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The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum cha- perone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

Per- and poly-fluoroalkyl substances (PFASs) are persistent and bioaccumulative compounds with adverse impacts on the environment and human health. Diet is one of the main sources of exposure to PFASs. Recently, the EFSA established a tolerable weekly intake (TWI) limit (4.4 ng/kg b.w.) for a mixture of the four major PFASs. Eggs and egg products can contribute to this intake, with their contamination possibly dependent on the husbandry system. Monitoring Italian eggs from backyard chickens revealed a relatively uniform PFAS contamination, with perfluoro-1-octanesulfonate being the most abundant. Contamination was detected to be significantly higher in eggs from backyard chickens than in eggs from commercial laying hens, consistent with a previous Italian study. According to the recently set TWI value, the consumption of eggs from backyard chickens could contribute significantly to dietary intake of PFASs (up to 29% of the TWI in children, considering the lower bound approach).

Perfluoroalkyl substances (PFASs) are a family of persistent pollutants of anthropic origin which can reach humans mainly through diet, causing potentially dangerous effects on health. Fish and fishery products are a major source of exposure, but intra- and inter-specific contamination can be extremely variable. In the present study a single species monitoring of the presence of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the two main compounds of the family, was performed on 140 farmed and wild caught European sea basses (Dicentrarchus labrax) from different places in the Mediterranean area. The results highlight a strong correlation between the level of contamination and the origin of fishes, if wild or farmed: on average, wild caught sea basses (PFOS: 112-12,405 ng/kg, median 1345 ng/kg; PFOA: 9–487 ng/kg, median 28 ng/kg) showed higher levels than intensively farmed sea basses (PFOS: 11–105 ng/kg, median 32 ng/kg; PFOA: 9–51 ng/kg, median 21 ng/kg). Significant differences among the various rearing systems were also observed, with extensively-farmed subjects presenting relatively higher levels of both compounds compared to intensively farmed. Moreover, a certain variability among wild fish caught from different sampling sites was observed, confirming that PFASs contamination, which reached in some cases noticeable concentrations, might be influenced by the geographical origin.

Graphical abstract, Although budesonide is frequently used in veterinary medicine for the treatment of canine respiratory and bowel inflammatory diseases, knowledge is lacking regarding its kinetics in this species. We developed and validated a liquid chromatography–tandem mass spectrometry method for the determination of budesonide and its metabolite 16α-hydroxyprednisolone in dog plasma. The analytes were extracted by solid phase extraction and analysis was performed by high performance liquid chromatography–tandem mass spectrometry, with positive electrospray ionization.•This method allows budesonide and one of its main metabolites to be simultaneously quantified in dog plasma at fairly low concentrations.•The proposed protocol is very easy and fast to execute, without compromising analytical performances.•A small amount (0.5 mL) of plasma is required, making this approach suitable for pharmacokinetic studies also in small sized dogs.

The aim of this study was to determine the presence and the level of contamination of the most important mycotoxins (deoxynivalenol, fumonisin B1 and B2, aflatoxin B1, B2, G1 and G2, ochratoxin A and zearalenone) in 48 samples of extruded dry dog food found in the Italian market (24 samples from standard economy lines, 24 of premium lines). Analyses were performed using ultra-performance liquid chromatography coupled to tandem mass spectrometry. Although the concentrations of the mycotoxins in all samples proved to respect the European legislation with regards to animal feed, the analyses revealed a substantial presence of deoxynivalenol, fumonisins and ochratoxin A, with values above the limit of quantification (5 µg/kg) in 100%, 88% and 81% of the samples, respectively. In contrast, aflatoxins and zearalenone contamination proved to be very modest, with 88% and 75% of the samples, respectively, showing concentrations below the corresponding limit of quantification (5 µg/kg for aflatoxins and 10 µg/kg for zearalenone). Moreover, despite a very heterogeneous contamination, the concentration of fumonisins and ochratoxin A was significantly higher in standard foods than in premium ones (491 vs. 80.2 µg/kg dry matter for fumonisin B1; 113 vs. 38.5 µg/kg dry matter for fumonisin B2; 599 vs. 103 µg/kg dry matter for total fumonisins; 23.8 vs. 13.0 µg/kg dry matter for ochratoxin A; P < 0.001). Furthermore, a simultaneous presence of different mycotoxins (at concentrations higher than their limit of quantification) was observed in most of the pet foods analyzed; in particular, 19% of the samples were contaminated by no fewer than two different types of mycotoxins, 52% by three, 25% by four and 2% by all the mycotoxins evaluated. These results revealed the need for further investigation into the potential risk deriving from chronic exposure to low doses of the different types of mycotoxins that pet species are subject to today.

