The Reflux Symptom Score-12: Cross-cultural Adaptation and Validation for European Portuguese Speakers With Laryngopharyngeal Reflux.

OBJECTIVES: To translate and cross-culturally adapt the 12-item reflux symptom score (RSS-12) to European Portuguese (EP) and determine its clinimetric properties for symptomatic individuals with laryngopharyngeal reflux (LPR). STUDY DESIGN: Multinational cross-sectional cohort study. METHODS: The English RSS-12 was cross-culturally adapted according to the recommendations of the international guidelines. The validation study included the completion of the RSS-12, reflux symptom index, and voice handicap index by symptomatic and asymptomatic subjects with LPR. The RSS-12 was completed a second time by symptomatic subjects. Nine clinimetric properties were analyzed according to the international guidelines for validation of patient-reported outcome measures. RESULTS: The EP RSS-12 is equivalent to the English version (content, depth, and scoring). A total of 155 adults (84 with LPR symptoms) aged 21-78 years participated in the validation study. Statistical analyses revealed high internal consistency (Cronbach alpha >0.90), high test-retest reliability (intraclass correlation coefficient > 0.70, P < 0.001), low measurement error (Standard measure error of 5.21 for RSS and 1.59 for quality of life), good content validity (omission data <1% and item-total correlations > 0.652), good construct validity (61.9% of the total item variance with moderate item loadings), strong concurrent validity with reflux symptom index (rp = 0.772, P < 0.001) and moderate validity with voice handicap index (rp = 0.531, P < 0.001), and significantly known-groups validity (P < 0.001). The EP RSS-12 showed cross-cultural validity with French and Persian versions and high predictive validity with a cut-off value >8 for a sensitivity of 91.7% and a specificity of 91.5%. CONCLUSIONS: The EP RSS-12 retained the features of the English version and is a reliable and valid patient-reported outcome measure for EP individuals with LPR in the study.

Reconstruction of full antibody sequences in NGS datasets and accurate VL:VH coupling by cluster coordinate matching of non-overlapping reads.

Next-generation sequencing (NGS) is an indispensable tool in antibody discovery projects. However, the limits on NGS read length make it difficult to reconstruct full antibody sequences from the sequencing runs, especially if the six CDRs are randomized. To overcome that, we took advantage of Illumina's cluster mapping capabilities to pair non-overlapping reads and reconstruct full Fab sequences with accurate VL:VH pairings. The method relies on in silico cluster coordinate information, and not on extensive in vitro manipulation, making the protocol easily deployable and less prone to PCR-derived errors. This work maintains the throughput necessary for antibody discovery campaigns, and a high degree of fidelity, which potentiates not only phage-display and synthetic library-based discovery methods, but also the NGS-driven analysis of naïve and immune libraries.

Precisely spun super rotors.

Improved optical control of molecular quantum states promises new applications including chemistry in the quantum regime, precision tests of fundamental physics, and quantum information processing. While much work has sought to prepare ground state molecules, excited states are also of interest. Here, we demonstrate a broadband optical approach to pump trapped SiO+ molecules into pure super rotor ensembles maintained for many minutes. Super rotor ensembles pumped up to rotational state N = 67, corresponding to the peak of a 9400 K distribution, had a narrow N spread comparable to that of a few-kelvin sample, and were used for spectroscopy of the previously unobserved C2Π state. Significant centrifugal distortion of super rotors pumped up to N = 230 allowed probing electronic structure of SiO+ stretched far from its equilibrium bond length.

Stability liabilities of biotherapeutic proteins: Early assessment as mitigation strategy.

