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In this work, the first phytochemical analysis ever performed on a whole cone of Araucaria bidwillii Hook. is presented. This was carried out by means of column chromatography, NMR spectroscopy and MS spectrometry on the female cone, evidencing the presence of forty metabolites, thirty-three of which are primary whilst the remaining are non-volatile secondary. A chemophenetic evaluation was also performed reporting seven new compounds for the species i.e. β-sitosterol ( 5 ), 15-agathic acid methyl ester ( 6 ), methyl ( E )-communate ( 7 ), shikimic acid ( 8 ), gallic acid ( 9 ), p -hydroxy-benzoic acid ( 10 ) and quinic acid ( 11 ). Throughout this evaluation some important qualitative phytochemical differences among this female cone, its seeds and the few other studied cones of the species comprised in the family, were evidenced. These whole results provide more information on the phytochemistry of this organ and of the species, in general, but also underline the need to perform more studies.

The increasing use of chiral agrochemicals sold as racemic formulations raises concern for the negative impacts that inactive enantiomers can have on aquatic life and human health. The present work just focuses on the determination of ten chiral pesticides in river water samples by applying a ferrofluid-based microextraction followed by their stereoselective liquid chromatography analysis. To develop the ferrofluid, magnetite nanoparticles were prepared and coated with oleic acid and then dispersed in a hydrophobic natural deep eutectic solvent (NaDES), composed of L-menthol and thymol (1:1). The stable colloidal dispersion was characterised by scanning electron microscopy, thermogravimetric analysis, energy-dispersive X-ray spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy. The analyte microextraction from 5 ml river samples was performed using 50 µl of ferrofluid, while acidified acetonitrile (150 µl) was used to break down the ferrofluid and solubilise the NaDES containing the analytes. All the extracts were analysed by high-performance liquid chromatography-tandem mass spectrometry. For each analyte, the baseline separation of isomers was achieved on a Lux i-Amylose-3 column (amylose tris(3-chloro-5-methylphenylcarbamate) working in reversed-phase mode; the combination with mass spectrometry detection allows the overall separation of 24 isomers (ten chiral analytes among which eight containing a single (one) chiral centre, one with two chiral centres and the last one existing in four stereoisomeric forms, due to the presence of two regioisomers with a chiral carbon) within 37 min. The method showed very good figures of merit in terms of recoveries (77.7-97.5%), intra-day and inter-day precision (2.7-7.7% and 6.9-14.9%, respectively), limit of detection (0.01-0.35 µg/L), limit of quantitation (0.03-1.20 µg/L), linear dynamic range, and intra-day and inter-day accuracy (1.2-14.8% and 1.8-15.0%, respectively). The presented method was able to detect 14 out of 24 isomers at the preventive limit established by the Italian legislation for single pesticide (that for a chiral pesticide is the sum of all its isomers) in surface waters, set at 0.1 µg/l. Finally, the method was evaluated using AGREEprep and ComplexGAPI metrics, compared with other ferrofluid-based methods, and applied to the analysis of water samples from two Italian rivers (the Nera River and the Tiber River), providing to be sustainable and reliable for the application to real river matrices., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Cigarette butts, one of the most common forms of litter in the world, represent a source of chemical and plastic pollution releasing thousands of toxic compounds and microfibers of cellulose acetate (CA). Besides the correct waste management, the recovery of CA from cigarette filters is a way to cushion their negative effects on the environment. Thus far, recycling strategies have been limited to industrial applications, while not many solutions have designed for water remediation. This work describes a strategy to valorize this harmful waste and to reverse its environmental impact, proposing a simple and effective procedure of reclamation of CA and its reuse to prepare a composite sorbent for the treatment of polluted water. The first step entails the washing of filters with hot water (T = 90 °C) and hot ethanol (T = 58-68 °C) to remove the impurities produced during cigarette burning, as verified by means of UV and attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectroscopy, thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC). The second step involves the use of the regenerated CA to prepare porous cylinder-shaped cryogels (15 mm × 10 mm) whose sorption properties are enhanced by the combination with AC (15 % w/w). The synthesis takes advantage of the sol-gel transition of the polymer dispersion (5 % w/V) in a solution acetone/water 5 mM in NH 3 (60/40, v/v) . After characterization by dynamic mechanical analysis (DMA), TGA, FT-IR, and scanning electron microscopy (SEM), the adsorption capability of the physical cryogel was studied in terms of treated environmental water volume, contact time and concentration of the selected pollutants. The results have shown that the proposed strategy is a low-cost way to recycle CA from cigarette butts and that the designed sorbent is a promising material for water treatment, allowing quick removal times and yields >79.6 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nanocomposite microbeads (average diameter = 10-100 µm) were prepared by a microemulsion-solidification method and applied to the magnetic solid-phase extraction (m-SPE) of fourteen analytes, among pesticides, drugs, and hormones, from human urine samples. The microbeads, perfectly spherical in shape to maximize the surface contact with the analytes, were composed of magnetic nanoparticles dispersed in a polylactic acid (PLA) solid bulk, decorated with multi-walled carbon nanotubes (mPLA@MWCNTs). In particular, PLA was recovered from filters of smoked electronic cigarettes after an adequate cleaning protocol. A complete morphological characterization of the microbeads was performed via Fourier-transform infrared (FTIR) spectroscopy, UV-Vis spectroscopy, thermogravimetric and differential scanning calorimetry analysis (TGA and DSC), scanning electron microscopy (SEM) and X-ray diffraction analysis (XRD). The recovery study of the m-SPE procedure showed yields ≥ 64%, with the exception of 4-chloro-2-methylphenol (57%) at the lowest spike level (3 µg L -1 ). The method was validated according to the main FDA guidelines for the validation of bioanalytical methods. Using liquid chromatography-tandem mass spectrometry, precision and accuracy were below 11% and 15%, respectively, and detection limits of 0.1-1.8 µg L -1 . Linearity was studied in the range of interest 1-15 µg L -1 with determination coefficients greater than 0.99. In light of the obtained results, the nanocomposite microbeads have proved to be a valid and sustainable alternative to traditional sorbents, offering good analytical standards and being synthetized from recycled plastic material. One of the main objectives of the current work is to provide an innovative and optimized procedure for the recycling of a plastic waste, to obtain a regular and reliable microstructure, whose application is here presented in the field of analytical chemistry. The simplicity and greenness of the method endows the procedure with a versatile applicability in different research and industrial fields., (© 2024. The Author(s).)

