Liquid chromatography-tandem mass spectrometry application, for the determination of extracellular hepatotoxins in Irish lake and drinking waters

A novel method for the determination of hepatotoxins; microcystins (MCs), and nodularin (Nod) in lake water and domestic chlorinated tap water has been developed using liquid chromatography hyphenated with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). Optimization of the mass spectrometer parameters and mobile-phase composition was performed to maximize the sensitivity and reproducibility of the method. Detection of the hepatotoxins was carried out using multiple reaction monitoring experiments, thus improving the selectivity of the method. A total ion chromatogram and a precursor ion scan on ion m/z 135 was also applied to all samples to detect unknown microcystins or microcystins for which there are no standards available. A comprehensive validation of the LC-ESI-MS/MS method was completed that took into account matrix effects, specificity, linearity, accuracy, and precision. Good linear calibrations were obtained for MC-LR (1-200 microg/L; R2=0.9994) in spiked lake and tap water samples (1-50 microg/L; R2=0.9974). Acceptable interday repeatability was achieved for MC-LR in lake water with RSD values (n=9) ranging from 9.9 (10 microg/L) to 5.1% (100 microg/L). Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked MCs and Nod samples; LOD=0.27 microg/L and LOQ=0.90 microg/L for MC-LR in the "normal linear range" and LOD=0.08 microg/L and LOQ=0.25 microg/L in the "low linear range" in both lake and chlorinated tap water. Similar results were obtained for a suite of microcystins and nodularin. This sensitive and rapid method does not require any sample preconcentration, including the elimination of solid-phase extraction (SPE) for the effective screening of hepatotoxins in water below the 1 microg/L WHO provisional guideline limit for MC-LR. Furthermore, SPE techniques are time-consuming, nonreproducible at trace levels, and offer poor recoveries with chlorinated water. The application of this LC-ESI-MS/MS method for routine screening of hepatotoxins in lake and chlorinated tap water (average Cl2=0.23 mg/L) is achieved and this study represents the first direct method for the screening of hepatotoxins in chlorinated tap water.     

Phospholipid Distribution and Phospholipid Fatty Acids of the Tropical Tunicates <Emphasis Type="Italic">Eudistoma</Emphasis> sp. and <Emphasis Type="Italic">Leptoclinides uniorbis</Emphasis>

Two tunicates, Eudistoma sp. and Leptoclinides uniorbis, collected from the tropical waters off Djibouti were investigated for lipids and phospholipid (PL) fatty acids. PL accounted for 38.2% of the total lipids in Eudistoma sp. and for 30.2% in L. uniorbis. PL classes were quantified by normal-phase high-performance liquid chromatography using an evaporative light-scattering detector and revealed essential differences. Eudistoma sp. contained mainly phosphatidylcholine (PC, 70.3% of total PL) and lysophosphatidylcholine (LPC, 11.9%) and was devoid of phosphatidylserine (PS), whereas the major PL of L. uniorbis was PS (59.1%) followed by PC (22.5%) and LPC (8.8%). Gas chromatography–mass spectrometry analyses of fatty acid (FA) derivatives revealed 38 FA in Eudistoma sp., and 35 FA in L. uniorbis, ranged from C₁₂ to C₂₄ chain lengths. Polyunsaturated FA accounted for 25.9% in Eudistoma sp. and for 32.3% in L. uniorbis. Interestingly, L. uniorbis contained a high percentage (16.7%) of the 20:5n-3 acid (8.9% in Eudistoma sp.) and the 18:4n-3 acid (4.1%). Significant levels of the 20:4n-6 acid were observed in both organisms (7.8 and 6.0% respectively). Eudistoma sp. contained the rare 20:3n-7 acid (2.3%) only recorded to date in hydrothermal vent animals. The cyclopropane dihydrosterculic acid was identified in both tunicates (0.7 and 0.5% respectively). These latter FA, together with some unusual branched saturated and monounsaturated FA, revealed the occurrence of associated bacteria in the tunicates. Another noticeable feature was a series of eight C₁₆ to C₁₈ aldehyde dimethylacetals revealing the presence of plasmalogens at 5.0% in Eudistoma sp. and 14.2% in L. uniorbis. The results of this study were compared with those previously published for other tunicates regarding mainly PL content and FA composition.     

