Inactivation of Human Salivary Glutathione Transferase P1-1 by Hypothiocyanite: A Post-Translational Control System in Search of a Role

Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.     

Spatially Distributed Effects of Mental Exhaustion on Resting-State FMRI Networks

Brain activity during rest is spatially coherent over functional connectivity networks called resting-state networks. In resting-state functional magnetic resonance imaging, independent component analysis yields spatially distributed network representations reflecting distinct mental processes, such as intrinsic (default) or extrinsic (executive) attention, and sensory inhibition or excitation. These aspects can be related to different treatments or subjective experiences. Among these, exhaustion is a common psychological state induced by prolonged mental performance. Using repeated functional magnetic resonance imaging sessions and spatial independent component analysis, we explored the effect of several hours of sustained cognitive performances on the resting human brain. Resting-state functional magnetic resonance imaging was performed on the same healthy volunteers in two days, with and without, and before, during and after, an intensive psychological treatment (skill training and sustained practice with a flight simulator). After each scan, subjects rated their level of exhaustion and performed an N-back task to evaluate eventual decrease in cognitive performance. Spatial maps of selected resting-state network components were statistically evaluated across time points to detect possible changes induced by the sustained mental performance. The intensive treatment had a significant effect on exhaustion and effort ratings, but no effects on N-back performances. Significant changes in the most exhausted state were observed in the early visual processing and the anterior default mode networks (enhancement) and in the fronto-parietal executive networks (suppression), suggesting that mental exhaustion is associated with a more idling brain state and that internal attention processes are facilitated to the detriment of more extrinsic processes. The described application may inspire future indicators of the level of fatigue in the neural attention system.     

Multi-Scale Analysis of the European Airspace Using Network Community Detection

We show that the European airspace can be represented as a multi-scale traffic network whose nodes are airports, sectors, or navigation points and links are defined and weighted according to the traffic of flights between the nodes. By using a unique database of the air traffic in the European airspace, we investigate the architecture of these networks with a special emphasis on their community structure. We propose that unsupervised network community detection algorithms can be used to monitor the current use of the airspace and improve it by guiding the design of new ones. Specifically, we compare the performance of several community detection algorithms, both with fixed and variable resolution, and also by using a null model which takes into account the spatial distance between nodes, and we discuss their ability to find communities that could be used to define new control units of the airspace.     

mulet (mlt) encodes a tubulin-binding cofactor E-like homolog required for spermatid individualization in Drosophila melanogaster

Spermatogenesis in all animal species occurs within a syncytium. Only at the very end of spermatogenesis are individual sperm cells resolved from this syncytium in a process known as individualization. Individualization in Drosophila begins as a membrane-cytoskeletal complex known as the individualization complex (IC) assembles around the sperm heads and proceeds down the flagella, removing cytoplasm from between the sperm tails and shrink-wrapping each spermatid into its own plasma membrane as it travels. The mulet (mlt) mutation results in severely disrupted ICs, indicating that the mlt gene product is required for individualization. Inverse PCR followed by cycle sequencing maps all known P-insertion alleles of mlt to two overlapping genes, CG12214 (the Drosophila tubulin-binding cofactor E-like homolog) and KCNQ (a large voltage-gated potassium channel). However, since the alleles of mlt map to the 5′-UTR of CG12214 and since CG12214 is contained within an intron of KCNQ, it was hypothesized that mlt and CG12214 are allelic. Indeed, CG12214 mutant testes exhibited severely disrupted ICs and were indistinguishable from mlt mutant testes, thus further suggesting allelism. To test this hypothesis, alleles of mlt were crossed to CG12214 in order to generate trans-heterozygous males. Testes from all trans-heterozygous combinations revealed severely disrupted ICs and were also indistinguishable from mlt mutant testes, indicating that mlt and CG12214 fail to complement one another and are thus allelic. In addition, complementation testing against null alleles of KCNQ verified that the observed individualization defect is not caused by a disruption of KCNQ. Finally, since a population of spermatid-associated microtubules known to disappear prior to movement of the IC abnormally persists during individualization in CG12214 mutant testes, this work implicates TBCE-like in the removal of these microtubules prior to IC movement. Taken together, these results identify mlt as CG12214 and suggest that the removal of microtubules by TBCE-like is a necessary pre-requisite for proper coordinated movement of the IC.     

