Simultaneous detection of infectious human echoviruses and adenoviruses by an in situ nuclease-resistant molecular beacon-based assay

A multiplex methodology using two nuclease-resistant molecular beacons that target specific genomic regions of adenovirus 2 and echovirus 17 during simultaneous infection in A549 cells is presented. Using fluorescence microscopy, visualization of enteroviral and adenoviral replication was possible within 3 h postinfection.     

Ostracoda and Mollusca biodiversity and hydrochemical features in Late Miocene brackish basins of Italy

Late Miocene brackish ostracods and molluscs collected in three Italian basins show noticeable differences in their taxonomic composition, despite their capability of dispersing across wide geographic areas. In the Venetian-Friulian Basin (northern Italy), the upper Tortonian sediments contain oligotypic ostracod assemblages including Hemicyprideis dacica dacica, Hemicytheria pejinovicensis, and Loxoconcha cf. L. josephi and few gastropods referable to Planorbidae and Stenothyroides, which are typical of the central Paratethys. In central Italy, the brackish ostracods and molluscs recovered from upper Tortonian-lower Messinian deposits from four Tuscan basins (Volterra-Radicondoli, Velona, Baccinello-Cinigiano, and Valdelsa) display high affinity at a generic level but strong endemicity at a specific level. At Cessaniti (southern Italy), the upper Tortonian unit contains oligotypic brackish ostracods and molluscs: Mediocytherideis (Sylvestra) posterobursa, Cyprideis ruggierii, Loxoconcha cf. L. biformata, and Zonocypris membranae quadricella characterise the ostracod fauna, while Granulolabium bicinctum and Hydrobia frauenfeldi are the dominant molluscs. The recovered ostracods have a strong affinity with brackish species from central and eastern Palaeo-Mediterranean areas, whereas the molluscs present a Paratethyan origin. Despite the fact that the basins are all brackish and partly coeval, the systematics of these assemblages highlights the absence of common species among the three studied areas. Geochemical analyses (stable isotopes and trace elements) are performed on ostracods, and ⁸⁷Sr/⁸⁶Sr ratios are established in molluscs and echinoids. The results suggest brackish environments with different compositions and origins of solutes in the three different areas. The Tuscan basins are characterised by brackish waters, with NaCl-enriched waters coming from aquifers of Triassic evaporite bedrock. The brackish deposits of the Venetian-Friulian Basin and Cessaniti are true marginal marine environments, although the northern basin may have been influenced by both the Paratethyan Sava Basin and the northern portion of the Palaeo-Mediterranean water bodies.     

Novel colicin Fy of Yersinia frederiksenii inhibits pathogenic Yersinia strains via YiuR-mediated reception, TonB import, and cell membrane pore formation

A novel colicin type, designated colicin Fy, was found to be encoded and produced by the strain Yersinia frederiksenii Y27601. Colicin Fy was active against both pathogenic and nonpathogenic strains of the genus Yersinia. Plasmid YF27601 (5,574 bp) of Y. frederiksenii Y27601 was completely sequenced. The colicin Fy activity gene (cfyA) and the colicin Fy immunity gene (cfyI) were identified. The deduced amino acid sequence of colicin Fy was very similar in its C-terminal pore-forming domain to colicin Ib (69% identity in the last 178 amino acid residues), indicating pore forming as its lethal mode of action. Transposon mutagenesis of the colicin Fy-susceptible strain Yersinia kristensenii Y276 revealed the yiuR gene (ykris001_4440), which encodes the YiuR outer membrane protein with unknown function, as the colicin Fy receptor molecule. Introduction of the yiuR gene into the colicin Fy-resistant strain Y. kristensenii Y104 restored its susceptibility to colicin Fy. In contrast, the colicin Fy-resistant strain Escherichia coli TOP10F' acquired susceptibility to colicin Fy only when both the yiuR and tonB genes from Y. kristensenii Y276 were introduced. Similarities between colicins Fy and Ib, similarities between the Cir and YiuR receptors, and the detected partial cross-immunity of colicin Fy and colicin Ib producers suggest a common evolutionary origin of the colicin Fy-YiuR and colicin Ib-Cir systems.     

Anti-superoxide and anti-peroxynitrite strategies in pain suppression

Superoxide (SO, O(2)·(-)) and its reaction product peroxynitrite (PN, ONOO(-)) have been shown to be important in the development of pain of several etiologies. While significant progress has been made in teasing out the relative contribution of SO and PN peripherally, spinally, and supraspinally during the development and maintenance of central sensitization and pain, there is still a considerable void in our understanding. Further research is required in order to develop improved therapeutic strategies for selectively eliminating SO and/or PN. Furthermore, it may be that PN is a more attractive target, in that unlike SO it has no currently known beneficial role. Our group has been at the forefront of research concerning the role of SO and PN in pain, and our current findings have led to the development of two new classes of orally active catalysts which are selective for PN decomposition while sparing SO. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees

‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘Ca. Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘Ca. Phytoplasma mali’ in order to develop sustainable approaches for managing AP.     