Understanding the Underlying Molecular Mechanisms of Meiotic Arrest during In Vitro Spermatogenesis in Rat Prepubertal Testicular Tissue

In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.     

Inactivation of Osteoblast PKC Signaling Reduces Cortical Bone Mass and Density and Aggravates Renal Osteodystrophy in Mice with Chronic Kidney Disease on High Phosphate Diet

Chronic kidney disease (CKD) frequently leads to hyperphosphatemia and hyperparathyroidism, mineral bone disorder (CKD-MBD), ectopic calcifications and cardiovascular mortality. PTH activates the osteoanabolic Gα_s/PKA and the Gα_q/11/PKC pathways in osteoblasts, the specific impact of the latter in CKD-MBD is unknown. We generated osteoblast specific Gα_q/11 knockout (KO) mice and established CKD-MBD by subtotal nephrectomy and dietary phosphate load. Bone morphology was assessed by micro-CT, osteoblast function by bone planar scintigraphy at week 10 and 22 and by histomorphometry. Osteoblasts isolated from Gα_q/11 KO mice increased cAMP but not IP3 in response to PTH 1-34, demonstrating the specific KO of the PKC signaling pathway. Osteoblast specific Gα_q/11 KO mice exhibited increased serum calcium and reduced bone cortical thickness and mineral density at 24 weeks. CKD Gα_q/11 KO mice had similar bone morphology compared to WT, while CKD Gα_q/11-KO on high phosphate diet developed decreased metaphyseal and diaphyseal cortical thickness and area, as well as a reduction in trabecular number. Gα_q/11-KO increased bone scintigraphic tracer uptake at week 10 and mitigated tracer uptake in CKD mice at week 22. Histological bone parameters indicated similar trends. Gα_q/11-KO in osteoblast modulates calcium homeostasis, bone formation rate, bone morphometry, and bone mineral density. In CKD and high dietary phosphate intake, osteoblast Gα_q/11/PKC KO further aggravates mineral bone disease.     

Milk fat globule membrane glycoproteins: Valuable ingredients for lactic acid bacteria encapsulation?

The membrane (Milk Fat Globule Membrane – MFGM) surrounding the milk fat globule is becoming increasingly studied for its use in food applications due to proven nutritional and technological properties. This review focuses first on current researches which have been led on the MFGM structure and composition and also on laboratory and industrial purification and isolation methods developed in the last few years. The nutritional, health benefits and techno-functional properties of the MFGM are then discussed. Finally, new techno-functional opportunities of MFGM glycoproteins as a possible ingredient for Lactic Acid Bacteria (LAB) encapsulation are detailed. The ability of MFGM to form liposomes entrapping bioactive compounds has been already demonstrated. One drawback is that liposomes are too small to be used for bacteria encapsulation. For the first time, this review points out the numerous advantages to use MFGM glycoproteins as a protecting, encapsulating matrix for bacteria and especially for LAB.     

Antioxidant activity and phenolic content of 16 raisin grape (Vitis vinifera L.) cultivars and selections

Six raisin grape cultivars and 10 new raisin grape selections were analyzed for antioxidant activity (ABTS assay) and for total and individual phenolic compounds. Samples were freeze–dried and values are reported on a dry weight basis. Antioxidant activity across the 16 samples ranged from 7.7 to 60.9 μmol Trolox/g DW, with A95-27 exhibiting the greatest activity. Total phenolic content, determined in gallic acid equivalents using the Folin–Ciocalteau assay, ranged from 316.3 to 1141.3 mg gallic acid/100 g DW and was strongly correlated (r = 0.990) with antioxidant results. Concentrations of individual phenolics were determined by HPLC. trans-Caftaric acid was the predominant compound in all samples. A95-15 contained the lowest concentration (153.5 μg/g DW) of caftaric acid, while Fiesta contained the highest concentration (598.7 μg/g DW). Selections A56-66, A95-15, and A95-27 had much higher levels of catechin (86.5–209.1 μg/g DW) and epicatechin (126.5–365.7 μg/g DW) than the other samples.     

