Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.     

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.     

Response of soil microbial biomass and activity in early restored lands in the northeastern Brazilian Atlantic Forest

Intensive land use of the Brazilian Atlantic Forest accelerated with the rise of sugar cane plantations in the northeastern part of Brazil. Consequently, many ecosystems were destroyed, including riparian forests. The number of studies of riparian restoration has increased but comparative studies on the belowground effects of common reforestation strategies are rare. Here, we compared soil microbial properties among four different land use types: native rainforest, sugar cane plantation, single species reforestation, and mixed species reforestation, each replicated at two spatially independent sites. Soil samples were taken in 2013 and 2014, that is 2 and 3 years after reforestation, respectively. In both years, land use types had a significant effect on basal respiration, microbial biomass, and specific respiration (whereas specific respiration was marginally affected in 2014). In 2013, basal respiration in sugar cane plantations was significantly lower (?65%) when compared to native forests. In 2014, basal respiration (+60%) and soil microbial biomass (+90%) were significantly higher in mixed species reforestation compared to sugar cane, whereas single species reforestation had comparable values as in sugar cane plantations. Our results indicate that soil microbial biomass and activity respond rapidly to land use change when mixed species reforestation is used. Thus, using mixed species reforestation may enhance the provisioning of ecosystem services already in the short term.     

A new role for the P2Y-like GPR17 receptor in the modulation of multipotency of oligodendrocyte precursor cells in vitro

Oligodendrocyte precursor cells (OPCs, also called NG2 cells) are scattered throughout brain parenchyma, where they function as a reservoir to replace lost or damaged oligodendrocytes, the myelin-forming cells. The hypothesis that, under some circumstances, OPCs can actually behave as multipotent cells, thus generating astrocytes and neurons as well, has arisen from some in vitro and in vivo evidence, but the molecular pathways controlling this alternative fate of OPCs are not fully understood. Their identification would open new opportunities for neuronal replace strategies, by fostering the intrinsic ability of the brain to regenerate. Here, we show that the anti-epileptic epigenetic modulator valproic acid (VPA) can promote the generation of new neurons from NG2⁺ OPCs under neurogenic protocols in vitro, through their initial de-differentiation to a stem cell-like phenotype that then evolves to “hybrid” cell population, showing OPC morphology but expressing the neuronal marker βIII-tubulin and the GPR17 receptor, a key determinant in driving OPC transition towards myelinating oligodendrocytes. Under these conditions, the pharmacological blockade of the P2Y-like receptor GPR17 by cangrelor, a drug recently approved for human use, partially mimics the effects mediated by VPA thus accelerating cells’ neurogenic conversion. These data show a co-localization between neuronal markers and GPR17 in vitro, and suggest that, besides its involvement in oligodendrogenesis, GPR17 can drive the fate of neural precursor cells by instructing precursors towards the neuronal lineage. Being a membrane receptor, GPR17 represents an ideal “druggable” target to be exploited for innovative regenerative approaches to acute and chronic brain diseases.     

The effects of metal ions on the structure and stability of the DNA gyrase B protein

The effects of mono- and divalent metal ions on the DNA gyrase B subunit, on its 43 kDa and 47 kDa domains, and on two mutants in the Toprim domain (D498A and D500C) were investigated by means of circular dichroism and protein melting experiments. Both types of metal ion, with the notable exception of Mn2+, did not affect the conformational properties of the enzyme subunit at room temperature, but were able to produce selective and differential effects on protein stability. In particular, monovalent (K+) ions increased the stability of the gyrase B structure, whereas destabilising effects were most prominent using Mn2+ as the metal ion. Ca2+ and Mg2+ produced comparable changes in the gyrase B melting profile. Additionally, we found that monovalent (K+) ions were more effective in the 43 kDa N-terminal domain where ATP binding occurs, whereas divalent ions caused large modifications in the conformational stability of the 47 kDa C-terminal domain. Our results on gyrase B mutants indicate that D498 interacts with Mn2+, whereas it has little effect on the binding of the other ions tested. A D500C mutation, in contrast, effectively impairs Mg2+ affinity, suggesting effective contacts between this ion and D500 in the wild-type enzyme. Hence, the sites of metal ion complexation within the Toprim domain are modulated by the nature of the ion species. These results suggest a double role played by metal ions in the catalytic steps involving DNA gyrase B. One has to do with direct involvement of cations complexed to the Toprim domain in the DNA cutting-rejoining process, the other, until now overlooked, is connected to the dramatic changes in protein flexibility produced by ion binding, which reduces the energy required for the huge conformational changes essential for the catalytic cycle to occur.     

