Mining massive amounts of transcript data for alternative splicing information is paramount to help understand how the maturation of RNA regulates gene expression. We developed an algorithm to cluster transcript data to annotated genes to detect unannotated splice variants. A higher number of alternatively spliced genes and isoforms were found compared to other alternative splicing databases. Comparison of human and mouse data revealed a marked increase, in human, of splice variants incorporating novel exons and retained introns. Previously unannotated exons were validated by tiling array expression data and shown to correspond preferentially to novel first exons. Retained introns were validated by tiling array and deep sequencing data. The majority of retained introns were shorter than 500 nt and had weak polypyrimidine tracts. A subset of retained introns matching small RNAs and displaying a high GC content suggests a possible coordination between splicing regulation and production of noncoding RNAs. Conservation of unannotated exons and retained introns was higher in horse, dog and cow than in rodents, and 64% of exon sequences were only found in primates. This analysis highlights previously bypassed alternative splice variants, which may be crucial to deciphering more complex pathways of gene regulation in human.

Endo-1,5-α-L-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. Two extracellular endo-1,5-α-L-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-L-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-L-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-L-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.

Sleep staging is a crucial step before the scoring the sleep apnoea, in subjects that are tested for this condition. These patients undergo a whole night polysomnography recording that includes EEG, EOG, ECG, EMG and respiratory signals. Sleep staging refers to the quantification of its depth. Despite the commercial sleep software being able to stage the sleep, there is a general lack of confidence amongst health practitioners of these machine results. Generally the sleep scoring is done over the visual inspection of the overnight patient EEG recording, which takes the attention of an expert medical practitioner over a couple of hours. This contributes to a waiting list of two years for patients of the Portuguese Health Service. In this work we have used a spectral comparison method called Itakura distance to be able to make a distinction between sleepy and awake epochs in a night EEG recording, therefore automatically doing the staging. We have used the data from 20 patients of Hospital Pulido Valente, which had been previously visually expert scored. Our technique results were promising, in a way that Itakura distance can, by itself, distinguish with a good degree of certainty the N2, N3 and awake states. Pre-processing stages for artefact reduction and baseline removal using Wavelets were applied.

Iron oxide magnetic nanoparticles {(MNPs)} were synthesized by the chemical co-precipitation method and coated with gum arabic {(GA)} by physical adsorption and covalent attachment. Cultures of mammalian cell lines {(HEK293}, {CHO} and {TE671)} were grown in the presence of uncoated and {GA-coated} {MNPs.} Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for {GA} coating of {MNPs} endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the {GA} was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures. Copyright © 2010 John Wiley & Sons, Ltd.

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force.

A software tool for the analysis of the High-Resolution Electrocardiogram (HR-ECG) for Arrhythmia detection is introduced. New algorithms based on Wavelet analysis are presented and compared with the classic Simson protocol over the P and QRS segments of the Electrocardiogram (EEG). A novel procedure based on a two step wavelet analysis and synthesis is performed in order to obtain a frequency description of the P, T or QRS segments. This frequency ?signature? is useful for the detection of otherwise asymptomatic Arrhythmia patients. The tool has been developed in Matlab, and deployed for a standalone C application.

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Rail corrugation is a phenomenon that leads to a waving in the rails with wavelengths typically between 3 cm and 100 cm and amplitude levels of several microns. The genesis of this waving is complex. Rail corrugation is a recognized problem that leads to excess vibration on the rails and vehicles to a point of reducing their life span and compromising safety. In urban areas excess vibration noise is also a problem. A software tool was developed to analyze accelerometer signals acquired in the boggies of rail vehicles in order to quantify the rail corrugation according to their frequency and amplitude. A wavelet packet methodology was used in this work and compared with the One Third Octave Filter (OTOF) power representations, which is currently used in the industry. It is shown that the former produces better results.

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The study focuses on the public participation during the decision-making process of the High Speed Rail Project (popularly known as TGV) in Portugal. The study analyzes the media references on the topic and its actors in a quantitative and qualitative way from September 2008 to November 2009 anchored in the 2009 Legislatives and Local elections. The work concludes that despite the political polarization around the High Speed Rail Project, contributing to bring it to public debate, it didn’t allow for an improvement in qualitative information that could allow the emergence of an active and informed citizen participation in the debate.

This paper analyses the application of the Innovation Scoring model and its results to the railway business branch of a foreign multinational. Results confirm some enrolment in non-core Research & Development connected to the commercial activity of selling trains. Two main determinants were found to support this enrolment: the impact of a new Portuguese law imposing R&D investment for public contracts; and a certain degree of openness in the multinational’s innovation strategy based on predictable growth for the Portuguese market. The study also confirms the usefulness of the Innovation Scoring model, leaving some suggestions for improvement such as tailoring for multi-decision centres, implementing it through a third party, reducing the number of questions and introduction of a product evaluation methodology.

Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.