Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Effect of accelerated rehabilitation on function after ankle sprain: randomised controlled trial

Objective: To compare an accelerated intervention incorporating early therapeutic exercise after acute ankle sprains with a standard protection, rest, ice, compression, and elevation intervention.

Design: Randomised controlled trial with blinded outcome assessor.

Setting: Accident and emergency department and university based sports injury clinic.

Participants: 101 patients with an acute grade 1 or 2 ankle sprain.

Interventions: Participants were randomised to an accelerated intervention with early therapeutic exercise (exercise group) or a standard protection, rest, ice, compression, and elevation intervention (standard group).

Main outcome measures: The primary outcome was subjective ankle function (lower extremity functional scale). Secondary outcomes were pain at rest and on activity, swelling, and physical activity at baseline and at one, two, three, and four weeks after injury. Ankle function and rate of reinjury were assessed at 16 weeks.

Results: An overall treatment effect was in favour of the exercise group (P=0.0077); this was significant at both week 1 (baseline adjusted difference in treatment 5.28, 98.75% confidence interval 0.31 to 10.26; P=0.008) and week 2 (4.92, 0.27 to 9.57; P=0.0083). Activity level was significantly higher in the exercise group as measured by time spent walking (1.2 hours, 95% confidence interval 0.9 to 1.4 v 1.6, 1.3 to 1.9), step count (5621 steps, 95% confidence interval 4399 to 6843 v 7886, 6357 to 9416), and time spent in light intensity activity (53 minutes, 95% confidence interval 44 to 60 v 76, 58 to 95). The groups did not differ at any other time point for pain at rest, pain on activity, or swelling. The reinjury rate was 4% (two in each group).

Conclusion: An accelerated exercise protocol during the first week after ankle sprain improved ankle function; the group receiving this intervention was more active during that week than the group receiving standard care.

The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations

Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 angstrom-long channel and at the opening of the 30 angstrom-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.

Novel photocatalyst-based colourimetric indicator for oxygen: Use of a platinum catalyst for controlling response times

The preparation and characterisation of a novel, UV-activated, solvent-based, colourimetric indicator for oxygen is described, comprising a redox dye (methylene blue, MB), semiconductor photocatalyst (Pt-TiO2), and a sacrificial electron donor (SED = glycerol), all dispersed/dissolved in a polymer medium (sulfonated polystyrene. SPS). Upon exposure to UVA light, the Pt-TiO2/MB/glycerol/SPS oxygen indicator is readily photobleached as the MB is converted into its oxygen-sensitive, leuco form, LMB. In contrast to its non-platinised TiO2 counterpart (TiO2/MB/glycerol/SPS oxygen indicator), the recovery of the original colour is faster (ca. 1.5 days cf. 5 days at 21 degrees C). This is due to the catalytic action of the 0.38 wt% platinum loaded onto the semiconductor photocatalyst. TiO2, on the oxidation of the photogenerated LMB by ambient O-2. Furthermore, by increasing the level of platinum loading, recovery times can be decreased further; e.g. a Pt-TiO2/MB/glycerol/SPS oxygen indicator with platinum level of 1.52 wt% recovers fully within 12 h. A study of the kinetics of recovery as a function of film thickness revealed the recovery step is not controlled by the diffusion of O-2 through the film, but instead dependent upon the slow rate of oxidation of LMB to MB by O-2 in the low dielectric polymer encapsulation medium. Other work showed this recovery is only moderately dependant upon temperatures above -10 degrees C and very sensitive to relative humidity above 30% RH. Potential uses of this UV light activated indicator are discussed briefly. (C) 2011 Elsevier B.V. All rights reserved.

Novel photocatalyst-based colourimetric indicator for oxygen

The preparation and characterisation of a novel, UV-activated solvent-based, colourimetric indicator for O-2 is described, comprising a redox dye (methylene blue, MB), semiconductor photocatalyst (TiO2), and a sacrificial electron donor (SED), all dispersed/dissolved in a polymer medium (sulfonated polystyrene, SPS). Upon exposure, the indicator is readily photobleached as the MB is converted into its oxygen-sensitive, leuco form, LMB. Unlike its water-based counterpart, the recovery of the original colour is very slow (ca. 5 days cf. 6 min), probably due to the largely hydrophobic nature of the polymer encapsulation medium. The kinetics of film photobleaching appear to fit very well, in terms of: irradiance, [TiO2] and [MB], to the usual Langmuir-Hinshelwood type equation associated with a photocatalytic process. The glycerol appears not only to function as a SED, but also a plasticizer and medium for dye dissolution. The kinetics of colour recovery of the photobleached film appear directly dependent upon the ambient level of O-2 but shows a more complex dependence upon the relative humidity, RH. The photobleached film does not recover any of its colour over a 24 h period if the RH

Water-based colourimetric optical indicators for the detection of carbon dioxide

Water-based colourimetric indicator films are shown to have increased operational lifetimes under ambient conditions compared to similar solvent-based counterparts. The response and sensitivity characteristics of a water-based, carbon dioxide-responsive ink are characterised and compared and contrasted to those of a similar solvent-based indicator. The changes in the response characteristics of the ink as a function of the amount of base (sodium hydrogen carbonate) and plasticizer (glycerol) contained in the ink are reported, as are the effects of varying ambient temperature and humidity. The ink is incorporated into a felt tip pen and applied to a number of different substrates, producing a distinct, reversible colour change on all tested surfaces, when a sufficient level of carbon dioxide is present. The possible application of the indicator is discussed briefly.