The use of efficient solar ovens can be a way for progressing ecofriendly technologies in the field of food preparation. In most performing direct solar ovens, the sun rays reach the foods directly, therefore, it becomes essential to check whether, in these conditions, foods can retain theirnutraceutical properties (antioxidants, vitamins and carotenoids). In the present research work, to investigate this issue, several foods (vegetables, meats, and a fish sample) were analyzed before and after cooking (traditional oven, solar oven, and solar oven with a UV filter). The content of lipophilic vitamins and carotenoids (analyzed via HPLC-MS) and the variation of total phenolic content (TPC), and antioxidant capacity (via Folin-Ciocalteu and DPPH assays) have evidenced that cooking with the direct solar oven allows to preserve some nutrients (i.e., tocopherols) and, sometimes, to enhance nutraceutical properties of vegetables (for example, solar oven-cooked eggplants showed 38% higher TPC compared to electric oven cooked sample) and meats. The specific isomerization of all-trans-β-carotene to 9-cis was also detected. The use of a UV filter is advisable to avoid UV drawbacks (for instance, a significant carotenoid degradation) without losing the beneficial effects of the other radiations.

The protection of the health and safety of workers in the agricultural sector requires the assessment of human exposure to pesticides through biomonitoring programs. In doing this, the health and safety of laboratory analysts should be also protected through the use of analytical procedures as safe as possible. According to Green Analytical Chemistry and Green Sample Preparation principles, the use of miniaturized extraction techniques such as dispersive liquid-liquid microextraction (DLLME) should be encouraged, with the aim of limiting the consumption of chemicals (solvents and reagents) and energy, as well as the production of wastes. Moreover, safer and more environmentally friendly alternatives to petroleum-derived solvents must be found. In this regard, the effort of researchers is focused on the development/identification of sustainable solvents, some of which have been ranked according to their disposal, environmental, safety, and health features. Here, we introduce the alternative use of isoamyl acetate ("banana oil") as sustainable extraction solvent. Compared with the trendy class of eutectic solvents (ESs), isoamyl acetate is advantageous because it is already in the liquid state, which fasters operations, and because it can be evaporated to dryness and reconstituted in a solvent compatible with the mobile chromatographic phase, which is not an option for ESs. The applicability of isoamyl acetate has been here evaluated for the first time to extract 12 pesticides of different polarities from urine matrix, by using a DLLME approach. The HPLC/MS validated method has proven to possess the sensitivity, precision and accuracy required to reliable bioanalytical methods. Its cheapness, simplicity and quickness make it an ideal sustainable choice for the screening of large numbers of samples in health monitoring programs involving people occupationally-exposed to pesticides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

