Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use.

The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg-1 to 13,000 µg STX equiv. kg-1 by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg-1 on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg-1) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.

Molecular characterization of water and surfactant AOT at nanoemulsion surfaces.

Nanoemulsions and microemulsions are environments where oil and water can be solubilized in one another to provide a unique platform for many different biological and industrial applications. Nanoemulsions, unlike microemulsions, have seen little work done to characterize molecular interactions at their surfaces. This study provides a detailed investigation of the near-surface molecular structure of regular (oil in water) and reverse (water in oil) nanoemulsions stabilized with the surfactant dioctyl sodium sulfosuccinate (AOT). Vibrational sum-frequency scattering spectroscopy (VSFSS) is used to measure the vibrational spectroscopy of these AOT stabilized regular and reverse nanoemulsions. Complementary studies of AOT adsorbed at the planar oil-water interface are conducted with vibrational sum-frequency spectroscopy (VSFS). Jointly, these give comparative insights into the orientation of interfacial water and the molecular characterization of the hydrophobic and hydrophilic regions of AOT at the different oil-water interfaces. Whereas the polar region of AOT and surrounding interfacial water molecules display nearly identical behavior at both the planar and droplet interface, there is a clear difference in hydrophobic chain ordering even when possible surface concentration differences are taken into account. This chain ordering is found to be invariant as the nanodroplets grow by Ostwald ripening and also with substitution of different counterions (Na:AOT, K:AOT, and Mg:AOT) that consequently also result in different sized nanoparticles. The results paint a compelling picture of surfactant assembly at these relatively large nanoemulsion surfaces and allow for an important comparison of AOT at smaller micellar (curved) and planar oil-water interfaces.

Kinetics and intermediates of the reaction of fully reduced Escherichia coli bo3 ubiquinol oxidase with O2.

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 µs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 µs, 30 µs, 42 µs, 470 µs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 µs O2 binding (3.8 × 10(7) M(-1) s(-1)), producing compound A, followed by faster (22 µs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 µs), producing the F intermediate and semiquinone. In the 470 µs step, the o3 F state is converted into the o3(3+) oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.