A. Toby A. Jenkins

A thin film detection/response system for pathogenic bacteria

This paper describes the modification of nonwoven fabric such that it responds by releasing an encapsulated antimicrobial from within an attached vesicle in response to two species of pathogenic bacteria (Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1), but does not respond to nonpathogenic Escherichia coli DH5alpha. This concept is based on the generalization that a majority of pathogenic bacteria secrete virulence factors such as toxins and lipases that actively damage cell membranes, typically observed as tissue damage around infected wounds, while nonpathogenic bacteria do not (or not at high concentration). The eventual aim of this work is to produce responsive dressings which release antimicrobials and change color only on infected wounds. This paper details preliminary approaches to achieving this goal, including vesicle-bacteria studies in aqueous suspension, and fluorescence imaging of fluorescein containing vesicles lysed by S. aureus and P. aeruginosa, but not by E. coli.

The Development of Boronic Acids as Sensors and Separation Tools

Synthetic receptors for diols that incorporate boronic acid motifs have been developed as new sensors and separation tools. Utilizing the reversible interactions of diols with boronic acids to form boronic esters under new binding regimes has provided new hydrogel constructs that have found use as dye-displacement sensors and electrophoretic separation tools; similarly, molecular boronic-acid-containing chemosensors were constructed that offer applications in the sensing of diols. This review provides a somewhat-personal perspective of developments in boronic-acid-mediated sensing and separation, placed in the context of the seminal works of others in the area, as well as offering a concise summary of the contributions of the co-authors in the area.

Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes

Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimers disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.

Boronate affinity saccharide electrophoresis: A novel carbohydrate analysis tool

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore‐assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5–1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore‐assisted carbohydrate electrophoresis of 2‐aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar‐containing molecules.

Boronic acid dendrimer receptor modified nanofibrillar cellulose membranes

Cellulose nanofibrils from sisal of typically 4–5 nm diameter and ca. 250 ± 100 nm length are reconstituted into thin films of ca. 6 µm thickness (or thicker freestanding films). Pure cellulose and cellulose composite films are obtained in a solvent evaporation process. A boronic acid appended dendrimer is embedded as a receptor in the nanofibrillar cellulose membrane. The number of boronic acid binding sites is controlled by varying the dendrimer content. The electrochemical and spectrophotometric properties of the nanocomposite membrane are investigated using the probe molecule alizarin red S. Pure cellulose membranes inhibit access to the electrode. However, the presence of boronic acid receptor sites allows accumulation of alizarin red S with a Langmuirian binding constant of ca. 6000 ± 1000 M−1. The 2-electron 2-proton reduction of immobilized alizarin red S is shown to occur in a ca. 60 nm zone close to the electrode surface. With a boronic acid dendrimer modified nanofibrillar cellulose composition of 96 wt% cellulose and 4 wt% boronic acid dendrimer, the analytical range for alizarin red S in aqueous acetate buffer pH 3 is approximately 10 µM to 1 mM.

Surface plasmon resonance shows that type IV pili are important in surface attachment by Pseudomonas aeruginosa

Type IV pili have been shown to play a role in the early stages of bacterial biofilm formation, but not in initial bacterial attachment. Here, using the surface analytical technique, surface plasmon resonance (SPR), we follow the attachment of the bacterium Pseudomonas aeruginosa in real time. In contrast to previous studies, we show that type IV pili mutants are defective in attachment. Both mutants lacking pili (pilA), and those possessing an overabundance of pili (pilT), showed reduced SPR measured attachment compared with the wild-type PAO1 strain. Both pil mutants also showed reduced pathogenicity in a model insect host, as measured by percentage mortality after 24 h. SPR revealed differences in the kinetics of attachment between pilA and pilT, differences obscured by endpoint assays using crystal violet stain. These results highlight the power of SPR in monitoring bacterial attachment in real time and also demonstrate an additional role for type IV pili beyond bacterial aggregation and micro-colony formation.

Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins

The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance.

This paper describes how the technique of surface plasmon resonance (SPR) can be utilized to follow (in real time) the attachment of Pseudomonas aeruginosa bacteria on bare gold and gold modified with a self‐assembled monolayer (SAM) of mercaptounadecanoic acid. We show that SPR is able to discriminate between the adsorption of live versus dead (thermally shocked) bacteria. Moreover, the SPR distinguishes between the adsorption of wild‐type versus mutant bacteria (single gene knockouts), the concentration of the bacterial suspension, and between bacteria adsorbing on SAM‐modified and bare gold. SPR is able to measure bacterial adsorption within seconds of the bacterial suspension being introduced. Finally, a qualitative correlation between results from SPR with a crystal violet staining assay for different mutant bacteria was observed.

Ionized gas (plasma) delivery of reactive oxygen species (ROS) into artificial cells

This study was designed to enhance our understanding of how reactive oxygen species (ROS), generated ex situ by ionized gas (plasma), can affect the regulation of signalling processes within cells. A model system, comprising of a suspension of phospholipid vesicles (cell mimics) encapsulating a ROS reporter, was developed to study the plasma delivery of ROS into cells. For the first time it was shown that plasma unequivocally delivers ROS into cells over a sustained period and without compromising cell membrane integrity. An important consideration in cell and biological assays is the presence of serum, which significantly reduced the transfer efficiency of ROS into the vesicles. These results are key to understanding how plasma treatments can be tailored for specific medical or biotechnology applications. Further, the phospholipid vesicle ROS reporter system may find use in other studies involving the application of free radicals in biology and medicine.

Surface plasmon resonance assay for real-time monitoring of somatic coliphages in wastewaters.

ABSTRACT The surface plasmon resonance (SPR) technique is a well-established method for the measurement of molecules binding to surfaces and the quantification of binding constants between surface-immobilized proteins and proteins in solution. In this paper we describe an extension of the methodology to study bacteriophage-bacterium interactions. A two-channel microfluidic SPR sensor device was used to detect the presence of somatic coliphages, a group of bacteriophages that have been proposed as fecal pollution indicators in water, using their host, Escherichia coli WG5, as a target for their selective detection. The bacterium, E. coli WG5, was immobilized on gold sensor chips using avidin-biotin and bacteriophages extracted from wastewater added. The initial binding of the bacteriophage was observed at high concentrations, and a separate, time-delayed cell lysis event also was observed, which was sensitive to bacteriophage at low concentrations. As few as 1 PFU/ml of bacteriophage injected into the chamber could be detected after a phage incubation period of 120 min, which equates to an approximate limit of detection of around 102 PFU/ml. The bacteriophage-bacterium interaction appeared to cause a structural change in the surface-bound bacteria, possibly due to collapse of the cell, which was observed as an increase in mass density on the sensor chip. These results suggest that this methodology could be employed for future biosensor technologies and for quantification of the bacteriophage concentration.