Sublethal doses of β-amyloid peptide abrogate DNA-dependent protein kinase activity

Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25-35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1-42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD.     

Structural mechanism of ATP-induced polymerization of the partition factor ParF: implications for DNA segregation

Segregation of the bacterial multidrug resistance plasmid TP228 requires the centromere-binding protein ParG, the parH centromere, and the Walker box ATPase ParF. The cycling of ParF between ADP- and ATP-bound states drives TP228 partition; ATP binding stimulates ParF polymerization, which is essential for segregation, whereas ADP binding antagonizes polymerization and inhibits DNA partition. The molecular mechanism involved in this adenine nucleotide switch is unclear. Moreover, it is unknown how any Walker box protein polymerizes in an ATP-dependent manner. Here, we describe multiple ParF structures in ADP- and phosphomethylphosphonic acid adenylate ester (AMPPCP)-bound states. ParF-ADP is monomeric but dimerizes when complexed with AMPPCP. Strikingly, in ParF-AMPPCP structures, the dimers interact to create dimer-of-dimer "units" that generate a specific linear filament. Mutation of interface residues prevents both polymerization and DNA segregation in vivo. Thus, these data provide insight into a unique mechanism by which a Walker box protein forms polymers that involves the generation of ATP-induced dimer-of-dimer building blocks.     

On the mechanisms underlying the depolarization block in the spiking dynamics of CA1 pyramidal neurons

Under sustained input current of increasing strength neurons eventually stop firing, entering a depolarization block. This is a robust effect that is not usually explored in experiments or explicitly implemented or tested in models. However, the range of current strength needed for a depolarization block could be easily reached with a random background activity of only a few hundred excitatory synapses. Depolarization block may thus be an important property of neurons that should be better characterized in experiments and explicitly taken into account in models at all implementation scales. Here we analyze the spiking dynamics of CA1 pyramidal neuron models using the same set of ionic currents on both an accurate morphological reconstruction and on its reduction to a single-compartment. The results show the specific ion channel properties and kinetics that are needed to reproduce the experimental findings, and how their interplay can drastically modulate the neuronal dynamics and the input current range leading to a depolarization block. We suggest that this can be one of the rate-limiting mechanisms protecting a CA1 neuron from excessive spiking activity.     

Taurine supplementation prevents morpho-physiological alterations in high-fat diet mice pancreatic β-cells

Taurine (Tau) is involved in beta (β)-cell function and insulin action regulation. Here, we verified the possible preventive effect of Tau in high-fat diet (HFD)-induced obesity and glucose intolerance and in the disruption of pancreatic β-cell morpho-physiology. Weaning Swiss mice were distributed into four groups: mice fed on HFD diet (36 % of saturated fat, HFD group); HTAU, mice fed on HFD diet and supplemented with 5 % Tau; control (CTL); and CTAU. After 19 weeks of diet and Tau treatments, glucose tolerance, insulin sensitivity and islet morpho-physiology were evaluated. HFD mice presented higher body weight and fat depots, and were hyperglycemic, hyperinsulinemic, glucose intolerant and insulin resistant. Their pancreatic islets secreted high levels of insulin in the presence of increasing glucose concentrations and 30 mM K⁺. Tau supplementation improved glucose tolerance and insulin sensitivity with a higher ratio of Akt phosphorylated (pAkt) related to Akt total protein content (pAkt/Akt) following insulin administration in the liver without altering body weight and fat deposition in HTAU mice. Isolated islets from HTAU mice released insulin similarly to CTL islets. HFD intake induced islet hypertrophy, increased β-cell/islet area and islet and β-cell mass content in the pancreas. Tau prevented islet and β-cell/islet area, and islet and β-cell mass alterations induced by HFD. The total insulin content in HFD islets was higher than that of CTL islets, and was not altered in HTAU islets. In conclusion, for the first time, we showed that Tau enhances liver Akt activation and prevents β-cell compensatory morpho-functional adaptations induced by HFD.     

On the use of possibility theory in uncertainty analysis of life cycle inventory

Purpose  

The purpose of this paper is to enhance the mathematical and physical understanding of practitioners of uncertainty analysis of life cycle inventory (LCI), on the application of possibility theory. The main questions dealt with are (1) clear definition of the terms—“necessity–possibility,” “probability,” “belief–plausibility,” and of their mutual relationships; (2) what justifies the substitution of classical probability for possibility; (3) mutual comparison of, and transformations in both senses between probability and possibility uncertainty measures; (4) how to construct meaningful input possibility measures from available probabilistic/statistic information; and (5) comparative analysis of the solutions of the problem of data uncertainty propagation in LCI, afforded, respectively, by probabilistic Monte Carlo simulation and possibilistic fuzzy interval arithmetic.