Development and Validation of a High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for the Simultaneous Determination of Irinotecan and Its Main Metabolites in Human Plasma and Its Application in a Clinical Pharmacokinetic Study

Irinotecan is currently used in several cancer regimens mainly in colorectal cancer (CRC). This drug has a narrow therapeutic range and treatment can lead to side effects, mainly neutropenia and diarrhea, frequently requiring discontinuing or lowering the drug dose. A wide inter-individual variability in irinotecan pharmacokinetic parameters and pharmacodynamics has been reported and associated to patients’ genetic background. In particular, a polymorphism in the UGT1A1 gene (UGT1A1*28) has been linked to an impaired detoxification of SN-38 (irinotecan active metabolite) to SN-38 glucuronide (SN-38G) leading to increased toxicities. Therefore, therapeutic drug monitoring of irinotecan, SN-38 and SN-38G is recommended to personalize therapy. In order to quantify simultaneously irinotecan and its main metabolites in patients’ plasma, we developed and validated a new, sensitive and specific HPLC–MS/MS method applicable to all irinotecan dosages used in clinic. This method required a small plasma volume, addition of camptothecin as internal standard and simple protein precipitation. Chromatographic separation was done on a Gemini C18 column (3 μM, 100 mm x 2.0 mm) using 0.1% acetic acid/bidistilled water and 0.1% acetic acid/acetonitrile as mobile phases. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring. The standard curves were linear (R² ≥0.9962) over the concentration ranges (10–10000 ng/mL for irinotecan, 1–500 ng/mL for SN-38 and SN-38G and 1–5000 ng/mL for APC) and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy, determined on three quality control levels for all the analytes, were always <12.3% and between 89.4% and 113.0%, respectively. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method was successfully applied to a pharmacokinetic study in metastatic CRC patients enrolled in a genotype-guided phase Ib study of irinotecan administered in combination with 5-fluorouracil/leucovorin and bevacizumab.     

Substrate-dependent modulation of the enzymatic catalytic activity: Reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.     

Data distribution in public veterinary service: health and safety challenges push for context-aware systems

Background

Today’s globalised and interconnected world is characterized by intertwined and quickly evolving relationships between animals, humans and their environment and by an escalating number of accessible data for public health. The public veterinary services must exploit new modeling and decision strategies to face these changes. The organization and control of data flows have become crucial to effectively evaluate the evolution and safety concerns of a given situation in the territory. This paper discusses what is needed to develop modern strategies to optimize data distribution to the stakeholders.

Main text

If traditionally the system manager and knowledge engineer have been concerned with the increase of speed of data flow and the improvement of data quality, nowadays they need to worry about data overflow as well. To avoid this risk an information system should be capable of selecting the data which need to be shown to the human operator. In this perspective, two aspects need to be distinguished: data classification vs data distribution. Data classification is the problem of organizing data depending on what they refer to and on the way they are obtained; data distribution is the problem of selecting which data is accessible to which stakeholder. Data classification can be established and implemented via ontological analysis and formal logic but we claim that a context-based selection of data should be integrated in the data distribution application. Data distribution should provide these new features: (a) the organization of situation types distinguishing at least ordinary vs extraordinary scenarios (contextualization of scenarios); (b) the possibility to focus on the data that are really important in a given scenario (data contextualization by scenarios); and (c) the classification of which data is relevant to which stakeholder (data contextualization by users).

Short conclusion

Public veterinary services, to efficaciously and efficiently manage the information needed for today’s health and safety challenges, should contextualize and filter the continuous and growing flow of data by setting suitable frameworks to classify data, users’ roles and possible situations.

     

Development and validation of a LC–MS/MS method for the determination of the novel oral 1,14 substituted taxane derivatives,IDN 5738 and IDN 5839, in mouse plasma and its application to the pharmacokinetic study

Two LC-ESI–MS and CID-MS/MS methods were developed and validated for pharmacokinetic studies of the novel oral taxane derivatives IDN 5738 and IDN 5839, used for preclinical evaluation in mice. The analysis requires 100 μL of plasma sample, involves the addition of an internal standard and protein precipitation with 0.1% HCOOH in acetonitrile. The HPLC separation was obtained on Sunfire C18 column and Selected Reaction Monitoring technique was used to quantify the taxanes. The recoveries were more than 90%; the methods were linear over the validated concentrations range of 25–1500 ng/mL for IDN 5738 and 25–5000 ng/mL for IDN 5839 and had a limit of detection of 0.14 and 0.25 ng/mL, respectively. The inter-day coefficient of variation (CV%) of the calibration standards ranged between 1.3 and 7.2% for IDN 5738 and between 0.0 and 9.0% for IDN 5839 and the mean accuracy was in the range 85.3–112.0% for IDN 5738 and between 80.0 and 111.0% for IDN 5839. Moreover, analysing quality control plasma samples on three different days, the methods resulted precise and accurate showing intra- and inter-day CV within 12% for both analytes, and accuracy of 92.0–113.3% and 85.9–105.7% for IDN 5738 and IDN 5839, respectively. With these methods, we studied for the first time, the pharmacokinetics of the two taxanes showing for both, good oral bioavailability (>50%).     

Cyclopeptide alkaloids from Scutia buxifolia Reiss

Scutianene E (1), 3,4,28-tris-epi-scutiaene E (2), 28-epi-scutianene E (3) and scutianene L (4), four neutral cyclopeptide alkaloids, were isolated from Scutia buxifolia Reiss, together with four known cyclopeptide alkaloids, scutianines B, C, D and E. Scutianenes 1-3 are diastereoisomeric compounds, with 3-hydroxyleucine as a β-hydroxy amino acid unit, which is connected to the styryl fragment via an ether bridge, β-phenylserine, as a common ring-bonded amino acid residue. Attached to the amino group of β-hydroxyamino acid is a side chain [trans-CH = CH-Ph]. The structures of the peptides were elucidated by means of spectroscopic analysis, including extensive 2D NMR studies. The stereochemistry for the diastereomeric 3,4,28-tris-epi-scutiaene E and 28-epi-scutianene E was confirmed by X-ray diffraction analysis of their O-acetyl derivatives.