A viologen-based UV indicator and dosimeter

A UV indicator/dosimeter based on benzyl viologen (BV2+) encapsulated in polyvinyl alcohol (PVA) is described. Upon exposure to UV light, the BV2+/PVA film turns a striking purple colour due to the formation of the cation radical, BV center dot+. The usual oxygen sensitivity of BV center dot+ is significantly reduced due to the very low oxygen permeability of the encapsulating polymer, PVA. Exposure of a typical BV2+/PVA film, for a set amount of time, to UVB light with different UV indices produces different levels of BV center dot+, as measured by the absorbance of the film at 550 nm. A plot of the change in absorbance at this wavelength, Delta Abs(550), as a function of UV index, UVI, produces a linear calibration curve which allows the film to be used as a UVB indicator, and a similar procedure could be employed to allow it to be used as a solar UVI indicator. A typical BV2+/PVA film generates a significant, semi-permanent (stable for > 24 h) saturated purple colour (absorbance similar to 0.8-0.9) upon exposure to sunlight equivalent to a minimal erythemal dose associated with Caucasian skin, i.e. skin type II. The current drawbacks of the film and the possible future use of the BV2+/PVA film as a personal solar UV dosimeter for all skin types are briefly discussed.

Molecular Logic Gates and Luminescent Sensors Based on Photoinduced Electron Transfer

The competition between Photoinduced electron transfer (PET) and other de-excitation pathways such as fluorescence and phosphorescence can be controlled within designed molecular structures. Depending on the particular design, the resulting optical output is thus a function of various inputs such as ion concentration and excitation light dose. Once digitized into binary code, these input-output patterns can be interpreted according to Boolean logic. The single-input logic types of YES and NOT cover simple sensors and the double- (or higher-) input logic types represent other gates such as AND and OR. The logic-based arithmetic processors such as half-adders and half-subtractors are also featured. Naturally, a principal application of the more complex gates is in multi-sensing contexts.

On the mechanism of the inhibition of transducin function by farnesylcysteine analogs

The gamma subunits of heterotrimeric G proteins are isoprenylated/methylated on their carboxy termini. The photoreceptor G protein, transducin, is farnesylated/methylated at this position. Since the isoprenyl group is required for G protein function, it is of great interest to determine the mechanism by which the farnesyl group of T gamma interacts with the other transducin subunits and/or the activated photoreceptor, rhodopsin. Farnesylcysteine derivatives (N-acetyl-S-farnesyl-L-cysteine and farnesylated peptides) have been previously shown to have effects on transducin activity at high concentrations. Here, an extensive survey is done of farnesylcysteine analogs and other lipid molecules, which are tested for their ability to inhibit GTP/GDP exchange in transducin catalyzed by photolyzed rhodopsin. These studies are carried out to determine the nature of the inhibition process. While it does not appear that these molecules exhibit the specificity which would characterize a ligand-receptor type mechanism, the results suggest that these compounds are not acting in a nonspecific detergent-like manner either. The most likely mode of action of farnesylcysteine analogs is that they interfere with the lipid-lipid based association of T alpha and T beta gamma through the lipid modifications present on each subunit.

Interfacial Properties of LiTFSI and LiPF6-Based Electrolytes in Binary and Ternary Mixtures of Alkylcarbonates on Graphite Electrodes and Celgard Separator

For a better understanding of the adsorption behavior of alkylcarbonate-based electrolytes on graphite electrodes and Celgard separator for Li-ion batteries applications, the interface parameters are determined by contact angle and surface tension measurements. The correlation between these parameters and chemical compositions made of alkyl carbonate with a varying nature of lithium salts (LiPF6 and LiTFSI) and volume fractions of binary and ternary mixtures containing propylene carbonate (PC), ethylene carbonate (EC), and dimethyl carbonate (DMC) is investigated. From the obtained contact angle and surface tension (?L) values for each liquid, the dispersive and polar components of the surface tension (?Ld and ?Lp) of the electrolyte and interfacial free energy between the solid and liquid (?SL) were then calculated using the Young’s equation. The variation of contact angle (?) and the surface tension, as well as the work of adhesion (WA) of binary PC/DMC mixtures on PP, PE, and PET model surfaces were also measured and commented as function of volume fraction of PC in DMC. Finally, the Zisman’s critical surface tension (?C) for studied surfaces was then obtained showing positives slopes of cos ? versus ?L. This behavior is explained by a relative higher adsorption of alkylcarbonates to the hydrogenated supports or graphite. These results are decisive to understand the performance of electrolyte/electrode material/separator interfaces in lithium-ion battery devices.

