Influence of chloride ions and pH level on the durability of high performance cement pastes

The resistance to chemical attack of low water to binder ratio pastes containing silica fume was studied by soaking small paste disks in three different pH controlled solutions, with or without sodium chloride, for periods of up to three months. The pastes were made using water to binder ratios of 0,25 and 0,38. The three solutions in which the paste disks were soaked were the following: 3% NaCl (by weight) at a pH level of 8,5,0% NaCl at 8,5, and 0% NaCl at 4,5. After three months of exposure, the results show that the pH level of the aggressive solution is the most important factor controlling the durability of cement pastes subjected to chemical attack. The total porosity and the depth of decalcification was found to increase with the decrease of the pH level. It was also found that the3water to binder ratio does not significantly affect the deterioration processes, but only influences the kinetics of these processes. The decrease of the water to binder ratio reduces significantly the rate of deterioration. Chloroaluminate crystals were observed only in the cement pastes having a water to binder ratio of 0,38.     

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis

Two different combinatorial mutagenesis experiments on the light-harvestingII (LH2) protein of Rhodobacter capsulatus indicate that heuristicrules relating sequence directly to phenotype are dependenton which sets or groups of residues are mutated simultaneously.Previously reported combinatorial mutagenesis of this chromogenicprotein (based on both phylogenetic and structural models) showedthat substituting amino acids with large molar volumes at Gly^ß31caused the mutated protein to have a spectrum characteristicof light-harvesting I (LH1). The six residues that underwentcombinatorial mutagenesis were modeled to lie on one side ofa transmembrane -helix that binds bacteriochlorophyll. In asecond experiment described here, we have not used structuralmodels or phylogeny in choosing mutagenesis sites. Instead,a set of six contiguous residues was selected for combinatorialmutagenesis. In this latter experiment, the residue substitutedat Gly^ß31 was not a determining factor in whetherLH2 or LH1 spectra were obtained; therefore, we conclude thatthe heuristic rules for phenotype prediction are context dependent.While phenotype prediction is context dependent, the abilityto identify elements of primary structure causing phenotypediversity appears not to be. This strengthens the argument forperforming combinatorial mutagenesis with an arbitrary groupingof residues if structural models are unavailable.     

Prediction of diffusion coefficients in cement-based materials on the basis of migration experiments

The chloride diffusion and migration coefficients of 15 different mortar mixtures were systematically compared. Test parameters included water/binder ratio (0.25 and 0.45), type of binder (ASTM type I, ASTM type III, and ASTM type V), use of silica fume and sand volume fractions (0%, 30%, and 50%). Test results indicate the various ways of evaluating chloride transport coefficients generally yield much different values. Test results also show that the assumption of non interacting diffusing flows, used in the mathematical treatment of diffusion and migration equations, is most probably incorrect.     

Résistance au gel-dégel du béton compacté au rouleau pour les barrages à base de liant Rolac

Ce texte présente les principaux résultats d'une étude sur la résistance au gel-dégel du béton compacté au rouleau à base de liant Rolac. Au total, 18 mélanges de béton compacté au rouleau ont été réalisés en laboratoire et soumis à des essais de gel dans l'air—dégel dans l'eau (norme ASTM C 666, procédure B). Les caractéristiques du réseau de vides d'air de tous les mélanges on été déterminées en suivant la méthode dite ‘Modified Point Count’ de la norme ASTM C457, en faisant la distinction entre les bulles d'air sphériques et les vides de compactage de forme irrégulière. Les résultats démontrent qu'il a été possible d'entraîner de l'air dans ces bétons (environ 1,5 à 2% de bulles sphériques) et que cette quantité d'air était suffisante pour protéger le béton contre le gel. En effet, tous les mélanges sans agent entraîneur d'air se sont détruits en moins de 50 cycles alors que tous les bétons fabriqués avec un agent entraîneur d'air ont passé le cap des 300 cycles de gel sans détérioration significative.     

Directed evolution of PDZ variants to generate high-affinity detection reagents

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.     

Recursive ensemble mutagenesis

We have developed a generally applicable experimental procedureto find functional proteins that are many mutational steps fromwild type. Optimization algorithms, which are typically usedto search for solutions to certain combinatorial problems, havebeen adapted to the problem of searching the ‘sequencespace’ of proteins. Many of the steps normally performedby a digital computer are embodied in this new molecular geneticstechnique, termed recursive ensemble mutagenesis (REM). REMuses information gained from previous iterations of combinatorialcassette mutagenesis (CCM) to search sequence space more efficiently.We have used REM to simultaneously mutate six amino acid residuesin a model protein. As compared to conventional CCM, one iterationof REM yielded a 30-fold increase in the frequency of ‘positive’mutants. Since a multiplicative factor of similar magnitudeis expected for the mutagenesis of additional sets of six residues,performing REM on 18 sites is expected to yield an exponential(30 000-fold) increase in the throughput of positive mutantsas compared to random [NN(G,C)]₁₈ mutagenesis.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.     

Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase

Directed evolution has become an important enabling technologyfor the development of new enzymes in the chemical and pharmaceuticalindustries. Some of the most interesting substrates for theseenzymes, such as polymers, have poor solubility or form highlyviscous solutions and are therefore refractory to traditionalhigh-throughput screens used in directed evolution. We combineddigital imaging spectroscopy and a new solid-phase screeningmethod to screen enzyme variants on problematic substrates highlyefficiently and show here that the specific activity of theenzyme galactose oxidase can be improved using this technology.One of the variants we isolated, containing the mutation C383S,showed a 16-fold increase in activity, due in part to a 3-foldimprovement in K_m. The present methodology should be applicableto the evolution of numerous other enzymes, including polysaccharide-modifyingenzymes that could be used for the large-scale synthesis ofmodified polymers with novel chemical properties.