Morpho-colorimetric characterization by image analysis to identify diaspores of wild plant species

Digital images of ex situ germplasm stored in the Sardinian Germplasm Bank (BG-SAR) were used for the application of image analysis techniques at the Stazione Sperimentale di Granicoltura per la Sicilia. The analysed accessions refer to 148 taxonomic units belonging to 102 genera and 47 families, typical of the Sardinian flora, and of the Mediterranean basin in general.The images of diaspores were acquired by a flatbed scanner and elaborated with a macro specially developed for the morphometric and colorimetric measurements. This method allowed carrying out a database for the characterization of autochthonous germplasm in entry to the bank and the realization of statistic classifiers for the discrimination of genera and species within the following families: Apiaceae, Boraginaceae, Caryophyllaceae, Cistaceae, Fabaceae and Scrophulariaceae. Such classifiers, based on the linear discriminant analysis (LDA) technique and checked by cross-validation, showed a performance included between 74.3% and 96.4%.In addition, for the genus Astragalus, it was possible to elaborate a classifier able to identify very similar taxa of a species complex, obtaining a performance between 83.7% and 100%. Such analysis proved the validity of the methodology also from the taxonomic point of view.Suggestions for subsequent methodological progress, which could offer applications in other research issues, such as ecological analysis, soil seed bank and archaeological botany are proposed.     

Calcium-Dependent Evoked Release of N[3H]Acetylaspartylglutamate from the Optic Pathway

N-Acetylaspartylglutamate (NAAG) is a neuropeptide localized to several putative glutamatergic neuronal systems, including the rodent optic pathway. To determine whether the peptide is released by depolarization, the superior colliculus of the rat was perfused with 2 microCi of [3H]NAAG, then with Krebs-bicarbonate buffer for 1 h, using a microdialysis system. Subsequently, 10-min fractions were collected and analyzed by HPLC for [3H]NAAG. Addition of 100 microM veratridine resulted in a several-fold increase in the evoked release of [3H]NAAG that was virtually abolished by coperfusion with Ca2+-free Krebs buffer containing 1 mM EGTA. When [3H]glutamate was used as the precursor, veratridine depolarization resulted in only an 80% increase in the release of [3H]NAAG. Prior enucleation of the right eye reduced the spontaneous release of [3H]NAAG by 50%, and the veratridine-evoked release by greater than 85%, from the left superior colliculus. These results suggest that NAAG is released upon depolarization and may serve as a neurotransmitter/neuromodulator in the optic tract.     

Energy-dispersive X-ray fluorescence analysis applied to biomonitoring on alps

Biological Trace Element Research - The results of a research in progress at the Istituto di Fisica Generale Applicata—University of Milan—on natural and anthropogenic elements'...     

Further studies on bilitranslocase,a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, α and β, of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is α₂-β.     

Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopamine levels in the nucleus accumbens shell in rats

Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here we show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes.     

Natriuretic Peptide-Guided Therapy in Chronic Heart Failure: A Meta-Analysis of 2,686 Patients in 12 Randomized Trials

Background

The role of cardiac natriuretic peptides in the management of patients with chronic heart failure (HF) remains uncertain. The purpose of this study was to evaluate whether natriuretic peptide-guided therapy, compared to clinically-guided therapy, improves mortality and hospitalization rate in patients with chronic HF.

Methodology/Principal Findings

MEDLINE, Cochrane, ISI Web of Science and SCOPUS databases were searched for articles reporting natriuretic peptide-guided therapy in HF until August 2012. All randomized trials reporting clinical end-points (all-cause mortality and/or HF-related hospitalization and/or all-cause hospitalization) were included. Meta-analysis was performed to assess the influence of treatment on outcomes. Sensitivity analysis was performed to test the influence of potential effect modifiers and of each trial included in meta-analysis on results. Twelve trials enrolling 2,686 participants were included. Natriuretic peptide-guided therapy (either B-type natriuretic peptide [BNP]- or N-terminal pro-B-type natriuretic peptide [NT-proBNP]-guided therapy) significantly reduced all-cause mortality (Odds Ratio [OR]:0.738; 95% Confidence Interval [CI]:0.596 to 0.913; p = 0.005) and HF-related hospitalization (OR:0.554; CI:0.399 to 0.769; p = 0.000), but not all-cause hospitalization (OR:0.803; CI:0.629 to 1.024; p = 0.077). When separately assessed, NT-proBNP-guided therapy significantly reduced all-cause mortality (OR:0.717; CI:0.563 to 0.914; p = 0.007) and HF-related hospitalization (OR:0.531; CI:0.347 to 0.811; p = 0.003), but not all-cause hospitalization (OR:0.779; CI:0.414 to 1.465; p = 0.438), whereas BNP-guided therapy did not significantly reduce all-cause mortality (OR:0.814; CI:0.518 to 1.279; p = 0.371), HF-related hospitalization (OR:0.599; CI:0.303 to 1.187; p = 0.142) or all-cause hospitalization (OR:0.726; CI:0.609 to 0.964; p = 0.077).

Conclusions/Significance

Use of cardiac peptides to guide pharmacologic therapy significantly reduces mortality and HF related hospitalization in patients with chronic HF. In particular, NT-proBNP-guided therapy reduced all-cause mortality and HF-related hospitalization but not all-cause hospitalization, whereas BNP-guided therapy did not significantly reduce both mortality and morbidity.     

Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.     

Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

The development and characterization of an E. coli O25B bioconjugate vaccine

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

     

Solution structures of the reduced and Cu(I) bound forms of the first metal binding sequence of ATP7A associated with Menkes disease

The coding sequence for the first N-terminal copper binding motif of the human Menkes disease protein (MNK1; residues 2-79) was synthesized, cloned, and expressed in bacteria for biochemical and structural studies. MNK1 adopts the betaalphabetabetaalphabeta fold common to all the metal binding sequences (MBS) found in other metal transport systems (e.g., the yeast copper chaperone for superoxide dismutase CCS, the yeast copper chaperone ATX1 bound to Hg(II), and most recently Cu(I), the bacterial copper binding protein, CopZ, and the bacterial Hg(II) binding protein MerP), although substantial differences were found in the metal binding loop. Similar to ATX1, MNK1 binds Cu(I) in a distorted linear bicoordinate geometry. As with MerP, MNK1 has a high affinity for both Hg(II) and Cu(I), although it displays a marked preference for Cu(I). In addition, we found that F71 is a key residue in the compact folding of MNK1, and its mutation to alanine results in an unfolded structure. The homologous residue in MerP has also been mutated with similar results. Finally, to understand the relationship between protein folding and metal affinity and specificity, we expressed a chimeric MBS with the MNK1 protein carrying the binding motif of MerP (CAAC-MNK1); this chimeric protein showed differences in structure and the dynamics of the binding site that may account for metal specificity.