BlastR--fast and accurate database searches for non-coding RNAs

We present and validate BlastR, a method for efficiently and accurately searching non-coding RNAs. Our approach relies on the comparison of di-nucleotides using BlosumR, a new log-odd substitution matrix. In order to use BlosumR for comparison, we recoded RNA sequences into protein-like sequences. We then showed that BlosumR can be used along with the BlastP algorithm in order to search non-coding RNA sequences. Using Rfam as a gold standard, we benchmarked this approach and show BlastR to be more sensitive than BlastN. We also show that BlastR is both faster and more sensitive than BlastP used with a single nucleotide log-odd substitution matrix. BlastR, when used in combination with WU-BlastP, is about 5% more accurate than WU-BlastN and about 50 times slower. The approach shown here is equally effective when combined with the NCBI-Blast package. The software is an open source freeware available from www.tcoffee.org/blastr.html.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

Retrospective investigation of an influenza A/H1N1pdm outbreak in an Italian military ship cruising in the Mediterranean Sea, May-September 2009

Background

Clinical surveillance may have underestimated the real extent of the spread of the new strain of influenza A/H1N1, which surfaced in April 2009 originating the first influenza pandemic of the 21^st century. Here we report a serological investigation on an influenza A/H1N1pdm outbreak in an Italian military ship while cruising in the Mediterranean Sea (May 24-September 6, 2009).

Methods

The contemporary presence of HAI and CF antibodies was used to retrospectively estimate the extent of influenza A/H1N1pdm spread across the crew members (median age: 29 years).

Findings

During the cruise, 2 crew members fulfilled the surveillance case definition for influenza, but only one was laboratory confirmed by influenza A/H1N1pdm-specific RT-PCR; 52 reported acute respiratory illness (ARI) episodes, and 183 reported no ARI episodes. Overall, among the 211 crew member for whom a valid serological result was available, 39.3% tested seropositive for influenza A/H1N1pdm. The proportion of seropositives was significantly associated with more crowded living quarters and tended to be higher in those aged <40 and in those reporting ARI or suspected/confirmed influenza A/H1N1pdm compared to the asymptomatic individuals. No association was found with previous seasonal influenza vaccination.

Conclusions

These findings underline the risk for rapid spread of novel strains of influenza A in confined environment, such as military ships, where crowding, rigorous working environment, physiologic stress occur. The high proportion of asymptomatic infections in this ship-borne outbreak supports the concept that serological surveillance in such semi-closed communities is essential to appreciate the real extent of influenza A/H1N1pdm spread and can constitute, since the early stage of a pandemic, an useful model to predict the public health impact of pandemic influenza and to establish proportionate and effective countermeasures.     

Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

Cholesterol photosensitized oxidation in food and biological systems

Lipid oxidation is one of the main chemical degradations occurring in biological systems and leads to the formation of compounds that are related to aging and various chronic and degenerative diseases. The extent of oxidation will depend on the presence of antioxidants/pro-oxidants, the unsaturation degree of fatty acids, and environmental conditions. Lipid oxidation can also affect other molecules that have double bonds in their chemical structures, such as cholesterol. Cholesterol oxidation products (COPs) have been studied in depth, because of their negative and controversial biological effects. The formation of COPs can be particularly favored in the presence of light and photosensitizers, since they generate excited singlet oxygen that rapidly reacts with the double bond by a non radical mechanism and without any induction period. The present review intends to provide an overall and critical picture of cholesterol photosensitized oxidation in food and biological systems, and its possible impact on human health and well-being.     

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).