A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Investigation of flux in flat-plate modules for membrane distillation

A theoretical model for predicting the behaviour of membrane distillation by incorporating mass and heat transfer equations has been used to find permeate fluxes, and has been validated experimentally. The model accurately predicts mass and heat transfer. The main work studied the effect of module design using a flat-plate module in laminar flow conditions. Areas of investigation included the use of channels across the membrane surface, decreasing the available membrane surface area, and widening the inlet and outlet channels. The work showed that widening the channels increased the flux. Increased flux was also obtained by the use of channels on the permeate side, though not on the feed side.

Acoustic scattering by an inhomogeneous layer on a rigid plate

The problem of scattering of time-harmonic acoustic waves by an inhomogeneous fluid layer on a rigid plate in R2 is considered. The density is assumed to be unity in the media: within the layer the sound speed is assumed to be an arbitrary bounded measurable function. The problem is modelled by the reduced wave equation with variable wavenumber in the layer and a Neumann condition on the plate. To formulate the problem and prove uniqueness of solution a radiation condition appropriate for scattering by infinite rough surfaces is introduced, a generalization of the Rayleigh expansion condition for diffraction gratings. With the help of the radiation condition the problem is reformulated as a system of two second kind integral equations over the layer and the plate. Under additional assumptions on the wavenumber in the layer, uniqueness of solution is proved and the nonexistence of guided wave solutions of the homogeneous problem established. General results on the solvability of systems of integral equations on unbounded domains are used to establish existence and continuous dependence in a weighted norm of the solution on the given data.

Interaction of adult human neural crest-derived stem cells with a nanoporous titanium surface is sufficient to induce their osteogenic differentiation

Osteogenic differentiation of various adult stem cell populations such as neural crest-derived stem cells is of great interest in the context of bone regeneration. Ideally, exogenous differentiation should mimic an endogenous differentiation process, which is partly mediated by topological cues. To elucidate the osteoinductive potential of porous substrates with different pore diameters (30 nm, 100 nm), human neural crest-derived stem cells isolated from the inferior nasal turbinate were cultivated on the surface of nanoporous titanium covered membranes without additional chemical or biological osteoinductive cues. As controls, flat titanium without any topological features and osteogenic medium was used. Cultivation of human neural crest-derived stem cells on 30 nm pores resulted in osteogenic differentiation as demonstrated by alkaline phosphatase activity after seven days as well as by calcium deposition after 3 weeks of cultivation. In contrast, cultivation on flat titanium and on membranes equipped with 100 nm pores was not sufficient to induce osteogenic differentiation. Moreover, we demonstrate an increase of osteogenic transcripts including Osterix, Osteocalcin and up-regulation of Integrin β1 and α2 in the 30 nm pore approach only. Thus, transplantation of stem cells pre-cultivated on nanostructured implants might improve the clinical outcome by support of the graft adherence and acceleration of the regeneration process.