As many as 40% of all plant protection products currently used contain chiral active ingredients. Enantiomers of the same pesticide have identical physicochemical properties in an isotropic medium, but they may display different activity and toxicity because of their interaction with enzymes or other naturally occurring asymmetric molecules. This difference may also lead to variations in biotic degradation rates, making one enantiomer more persistent than the other in natural and agricultural environments. In terms of methodological aspects, this critical review describes the most used chiral stationary phases for HPLC enantioseparations of chiral pesticides, pinpointing their strengths and weaknesses. As far as their applicability is concerned, most research has been carried out by means of columns based on derivatized amylose/cellulose due to their rather universal analyte coverage. The chromatographic compatibility with sensitive detection techniques, such as mass spectrometry, has allowed the trace analysis of stereoisomers, revealing ubiquitous occurrence of some chiral pesticides in surface waters, sediments, plants, agricultural soils, roots, fruit and vegetables. The study of their distribution and degradation in various environmental compartments and agricultural soil-plant systems has highlighted the enrichment with one enantiomer over the other in certain matrices following the enantioselective dissipation catalysed by microorganisms or plant enzymes as well as the phenomenon of chiral inversion in some cases. Irrespective of the reliability of a chiral method, such investigations are often hindered by the lack of pure standards of single enantiomers, which makes it difficult to identify their stereochemical configuration and requires precise strategies of quantification. Surely, the research in this field has been grown over the last few years due to the necessity of assessing and limiting risks related to exposure to chiral pesticides, which can be considered emerging contaminants in all aspects., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: member of the Editorial Advisory Board of JCA, A.G. member of the Editorial Advisory Board of JCA, B.C. Editor of JCA, S.F., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A safe and environmentally friendly material for corrosion removal from metals is proposed in this article. Electrochemically corroded copper was selected as a target material, and a deep eutectic solvent (DES) composed of choline chloride and ascorbic acid, in a molar ratio of 2:1, was developed to this end. Aqueous solutions of the DES with a concentration above 70 wt% were found to be effective in the dissolution of patina and less aggressive towards other materials such as CaCO 3, which is the main component of limestone. These concentrated DES solutions were integrated with either cotton swabs or cellulose-based membranes and used for the cleaning of electrochemically corroded copper. The membrane containing 80 wt% DES aqueous solution exhibited the most desirable cleaning ability in terms of speed and area selectivity. X-ray diffraction analysis of the corroded copper before and after the application of the membrane was performed to demonstrate the successful corrosion removal.

A green solvent-based DLLME/HPLC-MS method for the determination of 19 pesticides in wine samples has been developed. The extractant solvent is a hydrophobic eutectic mixture composed of L-menthol and butylated hydroxytoluene in a molar ratio of 3:1. The endogenous ethanol of wine has been used as dispersive solvent, in order to avoid the solidification of the extracts under 19 °C. The mobile phase composition, the elution gradient and the sample injection volume were optimized in order to make this hydrophobic mixture compatible with conventional reversed phase chromatography and electrospray ionization. The method was validated in matrix, using a wine free from the target compounds. Average recovery as high as 80%, precision between 3 and 14%, and limits of detection and quantification much lower than the maximum residue levels (MRLs) for grapes and wines fixed by the EU regulation, make this multiresidue method fitted for the purpose, with the further advantages of being quick, cheap and in compliance with the green analytical chemistry. From the analysis of 11 commercial wines it was found that just in a bio sample the target compounds were not detectable or lower than quantification limit; as for the other samples, the most widespread and abundant pesticides were methoxyfenozide and boscalid, but their levels were much lower than the relative MRLs.