Identification of molecular liabilities and implementation of mitigation strategies are key aspects that need to be considered by pharmaceutical companies developing therapeutic proteins. In the field of monoclonal antibodies, an efficient and streamlined process known as developability assessment is used for the selection of the "fittest" candidate. Other protein modalities, have in most cases only a limited number of possible candidates, requiring a paradigm change to a concept of candidate enabling. The assessment of liabilities at early project phases with the possibility to re-engineer candidates becomes essential for the success of these projects. Each protein possesses a unique stability profile resulting from the interplay of conformational, colloidal, chemical and physical stability attributes. All of these attributes strongly depend on external factors. Conformational and colloidal stability profiles of three non-immunoglobulin domain based proteins, namely Carbonic anhydrase, Ovalbumin and Thyroglobulin, and of two monoclonal antibodies were assessed in dependence of solution pH, ionic strength and varying buffering agents. The impact of screened external factors on proteins' stability attributes varied significantly, indicating presence of molecule specific liabilities. Screening of such a broad space of conditions at early project phases is only feasible using low-material consuming, high-throughput analytical methods as exemplified in this study.

Computational mechanistic investigation of the Fe + CO2→ FeO + CO reaction.

We report a computational study of the mechanism and determination of the rate constants of the Fe + CO2→ FeO + CO reaction, in the 1000-3000 K temperature range, at the CCSD(T)/CBS//B3LYP/def2-TZVP level of theory. The overall rate constant was obtained by a Kinetic Monte Carlo simulation. The calculated rate constant, at 2000 K, is 9.72 × 10-13 cm3 molecule-1 s-1, in agreement with experimental measurements: 2.97 × 10-13 cm3 molecule-1 s-1 [A. Giesen et al., Phys. Chem. Chem. Phys., 2002, 4, 3665] and 1.13 × 10-13 cm3 molecule-1 s-1 [V. N. Smirnov, Kinet. Catal., 2008, 49, 607]. Our study shows that this reaction follows a complex mechanism, with multiple reaction paths contributing to the overall rate, and that CCSD(T) accurately describes this transition metal reaction.

Insulin and ß-adrenergic receptors mediate lipolytic and anti-lipolytic signalling that is not altered by type 2 diabetes in human adipocytes.

Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and ß-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway ß-adrenergic receptor signalling, via cAMP and PI3Kα, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and ß-adrenergic signalling control phosphorylation of FOXO1. The dose-response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and ß-adrenergic stimulation - in human adipocytes - that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.

Scenedesmus obliquus in poultry wastewater bioremediation.

Wastewater biological treatment with microalgae can be an effective technology, removing nutrients and other contaminants while reducing chemical oxygen demand. This can be particularly interesting for the meat producing industry which produces large volumes of wastewater from the slaughtering of animals and cleaning of their facilities. The main purpose of this research was the treatment of poultry wastewater using Scenedesmus obliquus in an economical and environmentally sustainable way. Two wastewaters were collected from a Portuguese poultry slaughterhouse (poultry raw - PR and poultry flocculated - PF) and the bioremediation was evaluated. The performance of microalga biomass growth and biochemical composition were assessed for two illumination sources (fluorescent vs LEDs). S. obliquus achieved positive results when grown in highly contaminated agro-industrial wastewater from the poultry industry, independently of the light source. The wastewater bioremediation revealed results higher than 97% for both ammonium and phosphate removal efficiency, for a cultivation time of 13 days. The saponifiable matter obtained from the biomass of the microalga cultures was, on average, 11% and 27% (m/malga) with PR and PF wastewater, respectively. In opposition, higher sugar content was obtained from microalgae biomass grown in PR wastewater (average 34% m/malga) in comparison to PF wastewater (average 23% m/malga), independently of the illumination source. Therefore, biomass obtained with PR wastewater will be more appropriate as a raw material for bioethanol/biohydrogen production (higher sugar content) while biomass produced in PF wastewater will have a similar potential as feedstock for both biodiesel and bioethanol/biohydrogen production (similar lipid and sugar content).

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy.

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [ Di Luca , Proc. Natl. Acad. Sci. U.S.A. , 2017 , 114 , E6314 - E6321 ].

