Anthrax toxin-induced rupture of artificial lipid bilayer membranes.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Theory for polymer analysis using nanopore-based single-molecule mass spectrometry.

Nanometer-scale pores have demonstrated potential for the electrical detection, quantification, and characterization of molecules for biomedical applications and the chemical analysis of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theoretical models that incorporate the interactions between chemicals (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) molecules in a single alpha-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a reduction in the mobile cation concentration inside the pore and an increase in the affinity between the polymer and the pore. The model was used to estimate the free energy of formation for K(+)-PEG inside the nanopore (approximately -49.7 meV) and the free energy of PEG partitioning into the nanopore ( approximately 0.76 meV per ethylene glycol monomer). The results suggest that rational, physical models for the analysis of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chemical and biological applications.

Sizing the Bacillus anthracis PA63 channel with nonelectrolyte poly(ethylene glycols).

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA(63)). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus alpha-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (approximately 1400 g/mol). The results suggest that the limiting diameter of the PA(63) pore is <2 nm, which is consistent with an all-atom model of the PA(63) channel and previous experiments using large ions.

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip.

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.

In vitro cytotoxicity of nitric oxide-releasing sol-gel derived materials.

The cytotoxicity of bare and PU-coated nitric oxide (NO)-releasing sol-gel derived materials (sol-gels) was investigated using L929 mouse fibroblasts in both direct and indirect contact models to differentiate between the biological impact of the sol-gel matrix and NO release. The flux of NO was varied up to 150 pmol cm(-2) s(-1) using N-(6-aminohexyl)-aminopropyltrimethoxysilane (balance iso-butyltrimethoxysilane) diazeniumdiolate (NO donor)-modified sol-gels. The addition of a polyurethane (PU) outer membrane greatly improved the stability of the sol-gel matrix without significantly suppressing the NO flux. Direct contact studies demonstrated a cytotoxic effect that was dependent on the aminosilane content of the sol-gel. The use of the thin PU overcoat eliminated this effect. A direct cytotoxicity dependence of NO release for L929 fibroblasts was discovered from indirect contact studies, where 24 h exposure to NO fluxes in excess of 50 pmol cm(-2) s(-1) was cytotoxic.

Nitric oxide-releasing sol-gels as antibacterial coatings for orthopedic implants.

To assess the benefits of nitric oxide (NO)-releasing sol-gels as potential antibacterial coatings for orthopedic devices, medical-grade stainless steel is coated with a sol-gel film of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane. Upon converting the diamine groups in these films to diazeniumdiolate NO donors, the NO release from the sol-gel-coated stainless steel is evaluated at both ambient and physiological temperature. Sol-gel films incubated at 25 degrees C have a lower NO flux over the first 24 h compared to those at 37 degrees C, but release more than five times longer. The bacterial adhesion resistance of NO-releasing coatings is evaluated in vitro by exposing bare steel, sol-gel, and NO-releasing sol-gel-coated steel to cell suspensions of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis at 25 degrees C and 37 degrees C. Cell adhesion to bare and sol-gel-coated steel is similar, while NO-releasing surfaces have significantly less bacterial adhesion for all species and temperatures investigated.

Inhibition of implant-associated infections via nitric oxide release.

The in vivo antibacterial activity of nitric oxide (NO)-releasing xerogel coatings was evaluated against an aggressive subcutaneous Staphylococcus aureus infection in a rat model. The NO-releasing implants were created by coating a medical-grade silicone elastomer with a sol-gel-derived (xerogel) film capable of storing NO. Four of the bare or xerogel-coated silicone materials were subcutaneously implanted into male rats. Ten rats were administered 10 microl of a 10(8) cfuml(-1)S. aureus colony directly into the subcutaneous pocket with the implant prior to wound closure. Infection was quantitatively and qualitatively evaluated after 8d of implantation with microbiological and histological methods, respectively. A 82% reduction in the number of infected implants was achieved with the NO-releasing coating. Histology revealed that the capsule formation around infected bare silicone rubber controls was immunoactive and that a biofilm may have formed. Capsule formation in response to NO-releasing implants had greater vascularity in comparison with uninoculated or untreated controls. These results suggest that NO-releasing coatings may dramatically reduce the incidence of biomaterial-associated infection.

Poly(vinyl chloride)-coated sol-gels for studying the effects of nitric oxide release on bacterial adhesion.

Nitric oxide (NO) releasing sol-gel materials coated with poly(vinyl chloride) (PVC) films exhibit increased stability at ambient and physiological temperatures. The polymer overcoat, however, reduces the NO fluxes by 5-35% over the initial week of release. The variation in NO fluxes between unmodified and PVC-coated sol-gels is negligible after 7 days. The PVC polymeric layer provides controlled surface chemistry for systematic studies of the effects of NO release on bacterial adhesion. As an example, the adhesion of Pseudomonas aeruginosa and Proteus mirabilis at PVC-coated NO-releasing sol-gels is investigated. A direct NO dependence on the reduction of P. aeruginosa adhesion is observed for NO fluxes up to 20 pmol cm(-2) s(-1). Although decreased by 50% in the presence of NO release, P. mirabilis adhesion does not appear to correlate to the flux of NO release. PVC-coated NO-releasing sol-gels may prove useful for studying the effects of localized NO release on other biological and chemical systems.