A novel chromatographic application in chiral separation by using the nano-LC technique is here reported. The chiral recognition of 12 antifungal drugs was obtained through a 75 µm I.D. fused-silica capillary, which was packed with a CSP-cellulose 3,5-dichlorophenylcarbamate (CDCPC), by means of a lab-made slurry packing procedure. The mobile phase composition and the experimental conditions were optimized in order to find the optimum chiral separation for some selected racemic mixtures of imidazole and triazole derivatives. Some important parameters, such as retention faction, enantioresolution, peak efficiency, and peak shape, were investigated as a function of the mobile phase (pH, water content, type and concentration of both the buffer and the organic modifier, and solvent dilution composition). Within one run lasting 25 min, at a flow rate of approximately 400 nL min -1 , eight couples of enantiomers were baseline-resolved and four of them were separated in less than 25 min. The method was then applied to milk samples, which were pretreated using a classical dispersive liquid-liquid microextraction technique preceded by protein precipitation. Finally, the DLLME-nano-LC-UV method was validated in a matrix following the main FDA guidelines for bioanalytical methods.

Glyphosate is the most commonly used herbicide worldwide. Its improper use during recent decades has resulted in glyphosate contamination of soils and waters. Fungal bioremediation is an environmentally friendly, cost effective, and feasible solution to glyphosate contamination in soils. In this study, several saprotrophic fungi isolated from agricultural environments were screened for their ability to tolerate and utilise Roundup in different cultural conditions as a nutritional source. Purpureocillium lilacinum was further screened to evaluate the ability to break down and utilise glyphosate as a P source in a liquid medium. The dose-response effect for Roundup, and the difference in toxicity between pure glyphosate and Roundup were also studied. This study reports the ability of several strains to tolerate 1 mM and 10 mM Roundup and to utilise it as nutritional source. P. lilacinum was reported for the first time for its ability to degrade glyphosate to a considerable extent (80%) and to utilise it as a P source, without showing dose-dependent negative effects on growth. Pure glyphosate was found to be more toxic than Roundup for P. lilacinum . Our results showed that pure glyphosate toxicity can be only partially addressed by the pH decrease determined in the culture medium. In conclusion, our study emphasises the noteworthy potential of P. lilacinum in glyphosate degradation.

The use of psychoactive substances is a serious problem in today's society and reliable methods of analysis are necessary to confirm their occurrence in biological matrices. In this work, a green sample preparation technique prior to HPLC-MS analysis was successfully applied to the extraction of 14 illicit drugs from urine samples. The isolation procedure was a dispersive liquid-liquid microextraction based on the use of a low transition temperature mixture (LTTM), composed of choline chloride and sesamol in a molar ratio 1:3 as the extracting solvent. This mixture was classified as LTTM after a thorough investigation carried out by FTIR and DSC, which recorded a glass transition temperature at -71 °C. The extraction procedure was optimized and validated according to the main Food and Drug Administration (FDA) guidelines for bioanalytical methods, obtaining good figures of merit for all parameters: the estimated lower limit of quantitation (LLOQ) values were between 0.01 µg L -1 (bk-MMBDB) and 0.37 µg L -1 (PMA); recoveries, evaluated at very low spike levels (in the ng-µg L -1 range), spanned from 55% (MBDB) to 100% (bk-MMBDB and MDPV); finally, both within-run and between-run precisions were lower than 20% (LLOQ) and 15% (10xLLOQ).

Solid-phase extraction (SPE) coupled to LC/MS/MS analysis is a valid approach for the determination of organic micropollutants (OMPs) in liquid samples. To remove the greatest number of OMPs from environmental matrices, the development of innovative sorbent materials is crucial. Recently, much attention has been paid to inorganic nanosystems such as graphite-derived materials. Graphene oxide has been employed in water-purification processes, including the removal of several micropollutants such as dyes, flame retardants, or pharmaceutical products. Polysaccharides have also been widely used as convenient media for the dispersion of sorbent materials, thanks to their unique properties such as biodegradability, biocompatibility, nontoxicity, and low cost. In this work, chitosan-graphene oxide (CS_GO) composite membranes containing different amounts of GO were prepared and used as sorbents for the SPE of pesticides. To improve their dimensional stability in aqueous medium, the CS_GO membranes were surface crosslinked with glutaraldehyde. The composite systems were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, swelling degree, contact angle, and mechanical measurements. As the GO content increased, a decrease in surface homogeneity, an improvement of mechanical properties, and a reduction of thermal stability of the CS-based membranes were observed. The increased dimensional stability in water, together with the presence of high GO amounts, made the prepared composite membranes more efficacious than the ones based just on CS in isolating and preconcentrating different hydrophilic/hydrophobic pollutants.