The key role of glutamate 172 in the mechanism of type II NADH:quinone oxidoreductase of Staphylococcus aureus.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane bound enzymes that deliver electrons to the respiratory chain by oxidation of NADH and reduction of quinones. In this way, these enzymes also contribute to the regeneration of NAD+, allowing several metabolic pathways to proceed. As for the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, the enzymatic mechanism of NDH-2s is still little explored and elusive. In this work we addressed the role of the conserved glutamate 172 (E172) residue in the enzymatic mechanism of NDH-2 from Staphylococcus aureus. We aimed to test our earlier hypothesis that E172 plays a key role in proton transfer to allow the protonation of the quinone. For this we performed a complete biochemical characterization of the enzyme's variants E172A, E172Q and E172S. Our steady state kinetic measurements show a clear decrease in the overall reaction rate, and our substrate interaction studies indicate the binding of the two substrates is also affected by these mutations. Interestingly our fast kinetic results show quinone reduction is more affected than NADH oxidation. We have also determined the X-ray crystal structure of the E172S mutant (2.55Ǻ) and compared it with the structure of the wild type (2.32Ǻ). Together these results support our hypothesis for E172 being of central importance in the catalytic mechanism of NDH-2, which may be extended to other members of the tDBDF superfamily.

Evaluation of Marine Microalga Diacronema vlkianum Biomass Fatty Acid Assimilation in Wistar Rats.

Diacronema vlkianum is a marine microalgae for which supposed health promoting effects have been claimed based on its phytochemical composition. The potential use of its biomass as health ingredient, including detox-shakes, and the lack of bioavailability studies were the main concerns. In order to evaluate the microalgae-biomass assimilation and its health-benefits, single-dose (CD1-mice) studies were followed by 66-days repeated-dose study in Wistar rats with the highest tested single-dose of microalgae equivalent to 101 mg/kg eicosapentaenoic acid + docosahexaenoic acid (EPA+DHA). Microalgae-supplementation modulated EPA and docosapentaenoic acid enrichment at arachidonic acid content expenditure in erythrocytes and liver, while increasing EPA content of heart and adipose tissues of rats. Those fatty acid (FA) changes confirmed the D. vlkianum-biomass FA assimilation. The principal component analyses discriminated brain from other tissues, which formed two other groups (erythrocytes, liver, and heart separated from kidney and adipose tissues), pointing to a distinct signature of FA deposition for the brain and for the other organs. The improved serum lipid profile, omega-3 index and erythrocyte plasticity support the cardiovascular benefits of D. vlkianum. These results bolster the potential of D. vlkianum-biomass to become a "heart-healthy" food supplement providing a safe and renewable source of bioavailable omega-3 FA.

Photophysical properties and the NO photorelease mechanism of a ruthenium nitrosyl model complex investigated using the CASSCF-in-DFT embedding approach.

A complete state-averaged active space self-consistent field (SA-CASSCF) calculation by means of the SA-CASSCF(18,14)-in-BP86 Miller-Manby embedding approach was performed to explore the ground and excited electronic states of the trans-[RuCl(NO)(NH3)4]2+ complex. Insights into the NO photodissociation mechanism and Ru-NO bonding properties are provided. In addition, spin-orbit (SO) interactions were taken into account to describe and characterize the spin-forbidden transitions observed at the low-energy regions of the trans-[RuCl(NO)(NH3)4]2+ UV-Vis spectrum. The SA-CASSCF(18,14)-in-BP86 electronic spectrum is in great agreement with the experimental data of Schreiner [Schreiner et al., Inorg. Chem., 1972, 11, 880].

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases.

Type II NADH:quinone oxidoreductase family: phylogenetic distribution, structural diversity and evolutionary divergences.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins, crucial for the catabolic metabolism, because they contribute to the maintenance of the NADH/NAD+ balance. In several pathogenic bacteria and protists, NDH-2s are the only enzymes performing respiratory NADH:quinone oxidoreductase activity. For this reason and for being considered absent in mammals, NDH-2s were proposed as suitable targets for novel antimicrobial therapies. We selected all sequences of genes encoding NDH-2s from fully sequenced genomes present in the KEGG database. These genes were present in 61% of the 1805 species belonging to Eukarya (83%), Bacteria (60%) and Archaea (32%). Notably sequences from mammal species including humans were retrieved in our selection as NDH-2s. The data obtained and the already available information allowed systematizing several properties of NDH-2s: (i) the existence of additional sequence motifs with putative regulatory functions, (ii) specificity towards NADH or NADPH and (iii) the type of quinone binding motif. We observed that NDH-2 family distribution is not congruent with the taxonomic tree, suggesting different origins for the eukaryotic sequences and possible lateral gene transfer among prokaryotes. We note the absence of genes coding for NDH-2 in anaerobic phyla and the presence of multiple copies in several genomes, specifically in cyanobacteria. These observations inspired us to propose a metabolic hypothesis for the appearance of NDH-2s.

