Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes

The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.     

Role of the shape of various bacteria in their separation by Microthermal Field-Flow Fractionation

The steady-state movement of the spherical and non-spherical particles, such as prolate or oblate rotational ellipsoids, cylinders, or parallelepipeds, suspended in a liquid and exposed to a unidirectional temperature gradient, is analyzed theoretically. The differences in the ratios of the rotational to translational diffusion coefficients of the non-spherical to spherical particles, the heterogeneity of thermal conductivity of the particle body, and the heterogeneity in surface chemical nature make possible to separate the particles according to differences in shape. Preliminary experimental separations of Gram-positive and Gram-negative, nearly spherical and rod-shaped bacteria performed by Microthermal Field-Flow Fractionation confirmed that the fractionation of the cells according to differences in shape is possible.     

Affinity capillary electrophoresis and density functional theory employed for the characterization of hexaarylbenzene-based receptor complexation with alkali metal ions

In this study, affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations were combined to investigate non-covalent binding interactions between the hexaarylbenzene-based receptor (R) and alkali metal ions, Rb(+) and Cs(+) , in methanol. The apparent binding (stability) constants (K(b) ) of the complexes of receptor R with alkali metal ions in the methanolic medium were determined by ACE from the dependence of effective electrophoretic mobility of the receptor R on the concentration of Rb(+) and Cs(+) ions in the BGE using a non-linear regression analysis. The receptor R formed relatively strong complexes both with rubidium (log K(b) =4.04±0.21) and cesium ions (log K(b) =3.72±0.22). The structural characteristics of the above alkali metal ion complexes with the receptor R were described by ab initio density functional theory calculations. These calculations have shown that the studied cations bind to the receptor R because they synergistically interact with the polar ethereal fence and with the central benzene ring via cation-π interaction.     

Magnetic and electrokinetic manipulations on a microchip device for bead-based immunosensing applications

The combination of electrophoretic and magnetic manipulations with electrochemical detection for a versatile microfluidic and bead-based biosensing application is demonstrated. Amperometric detection is performed in an off-channel setup by means of a voltammetric cell built at the microchannel outlet and using a gold working electrode. Superparamagnetic particles are introduced and handled inside the channel by means of an external permanent magnet in combination with the electrogenerated flow which allows reproducible loading. The specific detection of phenol as electroactive alkaline phosphatase product is used in this study as proof of concept for a sensitive protein quantification. Characterizations and optimization of different parameters have been carried out in order to achieve the best detection signal. The applicability of the device has been finally demonstrated by the detection of rabbit IgG as model protein after an immunoassay performed on magnetic particles as immobilization platform. A comparison between the electrochemical detection using the developed device and the optical standard detection revealed similar performances with, however, extremely lower amount of reagent used and shorter analysis time. The developed electrophoretic- and magnetic-based chip may open the way to several other biosensing applications with interest not only for other proteins but also for DNA analysis, cell counting, and environmental control.     

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.     

High-performance hydrogen production and oxidation electrodes with hydrogenase supported on metallic single-wall carbon nanotube networks

We studied the electrocatalytic activity of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaH2ase) immobilized on single-wall carbon nanotube (SWNT) networks. SWNT networks were prepared on carbon cloth by ultrasonic spraying of suspensions with predetermined ratios of metallic and semiconducting nanotubes. Current densities for both proton reduction and hydrogen oxidation electrocatalytic activities were at least 1 order of magnitude higher when hydrogenase was immobilized onto SWNT networks with high metallic tube (m-SWNT) content in comparison to hydrogenase supported on networks with low metallic tube content or when SWNTs were absent. We conclude that the increase in electrocatalytic activities in the presence of SWNTs was mainly due to the m-SWNT fraction and can be attributed to (i) substantial increases in the active electrode surface area, and (ii) improved electronic coupling between CaH2ase redox-active sites and the electrode surface.     

Resonant inelastic x-ray scattering at the limit of subfemtosecond natural lifetime

We present measurements of the resonant inelastic x-ray scattering (RIXS) spectra of the CH(3)I molecule in the hard-x-ray region near the iodine L(2) and L(3) absorption edges. We show that dispersive RIXS spectral features that were recognized as a fingerprint of dissociative molecular states can be interpreted in terms of ultrashort natural lifetime of ～200 attoseconds in the case of the iodine L-shell core-hole. Our results demonstrate the capacity of the RIXS technique to reveal subtle dynamical effects in molecules with sensitivity to nuclear rearrangement on a subfemtosecond time scale.     

High-throughput age synchronisation of Caenorhabditis elegans

We present a passive microfluidic strategy for sorting adult C. elegans nematodes on the basis of age and size. The separation mechanism takes advantage of phenotypic differences between 'adult' and 'juvenile' organisms and their behaviour in microfluidic architectures. In brief, the microfluidic device allows worms to sort themselves in a passive manner.