The phosphoglyceride/cholesterol ratio of membrane lipids can be approximately determined by gas chromatography by direct injection into a short capillary column of the lipid material extracted from tissues according, for instance, to the Folch procedure. However, as a result of the pyrolysis of the injected material, in the course of the separation acidic substances are produced that deactivate the stationary phase. With the aid of a suitable scavenger, such as diethylenetriamine, it is possible to regenerate the column activity.

1. 1. The seasonal variations in the fatty acid composition of the total lipids were studied in four selected tissues of Scapharca inaequivalvis : gills, I; mantle, II; foot together with digestive diverticula, III and haemolymph, IV. 2. 2. Forty-eight main fatty acids were found. Of these 30 were identified through the mass spectra of the corresponding methyl esters and/or by the increase in the gas chromatographic peak with suitable standards. 3. 3. The remaining fatty acids were characterized exclusively by means of the corresponding ECL values. 4. 4. The constituents identified are those that have normally been found in bivalve molluscs and phytoplankton. 5. 5. The qualitative composition of the fatty acid mixtures from I, II and III is very similar and clearly differs from that of IV. 6. 6. Fluctuations in the percentages of the single constituents were observed over the course of the year. 7. 7. A percentage increase in the unsaturated constituents was noted in the cold months. 8. 8. The percentage composition variations in the fatty acids from the lipid extracts of the different tissues and from the whole organism would seem to be linked to the feeding—growth, nonfeeding—maturation cycle of the mollusc.

The chemism of the transmethylation of neutral and/or polar glycerides, carried out using methanolic NaBH 4 in the presence of 2N NaOH added, is discussed. One millilitre of the reagent proposed here enables quantitative transmethylation of 2 mg of neutral lipid and up to 300 mg of polar lipid. The reaction is complete within 20 min at room temperature. This reagent seems particularly useful for the study of the fatty acid composition of high unsaturated lipids or when studying the structure of lipid oxidation products.

1. 1. The anatomical distribution of the sterols and their seasonal variations were studied in four tissues of the mollusc Scapharca inaequivalvis (Bruguiere): gills (I); mantle (II) foot together with digestive diverticula (III) and haemolymph (IV). 2. 2. The qualitative composition of the sterol mixtures extracted monthly from the four tissues was strictly constant. Each mixture contained ten constituents, one of which was not identified, while the others were 3-β-hydroxy sterols, common in marine molluscs and phytoplankton. No Δ 5,7 sterols were found. 3. 3. It was observed that the distribution of the sterols in the tissues considered is notably uniform. This could mean that all the sterols are equally important for the correct functioning of each organ.

1. 1. An investigation was carried out on the bivalve molluscs Venus gallina and Scapharca inaequivalvis (Bruguiere) with the aim of evaluating the seasonal variations of the sterols and of the fatty acids present in the lipid fraction. 2. 2. The samples were collected monthly from the Adriatic Sea, near Cesenatico, at a distance of approx. 500 m from the shore, where the water is approx. 3.5–4 m deep, the average salinity is approx. 31% and the temperature of the water goes from a minimum of roughly 8°C in the winter up to a maximum of approx. 24°C in the summer. 3. 3. The sterols, as well as the fatty acids, extracted from the tissues of V. gallina are practically the same as those isolated from the tissues of S. inaequivalvis and coincide with those found previously in other bivalve molluscs. 4. 4. This confirms the close link between the lipid fraction of the molluscs and their diet, which in this area consists mainly of bentonic diatoms or, alternatively, of plantonic dinoflagellates that have settled on the sea-bed. 5. 5. Some quantitative variations occurring in the composition of the constituents analysed would seem to depend more on internal factors, typical of the two different species of bivalve molluscs, rather than on different diet.

This report discusses the analytical procedure by which it is possible to isolate and identify the oxidation product of cellular and subcellular membrane lipids. The key point of this procedure is the method used for the transmethylation of the lipid material isolated from tissues. In effect, both the conversion of the glycerides into methyl esters and the reduction of the hydroperoxyl groups into the corresponding hydroxyl groups is performed in one step, without breaking any oxirane rings that may be present. The methyl esters containing functional groups introduced by oxidative processes are separated from the non-modified ones by preparative TLC and are identified by GLC and GC-MS.