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Contaminated water with hexavalent chromium Cr(VI) is a serious environmental problem. This study aimed to evaluate the Cr(VI) removal by zero valent iron nanoparticles (nZVI) reduction process and the impact of Cr(VI), nZVI and combined treatment with nZVI and Cr(VI) on tomato growth performance. To evaluate the Cr(VI) toxic effect on germination capability, seeds were exposed to increasing Cr(VI) concentrations up to 1000 mg L -1 . The inhibition of seed germination and the decrease of hypocotyl and root length started from Cr(VI) 5 mg L -1 . Under treatment with Cr(VI) + nZVI 5 mg L -1 , seed germination, hypocotyl and root length resulted significantly higher compared to Cr(VI) 5 mg L -1 treatment. The impact of only nZVI was investigated on chlorophyll and carotenoid in leaves; iron levels in leaves, roots, fruits and soil; carotenoid, fat-soluble vitamin and nicotianamine in mature fruits. A significant increase of leaf chlorophyll and carotenoids was observed after nZVI 5 mg L -1 treatment compared to controls. No significant variations were observed in carotenoids, fat-soluble vitamins and nicotianamine levels after treatment with nZVI 5 mg L -1 in mature fruits. For their ability to reduce Cr(VI) and to stimulate tomato growth, nZVI might to be considered as alternative for remediation purposes.

In recent years, great attention has been focused on rapid, selective, and environmentally friendly extraction methods to recover pigments and antioxidants from microalgae. Among these, supercritical fluid extraction (SFE) represents one of the most important alternatives to traditional extraction methods carried out with the use of organic solvents. In this study, the influence of parameters such as pressure, temperature, and the addition of a polar co-solvent in the SFE yields of carotenoids and fat-soluble vitamins from T. obliquus biomass were evaluated. The highest extraction of alpha-tocopherol, gamma-tocopherol, and retinol was achieved at a pressure of 30 MPa and a temperature of 40 °C. It was observed that overall, the extraction yield increased considerably when a preliminary step of sample pre-treatment, based on a matrix solid phase dispersion, was applied using diatomaceous earth as a dispersing agent. The use of ethanol as a co-solvent, under certain conditions of pressure and temperature, resulted in selectively increasing the yields of only some compounds. In particular, a remarkable selectivity was observed if the extraction was carried out in the presence of ethanol at 10 MPa and 40 °C: under these conditions, it was possible to isolate menaquinone-7, a homologous of vitamin K2, which, otherwise, cannot not recovered by using traditional extraction procedures.

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide-insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low-level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co-transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high-throughput genetic transformation of plants and algae., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Analysis of carotenoids is very complex and demanding in terms of both separation and detection. In this article, an analytical strategy relying on high-performance liquid chromatography-photodiode array detection-tandem mass spectrometry (HPLC-PAD-MS/MS) is presented for the large-scale screening of these phytochemicals. Separation was realized by means of Non-Aqueous Reversed Phase (NARP) chromatography on a triacontyl stationary phase kept at subambient temperature, using a mobile phase compatible with atmospheric pressure chemical ionization (APCI). The standards of 14 analytes were used to optimize the method and to predict the chromatographic behaviour of untargeted carotenoids. MS and MS/MS data, obtained during Information Dependent Acquisition (IDA) experiments, were utilized to set up a sensitive HPLC- selected reaction monitoring (SRM) method. Relative abundance between SRM ion currents (ion ratio) allowed the MS distinction of structural isomers (for example, bicyclic, monocyclic and acyclic isomers), while the identification of geometrical isomers was based on Q ratio and fine structure, as calculated from UV-vis spectra. The comparison of LC-PAD- SRM chromatograms, acquired after applying two different extraction procedures (matrix solid-phase dispersion and overnight cold saponification), allowed verifying that sweet peppers are a good source of xanthophylls, prevalently occurring as esterified forms. The overall strategy could identify more than 40 carotenoids in some sweet pepper varieties (Jolly, horn and sweet chili pepper) available on the Italian and European food market., (Copyright © 2018 Elsevier B.V. All rights reserved.)

