Saturation transfer difference (STD) NMR has emerged as one of the most popular ligand-based NMR techniques for the study of protein-ligand interactions. The success of this technique is a consequence of its robustness and the fact that it is focused on the signals of the ligand, without any need of processing NMR information about the receptor and only using small quantities of nonlabeled macromolecule. Moreover, the attractiveness of this experiment is also extendable to the classroom. In the context of a practical NMR class, this experiment is ideal to illustrate some fundamental NMR concepts, such as the nuclear Overhauser effect and relaxation in a multidisciplinary context, bridging chemistry and biochemistry with a taste of medicinal chemistry.

We use the readily available human serum albumin (HSA), 6-d,l-methyl-tryptophan (6-CH3-Trp), and 7- d,l-methyl-tryptophan (7-CH3-Trp) to introduce the STD-NMR experiment and to illustrate its applicability for ligand screening, mapping of binding moieties, and determination of the dissociation constant, in a context that can be explored or adapted to the student’s course level and topic (chemistry or biochemistry). We also cover the most important theoretical aspects of the STD experiment, calling attention to some of its limitations and drawbacks without a complex theoretical approach.

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the sigma(54)-RNA polymerase. We further demonstrate that the sigma(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with sigma(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the sigma(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.

We report a new strategy that combines a Forster Resonance Energy Transfer (FRET) based spectral codification tool with a single base extension (SBE) reaction for rapid and medium-throughput analysis of single nucleotide polymorphisms (SNPs). This strategy is based on the spectral codification - a donor (fluorophore labeled probe complementary to the region adjacent to an SNP) is used to induce specific FRET signatures from an acceptor fluorophore revealing the SNP variant. Using an SBE reaction and differently labeled ddNTPs, we can directly question each donor probe and retrieve information about which allele variant is present at that locus. The potential of the method is demonstrated by application to simultaneous questioning of two loci in the same reaction tube. Following calibration with all possible combinations of FRET pairs, an evaluation algorithm was calibrated so as to optimize base calling and allow unequivocal allele scoring with more than 80% confidence (for two simultaneous loci being questioned, one homo-and one heterozygous). In conclusion, this spectral codification approach may constitute a solution towards increasing throughput capability of single base extension based assays.

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 A, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

The majority of papers published in the last decades on European Union policy strongly stress the importance of the so-called Lisbon Strategy approved in the year 2000. The same applies to studies and reports on the shift of the European countries towards modernisation and restructuring policy in recent years. This EU development strategy defines a new direction for the coordination of national policies. But why has it become so important? One of the reasons is the fact that many of the papers are based on the concept of “knowledge society” as the key driver for an increased competitiveness of all political and economic regions of Europe. In this context, the term “knowledge” means the inter-linkage of education (including training, qualification, skills) and innovation (including research, information and communication). The use of the concept represents an important shift in the European strategy: further development would not only be based on investment in material infrastructures, but also more on the immaterial ground. However, this Lisbon Strategy was criticised by many politicians and opinion-makers in the first years of this century because the European structures were not prepared for such a quick change. At the same time, the focus for investment moved away from the traditional support of industrial sectors (manufacturing, agriculture and fisheries, construction) towards the “new economy” sectors. The vision of a knowledge society remained appealing also in a changing international context: the Middle East wars (Afghanistan, Iraq and Israel-Palestine) and the fast growth of the Chinese economy. However, the shadows of new recessions have strongly questioned the options made by the European Council. New challenges have emerged with the need to redefine collective strategies in terms of European development as set by the Lisbon strategy. “Europe 2020” is one more attempt to define a new strategy. But at present no clear path has been identifi

Nanotechnology has prompted researchers to develop new and improved materials aimed at biomedical applications with particular emphasis in diagnostics and therapy. Special interest has been directed at providing enhanced biomolecular diagnostics, including SNP detection gene expression profiles and biomarker characterisation. These strategies have focused on the development of nanoscale devices and platforms that can be used for single molecule characterisation of nucleic acid, DNA or RNA, and protein at an increased rate when compared to traditional techniques. Also, several advances have been reported on DNA analysis in real time, at both high resolution and very high throughputs, suitable for biomedical diagnostics. Here, we shall provide a review of available nanotechnology-based platforms for biomolecular recognition, and their application to molecular diagnostics and genome analysis, with emphasis on the use of noble metal nanoparticles for simple and specific analysis systems. Particular focus will be put on those already being translated into clinical settings. This article is part of a Special Issue entitled: Clinical Proteomics.

In this paper we report on the fabrication and performance of a portable and low cost optoelectronic platform integrating a double color tuned light emitting diode as light source, an amorphous/nanocrystalline silicon photodetector with a flat spectral response in the wavelength range from 520. nm to 630. nm and integrated electronic for signal acquisition and conditioning constituted by current to voltage converter, a filter and an amplification stage, followed by an analog to digital converter, with appropriate software for full automation to minimize human error. Incorporation of the double color tuned light emitting diode provides for a simple yet innovative solution to signal acquisition independently from the light intensity and/or solution concentration, while considerably decreasing production costs. Detection based on Au-nanoprobes constitutes the biorecognition step and allowed identification of specific sequences of Mycobacterium tuberculosis complex, namely Mycobacterium bovis and M. tuberculosis in biological samples.