Low pressure carbon dioxide solubility in pure electrolyte solvents for lithium-ion batteries as a function of temperature. Measurement and prediction

Experimental values for the carbon dioxide solubility in eight pure electrolyte solvents for lithium ion batteries – such as ethylene carbonate (EC), propylene carbonate (PC), dimethyl carbonate (DMC), ethyl methyl carbonate (EMC), diethyl carbonate (DEC), ?-butyrolactone (?BL), ethyl acetate (EA) and methyl propionate (MP) – are reported as a function of temperature from (283 to 353) K and atmospheric pressure. Based on experimental solubility data, the Henry’s law constant of the carbon dioxide in these solvents was then deduced and compared with reported values from the literature, as well as with those predicted by using COSMO-RS methodology within COSMOthermX software and those calculated by the Peng–Robinson equation of state implemented into Aspen plus. From this work, it appears that the CO2 solubility is higher in linear carbonates (such as DMC, EMC, DEC) than in cyclic ones (EC, PC, ?BL). Furthermore, the highest CO2 solubility was obtained in MP and EA solvents, which are comparable to the solubility values reported in classical ionicliquids. The precision and accuracy of the experimental values, considered as the per cent of the relative average absolute deviations of the Henry’s law constants from appropriate smoothing equations and from literature values, are close to (1% and 15%), respectively. From the variation of the Henry’s law constants with temperature, the partial molar thermodynamic functions of dissolution such as the standard Gibbs free energy, the enthalpy, and the entropy are calculated, as well as the mixing enthalpy of the solvent with CO2 in its hypothetical liquid state.

XOR-based hash functions

Bank conflicts can severely reduce the bandwidth of an interleaved multibank memory and conflict misses increase the miss rate of a cache or a predictor. Both occurrences are manifestations of the same problem: Objects which should be mapped to different indices are accidentally mapped to the same index. Suitable chosen hash functions can avoid conflicts in each of these situations by mapping the most frequently occurring patterns conflict-free. A particularly interesting class of hash functions are the XOR-based hash functions, which compute each set index bit as the exclusive-or of a subset of the address bits. When implementing an XOR-based hash function, it is extremely important to understand what patterns are mapped conflict-free and how a hash function can be constructed to map the most frequently occurring patterns without conflicts. Hereto, this paper presents two ways to reason about hash functions: by their null space and by their column space. The null space helps to quickly determine whether a pattern is mapped conflict-free. The column space is more useful for other purposes, e. g., to reduce the fan-in of the XOR-gates without introducing conflicts or to evaluate interbank dispersion in skewed-associative caches. Examples illustrate how these ideas can be applied to construct conflict-free hash functions.

An efficient particle filter for color-based tracking in complex scenes

In this paper, we introduce an efficient method for particle selection in tracking objects in complex scenes. Firstly, we improve the proposal distribution function of the tracking algorithm, including current observation, reducing the cost of evaluating particles with a very low likelihood. In addition, we use a partitioned sampling approach to decompose the dynamic state in several stages. It enables to deal with high-dimensional states without an excessive computational cost. To represent the color distribution, the appearance of the tracked object is modelled by sampled pixels. Based on this representation, the probability of any observation is estimated using non-parametric techniques in color space. As a result, we obtain a Probability color Density Image (PDI) where each pixel points its membership to the target color model. In this way, the evaluation of all particles is accelerated by computing the likelihood p(z|x) using the Integral Image of the PDI.

Non-parametric nonlinear system identification: A data-driven orthogonal basis function approach

In this paper, a data driven orthogonal basis function approach is proposed for non-parametric FIR nonlinear system identification. The basis functions are not fixed a priori and match the structure of the unknown system automatically. This eliminates the problem of blindly choosing the basis functions without a priori structural information. Further, based on the proposed basis functions, approaches are proposed for model order determination and regressor selection along with their theoretical justifications. © 2008 IEEE.

High-pressure volumetric properties of imidazolium-based ionic liquids: Effect of the anion

The densities of five imidazolium-based ionic liquids (ILs) (1-butyl-3-methylimidazolium tetrafluoroborate, [CiC4-Im][BF 4]; 1-butyl-3-methylimidazolium hexafluorophosphate, [CiC 4Im][PF6]; 1-butyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyl}imide, [C1C4Im][Tf 2N]; 1-ethyl-3-methylimidazoliumbis{(trifluoromethyl)sulfonyl}-imide, [C1C2Im][Tf2N]; l-ethyl-3-methylimidazolium ethylsulfate, [C1C2Im][EtSO4]) were measured as a function of temperature from (293 to 415) K and over an extended pressure range from (0.1 to 40) MPa using a vibratingtube densimeter. Knowledge of the variation of the density with temperature and pressure allows access to the mechanical coefficients: thermal expansion coefficient and isothermal compressibility. The effects of the anion and of the length of the alkyl chain on the imidazolium ring on the volumetric properties were particularly examined. The mechanical coefficients were compared with those of common organic solvents, water and liquid NaCl. Finally, a prediction model, based on an "ideal" volumetric behavior of the ILs, is proposed to allow calculation of the molar volume of imidazolium-based ionic liquids as a function of temperature. ©2007 American Chemical Society.