Terahertz Dipole Nanoantenna Arrays: Resonance Characteristics

Resonant dipole nanoantennas promise to considerably improve the capabilities of terahertz spectroscopy, offering the possibility of increasing its sensitivity through local field enhancement, while in principle allowing unprecedented spatial resolutions, well below the diffraction limit. Here, we investigate the resonance properties of ordered arrays of terahertz dipole nanoantennas, both experimentally and through numerical simulations. We demonstrate the tunability of this type of structures, in a range (～1–2 THz) that is particularly interesting and accessible by means of standard zinc telluride sources. We additionally study the near-field resonance properties of the arrays, finding that the resonance shift observed between near-field and far-field spectra is predominantly ascribable to ohmic damping.     

Discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-one-containing Phosphoinositide 3-kinase (PI3K) p110β isoform inhibitors through structure-based fragment optimisation

The discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-one-containing inhibitors of Phosphoinositide 3-kinases (PI3K) p110β isoform is reported. Structure-based optimisation of the original fragment hit resulted in lead compounds with improvements in ligand efficiency, lipophilicity efficiency, p110β potency and selectivity over p110α.     

Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products

The aim of this study was to investigate some probiotic properties of 42 wild Lactobacillus plantarum strains isolated from different Italian foods of animal origin. The strains were first screened for their antibiotic resistance profile (chloramphenicol, erythromycin, gentamicin, and tetracycline), subsequently they were tested for their in vitro resistance to lysozyme (100 mg L^?1), low pH (3.0, 2.5 and 2.0) and bile salts (0.3, 0.5 and 1.0 %). Moreover, agglutination property was studied (adhesion to Saccharomyces cerevisiae cells), as well as the presence of bsh and msa genes. The strains with the best characteristics were subjected to a further trial in order to evaluate their ability to survive to multiple stresses over time (lysozyme, low pH and bile salts) and the effect of these treatments on adhesion to yeast cells. All the strains were susceptible to chloramphenicol, erythromycin and gentamicin, while 6 strains were excluded from further evaluation because of their resistant phenotype against tetracycline. All the strains were able to grow in presence of lysozyme, as well as in MRS broth at pH 3.0. Only 4 strains showed a growth rate lower than 80 % when grown in MRS broth at pH 2.5, while a relevant growth rate decrease was observed after exposure to pH 2.0. Bile salts didn’t affect the viability of the L. plantarum cells. Twenty-one strains out of 33 tested strains were able to adhere to S. cerevisiae cells. Presence of both bsh and msa genes was detected in 6 strains. The strains resistant to all the stresses, positive to agglutination with S. cerevisiae and showing bsh and msa genes were selected for further evaluation and subjected to different stress treatments over time. The assessment of growth rates showed that exposure to lysozyme significantly increased low pH resistance in L. plantarum. This increase ranged from 2.35 to 15.57 %. The consequential lysozyme and low pH exposures didn’t affect the growth rate values after bile salts treatment, as well as the ability of the strains to adhere to yeast cells wasn’t modified by previous treatments (lysozyme, low pH and bile salts). The present work allows to increase knowledge about non starter lactic acid bacteria from Italian food products. The studied L. plantarum strains showed a good potential for their use as probiotic cultures. However, more in vivo tests are necessary to confirm this potentiality.     

A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala₃-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.     

ras-p21 Activates phospholipase D and A2, but not phospholipase C or PKC,in Xenopus laevis Oocytes

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42^MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.