Substrate-Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.

Silanol-Assisted Carbinolamine Formation in an Amine-Functionalized Mesoporous Silica Surface: Theoretical Investigation by Fragmentation Methods.

The aldol reaction catalyzed by an amine-substituted mesoporous silica nanoparticle (amine-MSN) surface was investigated using a large molecular cluster model (Si392O958C6NH361) combined with the surface integrated molecular orbital/molecular mechanics (SIMOMM) and fragment molecular orbital (FMO) methods. Three distinct pathways for the carbinolamine formation, the first step of the amine-catalyzed aldol reaction, are proposed and investigated in order to elucidate the role of the silanol environment on the catalytic capability of the amine-MSN material. The computational study reveals that the most likely mechanism involves the silanol groups actively participating in the reaction, forming and breaking covalent bonds in the carbinolamine step. Therefore, the active participation of MSN silanol groups in the reaction mechanism leads to a significant reduction in the overall energy barrier for the carbinolamine formation. In addition, a comparison between the findings using a minimal cluster model and the Si392O958C6NH361 cluster suggests that the use of larger models is important when heterogeneous catalysis problems are the target.

Respiratory complex I: A dual relation with H(+) and Na(+)?

Respiratory complex I couples NADH:quinone oxidoreduction to ion translocation across the membrane, contributing to the buildup of the transmembrane difference of electrochemical potential. H(+) is well recognized to be the coupling ion of this system but some studies suggested that this role could be also performed by Na(+). We have previously observed NADH-driven Na(+) transport opposite to H(+) translocation by menaquinone-reducing complexes I, which indicated a Na(+)/H(+) antiporter activity in these systems. Such activity was also observed for the ubiquinone-reducing mitochondrial complex I in its deactive form. The relation of Na(+) with complex I may not be surprising since the enzyme has three subunits structurally homologous to bona fide Na(+)/H(+) antiporters and translocation of H(+) and Na(+) ions has been described for members of most types of ion pumps and transporters. Moreover, no clearly distinguishable motifs for the binding of H(+) or Na(+) have been recognized yet. We noticed that in menaquinone-reducing complexes I, less energy is available for ion translocation, compared to ubiquinone-reducing complexes I. Therefore, we hypothesized that menaquinone-reducing complexes I perform Na(+)/H(+) antiporter activity in order to achieve the stoichiometry of 4H(+)/2e(-). In agreement, the organisms that use ubiquinone, a high potential quinone, would have kept such Na(+)/H(+) antiporter activity, only operative under determined conditions. This would imply a physiological role(s) of complex I besides a simple "coupling" of a redox reaction and ion transport, which could account for the sophistication of this enzyme. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction.

A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+)-protein complex, which is released by the quinone.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus.

In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents. In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0.873 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.8, b = 86.0, c = 269.9 Å, contained four monomers per asymmetric unit and diffracted to a resolution of 3.32 Å. A molecular-replacement solution was obtained and model building and refinement are currently under way.

Respiratory complex I from Escherichia coli does not transport Na(+) in the absence of its NuoL subunit.

We investigated H(+) and Na(+) transport by complex I from Escherichia coli devoid of the NuoL subunit, which is probably part of the ion translocating machinery. We observed that complex I devoid of the NuoL subunit still translocates H(+), although to a smaller extension than the complete version of complex I, but does not transport Na(+). Our results unequivocally reinforce the observation that E. coli complex I transports Na(+) in the opposite direction to that of the H+ and show that NuoL subunit is involved in the translocation of both ions by complex I.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.