Nitrate and sulfate: Effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep

Twenty male crossbred Texel lambs were used in a 2 × 2 factorial design experiment to assess the effect of dietary addition of nitrate (2.6% of dry matter) and sulfate (2.6% of dry matter) on enteric methane emissions, rumen volatile fatty acid concentrations, rumen microbial composition, and the occurrence of methemoglobinemia. Lambs were gradually introduced to nitrate and sulfate in a corn silage-based diet over a period of 4 wk, and methane production was subsequently determined in respiration chambers. Diets were given at 95% of the lowest ad libitum intake observed within one block in the week before methane yield was measured to ensure equal feed intake of animals between treatments. All diets were formulated to be isonitrogenous. Methane production decreased with both supplements (nitrate: -32%, sulfate: -16%, and nitrate+sulfate: -47% relative to control). The decrease in methane production due to nitrate feeding was most pronounced in the period immediately after feeding, whereas the decrease in methane yield due to sulfate feeding was observed during the entire day. Methane-suppressing effects of nitrate and sulfate were independent and additive. The highest methemoglobin value observed in the blood of the nitrate-fed animals was 7% of hemoglobin. When nitrate was fed in combination with sulfate, methemoglobin remained below the detection limit of 2% of hemoglobin. Dietary nitrate decreased heat production (-7%), whereas supplementation with sulfate increased heat production (+3%). Feeding nitrate or sulfate had no effects on volatile fatty acid concentrations in rumen fluid samples taken 24h after feeding, except for the molar proportion of branched-chain volatile fatty acids, which was higher when sulfate was fed and lower when nitrate was fed, but not different when both products were included in the diet. The total number of rumen bacteria increased as a result of sulfate inclusion in the diet. The number of methanogens was reduced when nitrate was fed. Enhanced levels of sulfate in the diet increased the number of sulfate-reducing bacteria. The number of protozoa was not affected by nitrate or sulfate addition. Supplementation of a diet with nitrate and sulfate is an effective means for mitigating enteric methane emissions from sheep.     

Psychiatric and psychological factors in patient decision making concerning antidepressant use.

The observation that the use of antidepressants has strongly increased during the past decade implies that on a micro level doctors and patients more often decide that antidepressants are the appropriate treatment. Therefore, it is important to increase insight into patients' decision making regarding the use of antidepressants. The decision making model used in the present study was based on A. Bandura's (1986) social cognitive theory. Two cohorts of patients were recruited and followed for 9 months. Among patients who use antidepressants (N = 166), the stronger pros and weaker cons of discontinuation and self-efficacy predicted more proximal goal intentions. Goal intentions predicted discontinuation after 9 months. Among patients who had used antidepressants in the past (N = 73), stronger pros of discontinuation and the weaker perceived functions of antidepressants predicted a more positive evaluation of their present state, compared with when they still used antidepressants. These temporal comparisons, in turn, predicted renewed use of antidepressants after 9 months. The results provide a framework for supporting and influencing decision making with regard to the use of antidepressants. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Biodegradation of porous versus non-porous poly(L-lactic acid) films

The influence of porosity on the degradation rate of poly(L-lactic acid) (PLLA) films was investigated in vitro and in vivo. Non-porous, porous and combi (porous with a non-porous layer) PLLA films were used. Changes in Mw, Mn, polydispersity (Mw/Mn) ratio, melting temperature (T _m), heat of fusion, tensile strength, E-modulus, mass and the remaining surface area of cross-sections of the PLLA films were measured. In general, during the degradation process, the porous film has the highest Mw, Mn, Mw/Mn ratio and T _m, while the non-porous film has the lowest. In contrast, the highest heat of fusion values were observed for the non-porous film, indicating the presence of relatively smaller molecules forming crystalline domains more easily. The tensile strength and E-modulus of the non-porous film decrease faster than those of the porous and the combi film. None of the three types of films showed massive mass loss in vitro nor a significant decrease in remaining polymer surface area in light microscopical sections in vitro and in vivo. Heavy surface erosion of the non-porous layer of the combi film was observed after 180 days, turning the combi film into a porous film. This is also indicated by the changes in tensile strength, Mw, Mw/Mn, T _m and heat of fusion as a function of time. It is concluded that non-porous PLLA degrades faster than porous PLLA. Thus, in our model, porosity is an important determinant of the degradation rate of PLLA films.     

Temperature-dependent fracture mechanisms in gel-spun hot-drawn ultra-high molecular weight polyethylene fibres

Tensile testing of gel-spun hot-drawn ultra-high molecular weight polyethylene (UHMWPE) fibres reveal a ductile-brittle transition temperature. Ductile fracture above the transition temperature is believed to be initiated by a stress-induced orthorhombic-hexagonal phase transition, whereas at lower temperatures brittle fracture occurs in the orthorhombic phase. SEM micrographs of the fracture surfaces of UHMWPE fibres fractured below the transition temperature show fibrillar fracture and cannot be distinguished from fibres fractured in a ductile way above the transition temperature. Received: 10 October 1997/Accepted: 31 October 1997     

Impact Fatigue Behaviour of Rubber‐Modified Glass Fibre Reinforced Polyamide 6

The impact fatigue behaviour of glass fibre reinforced polyamide 6 with and without impact modifier was studied. The accumulated damage was investigated by means of light microscopy, confocal laser scanning microscopy and scanning electron microscopy. The impact modifiers were found to increase the ductility of the matrix. In particular, the increased resistance to crack initiation and growth during biaxial impact fatigue was demonstrated. The rubber particles cavitate during impact testing and initiate local shearing, thereby reducing the amount of debonding at the fibre ends, as compared to the sample without toughness modifiers. The shearing of the matrix suppresses debonding, matrix flaws at the fibre ends and fibre pull‐out.     

The {alpha}/{beta} hydrolase fold

We have identified a new protein fold—the /ßhydrolase fold—that is common to several hydrolytic enzymesof widely differing phylogenetic origin and catalytic function.The core of each enzyme is similar: an /ß sheet, notbarrel, of eight ß-sheets connected by -helices. Theseenzymes have diverged from a common ancestor so as to preservethe arrangement of the catalytic residues, not the binding site.They all have a catalytic triad, the elements of which are borneon loops which are the best-conserved structural features inthe fold. Only the histidine in the nucleophile-histidine-acidcatalytic triad is completely conserved, with the nucleophileand acid loops accommodating more than one type of amino acid.The unique topological and sequence arrangement of the triadresidues produces a catalytic triad which is, in a sense, amirror-image of the serine protease catalytic triad. There arenow four groups of enzymes which contain catalytic triads andwhich are related by convergent evolution towards a stable,useful active site: the eukaryotic serine proteases, the cysteineproteases, subtilisins and the /ß hydrolase fold enzymes.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection

Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.     

Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.     

Safety effects of 30 Km/H zones in The Netherlands.

Since 1983, Dutch municipal authorities can institute a maximum speed of 30 km/h on roads or in zones within built-up areas. The safety effects of 30 km/h zones are supposed to be positive. In order to be sure about this, the Ministry of Transport has stimulated 15 municipalities to implement a 30 km/h zone and set up an evaluation of the safety effects of these zones. The evaluation research is coordinated by the Institute for Road Safety Research (SWOV). The evaluation concerns the changes in traffic flows, opinions of residents, conflicts, and accidents. This paper gives the results of the evaluation.