U-Net is a generic deep-learning solution for frequently occurring quantification tasks such as cell detection and shape measurements in biomedical image data. We present an ImageJ plugin that enables non-machine-learning experts to analyze their data with U-Net on either a local computer or a remote server/cloud service. The plugin comes with pretrained models for single-cell segmentation and allows for U-Net to be adapted to new tasks on the basis of a few annotated samples.

Thyreostats can be used fraudulently to promote a rapid increase in weight of breeding animals at low cost. Their severe toxicological effects impose the development of reliable analytical methods to be used in monitoring plans. This work describes an alternative approach to isolate residues of thiouracil, methyl-thiouracil, propyl-thiouracil, phenyl-thiouracil, tapazole and mercaptobenzimidazole from bovine muscle tissue. The developed procedure is based on the following three steps: i) matrix solid-phase dispersion with C 18 for the preliminary sample preparation; ii) subcritical water extraction (SWE) at 160°C and 100 bar; iii) clean-up on an Oasis HLB cartridge. The quantitative determination was performed by LC-MS/MS in dual polarity ionization by using internal standardization. The SWE-LC-MS/MS method was validated according to the identification criteria of the Commission decision 2002/657/EC. The relative recoveries ranged from 72 to 97%; within-lab reproducibility was less than 18%. The decision limit and the detection capability of all analytes were below the recommended concentration, set at 10 µg kg -1 , but the validation results demonstrated that this method could only be applied for screening of thiouracil and methyl-thiouracil. Besides the analytical advantages related to the use of water as solvent extraction, the procedure allowed significant removal of lipids, whose detrimental effects on instrumentation and MS sensitivity are well-known.

Adulteration of Mozzarella di Bufala Campana with cow milk is a common fraud because of the high price and limited seasonal availability of water buffalo milk. To identify such adulteration, this work proposes a novel approach based on the use of species-specific, low-molecular-weight biomarkers (LMWBs). Liquid chromatography-tandem mass spectrometry screening analyses identified β-carotene, lutein, and β-cryptoxanthin as LMWBs of cow milk, while ergocalciferol was found only in water buffalo milk. Adulterated mozzarellas were prepared in the laboratory and analyzed for the four biomarkers. Combined quantification of β-carotene and ergocalciferol enabled the detection of cow milk with a sensitivity threshold of 5% (w/w). The method was further tested by analyzing a certificated water buffalo mozzarella and several commercial products. This approach is alternative to conventional proteomic and genomic methods and is advantageous for routine operations as a result of its simplicity, speed, and low cost.

In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Amide-linked conjugates of indole-3-acetic acid (IAA) have been identified in most plant species. They function in storage, inactivation or inhibition of the growth regulator auxin. We investigated how the major known endogenous amide-linked IAA conjugates with auxin-like activity act in auxin signaling and what role ILR1-like proteins play in this process in Arabidopsis. We used a genetically encoded auxin sensor to show that IAA-Leu, IAA-Ala and IAA-Phe act through the TIR1-dependent signaling pathway. Furthermore, by using the sensor as a free IAA reporter, we followed conjugate hydrolysis mediated by ILR1, ILL2 and IAR3 in plant cells and correlated the activity of the hydrolases with a modulation of auxin response. The conjugate preferences that we observed are in agreement with available in vitro data for ILR1. Moreover, we identified IAA-Leu as an additional substrate for IAR3 and showed that ILL2 has a more moderate kinetic performance than observed in vitro. Finally, we proved that IAR3, ILL2 and ILR1 reside in the endoplasmic reticulum, indicating that in this compartment the hydrolases regulate the rates of amido-IAA hydrolysis which results in activation of auxin signaling.

Cellulose-based hydrogels, obtained by tuned, low-cost synthetic routes, are proposed as convenient gel electrolyte membranes. Hydrogels have been prepared from different types of cellulose by optimized solubilization and crosslinking steps. The obtained gel membranes have been characterized by infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, and mechanical tests in order to investigate the crosslinking occurrence and modifications of cellulose resulting from the synthetic process, morphology of the hydrogels, their thermal stability, and viscoelastic-extensional properties, respectively. Hydrogels liquid uptake capability and ionic conductivity, derived from absorption of aqueous electrolytic solutions, have been evaluated, to assess the successful applicability of the proposed membranes as gel electrolytes for electrochemical devices. To this purpose, the redox behavior of electroactive species entrapped into the hydrogels has been investigated by cyclic voltammetry tests, revealing very high reversibility and ion diffusivity.

