A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c7 from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e−/H+ transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e−/H+ coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens.

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.

Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of α-1,5-arabinooligosaccharides, at least up to four L-arabinosyl units. Although this system is the key transporter for α-1,5-arabinotriose and α-1,5-arabinotetraose, the results indicate that α-1,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear α-1,2- and α-1,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system.

The solid-phase synthesis (SPS) of a structurally complex glycopeptide, using Sieber amide resin, was monitored by high resolution magic angle spinning NMR, demonstrating the further application of this technique. A synthetic peptidoglycan derivative, a precursor of a biologically active PGN, known to be involved in the cellular recognition, was prepared by SPS. The synthesis involved the preparation of an N-alloc glucosamine moiety and the synthesis of a simple amino acid sequence L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. Last step consisted the coupling, on solid-phase, of the protected muramyl unit to the peptide chain. Proton spectra with good suppression of the polystyrene signals in swollen resin samples were obtained in DMF-d(7) as a solvent and by using a nonselective 1D TOCSY/DIPSI-2 scheme, thus allowing to follow the SPS without losses of compound and cleavage from the resin. The assignment of the proton spectra of the resin-bound amino acid sequence and of the bound glycopeptide was achieved through the combination of MAS COSY, TOCSY and NOESY.

Molecularly imprinted polymers (MIPs) of poly(ethylene glycol dimethacrylate) and poly(N-isopropylacrylamide-co-ethylene glycol dimethacrylate) were synthesized for the first time in supercritical carbon dioxide (scCO2), using Boc-l-tryptophan as template. Supercritical fluid technology provides a clean and one-step synthetic route for the preparation of affinity polymeric materials with sensing capability for specific molecules. The polymeric materials were tested as stationary \{HPLC\} phases for the enantiomeric separation of l- and d-tryptophan. \{HPLC\} results prove that the synthesized \{MIPs\} are able to recognize the template molecule towards its enantiomer which opens up potential applications in chromatographic chiral separation.

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.