The majority of papers published in the last decades on European Union policy strongly stress the importance of the so-called Lisbon Strategy approved in the year 2000. The same applies to studies and reports on the shift of the European countries towards modernisation and restructuring policy in recent years. This EU development strategy defines a new direction for the coordination of national policies. But why has it become so important? One of the reasons is the fact that many of the papers are based on the concept of “knowledge society” as the key driver for an increased competitiveness of all political and economic regions of Europe. In this context, the term “knowledge” means the inter-linkage of education (including training, qualification, skills) and innovation (including research, information and communication). The use of the concept represents an important shift in the European strategy: further development would not only be based on investment in material infrastructures, but also more on the immaterial ground. However, this Lisbon Strategy was criticised by many politicians and opinion-makers in the first years of this century because the European structures were not prepared for such a quick change. At the same time, the focus for investment moved away from the traditional support of industrial sectors (manufacturing, agriculture and fisheries, construction) towards the “new economy” sectors. The vision of a knowledge society remained appealing also in a changing international context: the Middle East wars (Afghanistan, Iraq and Israel-Palestine) and the fast growth of the Chinese economy. However, the shadows of new recessions have strongly questioned the options made by the European Council. New challenges have emerged with the need to redefine collective strategies in terms of European development as set by the Lisbon strategy. “Europe 2020” is one more attempt to define a new strategy. But at present no clear path has been identified. Whether the

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In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.

We are facing an era, where pressures on health costs are extremely high, and the reforms in health system are almost constant. But over time, one factor remains unchanged – Technology continues being the sustenance of health care. Manufacturers, clinicians, patients, diagnostic and therapeutic technicians, hospital managers, government leaders, among others, either in public or private sector, are increasingly demanding in the sustained seek for information that support its decisions. Those decisions are about different types of issues: if, or how the technology can be developed, whether a technology should or should not enter the market, whether to acquire and use certain technology, and so forth. Such demand is well implied in the growth and development of Health Technology Assessment (HTA). This specialised field is commonly understood according to the International Network of Agencies for Health Technology Assessment (INAHTA, 2003) as an multidisciplinary analysis and decisional field, which studies the implications of clinical, social, ethical and economic development, dissemination and use of health technologies, without neglecting its political analysis (Goodman, 2004). The political decisions made based on HTA reports should be based on scientific evidence, linking efforts between the technical, economic and political dimensions, resourcing to a participatory vision, so that we can translate the best possible decision (Novaes 2006). On the other hand, the success of these decisions depends critically on the skills of the researcher to convey wisdom and confidence in applying rules of argumentation (Grunwald, 2007). In this paper we analyse the technical and methodological aspects of HTA, seen as a tool for evaluating health procedures and techniques. And we analyse the needs for skills and qualifications development of the actors involved in this process.

This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.

We are facing an era, where pressures on health costs are extremely high, and the reforms in health system are almost constant. But over time, one factor remains unchanged – Technology continues being the sustenance of health care. Manufacturers, clinicians, patients, diagnostic and therapeutic technicians, hospital managers, government leaders, among others, either in public or private sector, are increasingly demanding in the sustained seek for information that support its decisions. Those decisions are about different types of issues: if, or how the technology can be developed, whether a technology should or should not enter the market, whether to acquire and use certain technology, and so forth. Such demand is well implied in the growth and development of Health Technology Assessment (HTA). This specialised field is commonly understood according to the International Network of Agencies for Health Technology Assessment (INAHTA, 2003) as an multidisciplinary analysis and decisional field, which studies the implications of clinical, social, ethical and economic development, dissemination and use of health technologies, without neglecting its political analysis (Goodman, 2004). The political decisions made based on HTA reports should be based on scientific evidence, linking efforts between the technical, economic and political dimensions, resourcing to a participatory vision, so that we can translate the best possible decision (Novaes 2006). On the other hand, the success of these decisions depends critically on the skills of the researcher to convey wisdom and confidence in applying rules of argumentation (Grunwald, 2007). In this paper we analyse the technical and methodological aspects of HTA, seen as a tool for evaluating health procedures and techniques. And we analyse the needs for skills and qualifications development of the actors involved in this process.

This working paper is based on the development of the Thesis Plan presented for the Units Project II and Project III at the 1st Winter School of PhD programme on Technology Assessment at FCT/UNL. It focuses the methodology analysis and includes empirical information elements, in order to understand how composite indicators of innovation can influence technology policy decisions. In order to test the hypotheses raised in the Thesis Plan, two separate phases were designed. On the first part, the work tests hypotheses 1 and partially 2, identifying the quality, depth and limitations of three famous complex indicator-based systems, namely the Science, Technology and Industry Scoreboard, the European Innovation Scoreboard 2008 and Innovation Union Scoreboard 2010. On the second phase, the remaining hypotheses are tested adding media databases analysis, which will provide complementary information to a set of interviews to policy makers, in order to understand the role of the composite indicators on technology decisions.

... CM Cordas (&) Á P . Raleiras Á F . Auche`re Á I. Moura Á JJG Moura ... de Quımica, Universidade Nova de Lisboa, 2859-516 Caparica, Portugal e-mail: cristina. cordas @dq.fct ... Present Address: P . Raleiras Department of Photochemistry and Molecular Science, PO Box 523, 75120 ...

ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1x10(-3) s(-1)), and an apparent dissociation constant (k(dapp)) of 227.4+/-25.7 muM and 112.3+/-8.7 muM was obtained in absence or presence of 20 muM V(10), respectively. Moreover, concentrations as low as 50 muM of decameric vanadate species (V(10)) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V(1)) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V(1) (0-200 muM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.

Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N(2)OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N(2)OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N(2)OR, or an electrostatic nature, in the case of P. denitrificans N(2)OR and A. cycloclastes N(2)OR. A set of well-conserved residues on the N(2)OR surface were identified as being part of the electron transfer pathway from the redox partner to N(2)OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N(2)OR sequence). Moreover, we built a model for Wolinella succinogenes N(2)OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N(2)OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N(2)OR domain is similar to that found in the other electron transfer complexes.

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

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The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.