (−0.27 to −0.06) per IQR increase in pollutant in summer but not in winter. Conclusion: Decreased global DNA methylation in whole blood was associated with exposure to NO2, PM10, PM2.5 and O3 at the home addresses of non- adults. Most effects were observed for the 5- to 30-day moving average exposures. Keywords: Air pollution, PM, Ozone, DNA methylation, Epigenetics, Human biomonitoring

The alkaline comet assay was used to investigate DNA damage levels in white blood cells of 45 normal healthy subjects. Therefore blood was sampled at four different periods, namely in February, June, August and November of the same year. Higher DNA damage levels were found in summertime, as well as higher levels of 1-hydroxypyrene in urine in this period. This suggests a higher exposure to polycyclic hydrocarbons in the summer compared with other periods of the year. The observed seasonal variation in DNA damage levels is in agreement with some, but in contradiction with other data. Seasonal variations in DNA damage levels can easily be explained by the existence of different confounders that may influence the results of a biomonitoring study. Besides sunlight and environmental pollution, also diet, allergy and physical exercise, for example, were already identified as important influencing factors. The investigation confirms that the blood sampling period is crucial in the planning and interpretation of biomonitoring studies.

The CALUX bioassay is at present the best screening method for dioxins and dioxin-like (dl) polychlorinated biphenyls (PCBs) in food and feed, and the only assay used in routine monitoring and during larger incidents. Furthermore, the use of bioassays in addition to chemical reference methods allows the discovery of novel contaminants with potentially adverse effects on human health. The CALUX bioassay shows a clear dose-related response both with dioxin standards and contaminated samples, but requires a clear strategy with respect to decision limits, necessary to deal with the many different action and tolerance limits in European Union (EU) legislation. In future, the CALUX bioassay will profit from further optimization, especially with respect to the clean-up procedure. As demonstrated in the limited number of interlaboratory studies performed so far, this should lead to an even more robust assay that can be easily introduced into less experienced laboratories. The present article discusses the different issues, based on some practical examples from the EU-DIFFERENCE project and gives recommendations for future studies.

Food-derived peptides can impact blood pressure through several mechanisms. However, their fate in. the gastrointestinal tract and bioavailability are difficult to assess because of their fast degradation and challenging analysis in physiologically relevant matrices. The aim of this study was to construct an in vitro bioavailability methodology in which luminal digestion is combined with Caco-2 cell transport. Egg ovalbumin hydrolysate, both in pure form and mixed with a food matrix, was used as a test case. Results indicate that a food matrix protected bioactive peptides from luminal digestion, especially in small intestine. Moreover, the Caco-2 absorption peak was extended over a longer time period (> 60 min) compared to the pure peptide solutions (similar to 15 min) which in total resulted in a 3-12 times higher absorption of the bioactive sequences after 60 min compared to fasted conditions. These results suggest further investigation is warranted towards peptide-based functional foods with improved gastro-intestinal stability and longer-term release in the blood.