“I think you just got mixed up”: confident peer tutors hedge to support partners’ face needs

During collaborative learning, computer-supported or otherwise, students balance task-oriented goals with the interpersonal goals of relationship-building. This means that in peer tutoring, some pedagogically beneficial behaviors may be avoided by peer tutors due to their likelihood to get in the way of relationship-building. In this paper, we explore how the interpersonal closeness between students in a peer tutoring dyad and the peer tutors’ instructional self-efficacy impacts those tutors’ delivery style of various tutoring moves, and explore the impact those tutoring move delivery styles have on their partners’ learning. We found that tutors with lower social closeness with their tutees provide more positive feedback to their tutee and use more indirect instructions and comprehension-monitoring, but this is only the case for tutors with greater tutoring self-efficacy. And in fact, those tutees solved more problems and learned more when their tutors hedged instructions and comprehension-monitoring, respectively. We found no effect of hedging for dyads with greater social closeness, on the other hand, suggesting that interpersonal closeness may reduce the face-threat of direct instructions and comprehension-monitoring, and hence reduce the need for indirectness, while tutors’ instructional self-efficacy allows tutors to use those moves without feeling threatened themselves. These results emphasize that designers of CSCL systems should understand the nature of how the interpersonal closeness between collaborating students intersects with those students’ self-efficacy to impact the use and delivery of their learning behaviors, in order to best support them in collaborating effectively.     

Oleuropein Prevents Neuronal Death,Mitigates Mitochondrial Superoxide Production and Modulates Autophagy in a Dopaminergic Cellular Model

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, primarily affecting dopaminergic neurons in the substantia nigra. There is currently no cure for PD and present medications aim to alleviate clinical symptoms, thus prevention remains the ideal strategy to reduce the prevalence of this disease. The goal of this study was to investigate whether oleuropein (OLE), the major phenolic compound in olive derivatives, may prevent neuronal degeneration in a cellular dopaminergic model of PD, differentiated PC12 cells exposed to the potent parkinsonian toxin 6-hydroxydopamine (6-OHDA). We also investigated OLE’s ability to mitigate mitochondrial oxidative stress and modulate the autophagic flux. Our results obtained by measuring cytotoxicity and apoptotic events demonstrate that OLE significantly decreases neuronal death. OLE could also reduce mitochondrial production of reactive oxygen species resulting from blocking superoxide dismutase activity. Moreover, quantification of autophagic and acidic vesicles in the cytoplasm alongside expression of specific autophagic markers uncovered a regulatory role for OLE against autophagic flux impairment induced by bafilomycin A1. Altogether, our results define OLE as a neuroprotective, anti-oxidative and autophagy-regulating molecule, in a neuronal dopaminergic cellular model.     

Citywide multi-grid urban flood modelling: the July 2012 flood in Beijing

Global flood management is a major issue for most cities which have to deal with worsening factors such as climate change and fast urban growth. Computer models have been used to model and understand urban flooding on a local scale in cities (25–50 km²). It has been practically impossible to model bigger cities in one go in sufficiently high resolution due to the heavy computations involved.

The present paper describes a new modelling approach for urban flooding which allows modelling on large city scale (1000 km²) while keeping sufficient resolution, e.g. 5 m or 10 m grid. The multicell approach is applied for the city of Beijing for July 21st, 2012 flood event. Model results are compared to testimonials from the 2012 event. Comparison to traditional 2D urban flood computations shows that the multicell approach is much faster than standard detailed models while keeping a suitable level of precision.