Objectives: To estimate an optimal follow-up (FU) interval for von Hippel-Lindau (VHL) patients with renal masses (RMs) by determining tumour growth rates from growth curves., Methods: Thirty lesions (47.6%) were classified as solid tumours (STs) and 33 (52.4%) as complex cysts (CCs). Variations in lesion volume over time were analyzed. For 53 lesions, we calculated the growth rate during the period when the volume of the lesion changed most rapidly, and called this the fast growth rate (FGR)., Results: The STs initially grew fast, followed by a period of slower growth. The CCs varied in volume over time, associated with variable amounts of their fluid component. The FGR correlated better with the latest volume for STs (r = 0.905) than for CCs (r = 0.780). An optimal FU interval between 3 and 12 months was derived by combining the FGR calculated from the curve with the latest volume measured., Conclusions: Analyzing growth curves and related kinetic parameters for RMs in VHL patients could be useful with a view to optimizing the subsequent FU interval and improving the active surveillance program., Key Points: • Measuring volume changes over time enables tumour growth curves to be charted. • Renal solid tumours increase in volume with a typical sigmoidal curve. • Complex cysts may increase and decrease in volume spontaneously over time. • The fast growth rate of solid tumours correlates with their latest volume. • The fast growth rate can orient the scheduling of subsequent follow-ups.

The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

Time-resolved quantitative analysis of auxin-mediated processes in plant cells is as of yet limited. By applying a synergistic mammalian and plant synthetic biology approach, we have developed a novel ratiometric luminescent biosensor with wide applicability in the study of auxin metabolism, transport, and signalling. The sensitivity and kinetic properties of our genetically encoded biosensor open new perspectives for the analysis of highly complex auxin dynamics in plant growth and development.

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Cellular auxin homeostasis is controlled at many levels that include auxin biosynthesis, auxin metabolism, and auxin transport. In addition to intercellular auxin transport, auxin homeostasis is modulated by auxin flow through the endoplasmic reticulum (ER). PIN5, a member of the auxin efflux facilitators PIN protein family, was the first protein to be characterized as an intracellular auxin transporter. We demonstrated that PIN8, the closest member of the PIN family to PIN5, represents another ER-residing auxin transporter. PIN8 is specifically expressed in the male gametophyte and is located in the ER. By combining genetic, physiological, cellular and biochemical data we demonstrated a role for PIN8 in intracellular auxin homeostasis. Although our investigation shed light on intracellular auxin transport in pollen, the physiological function of PIN8 still remains to be elucidated. Here we discuss our data taking in consideration other recent findings.

The plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxin-dependent transcriptional activity., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Background: Zenker's diverticulum (ZD) may be treated with a variety of endoscopic or open surgical techniques; the choice of treatment depends partly on the size of the diverticulum. The purpose of this study was to correlate ZD measurements obtained preoperatively and during surgery., Methods: From March 2006 to November 2008, 20 consecutive patients (19 males; median age 64.5 (range 37-88) years) with dysphagia secondary to ZD were enrolled for this study. All patients had preoperative barium radiography of the pharynx and esophagus, and diagnostic endoscopy. Ten patients underwent transoral stapling diverticulostomy and ten had open surgery. The depth of the ZD was measured on radiographic views, at endoscopy and during surgery, focusing on the distance from the top of the septum to the bottom of the pouch. The ZD dimensions obtained radiologically and endoscopically were compared with those found during surgery. Correlations and agreements between measurements were assessed using Pearson's correlation coefficients and method-comparison analysis, respectively., Results: The median depth of the ZD was 2.9 cm (mean 2.95 ± 1.12 cm; range 1.5-6 cm), 3.0 cm (mean 3.24 ± 1.27 cm; range 1.7-6.8 cm), and 3.0 cm (mean 2.99 ± 1.01 cm; range 1.5-6 cm) when measured during surgery, radiology, and endoscopy, respectively. The correlation and agreement between the radiographic and surgical ZD measurements were good, whereas those between the endoscopic and surgical measurements were poor., Conclusions: These findings confirm that preoperative barium radiography is mandatory in order to choose the most appropriate surgical treatment for ZD.