Membrane potentials, oxidative stress and the dispersal response of bacterial biofilms to 405 nm light.

The majority of chronic infections are caused by biofilms, which have higher levels of antibiotic resistance than planktonic growth. Violet-blue 405 nm light has recently emerged as a novel bactericide, but limited studies have been conducted on its effectiveness against biofilms. We found that in response to 405 nm light both Pseudomonas aeruginosa and Bacillus subtilis biofilms exhibited cell dispersal and membrane potential hyperpolarisations. The response to 405 nm light depended on the stage of biofilm growth. The use of reactive oxygen species scavengers reduced membrane hyperpolarisation and biofilm dispersal in response to 405 nm light. This is the first time that membrane potential hyperpolarisations have been linked with photooxidative stress in bacteria and with biofilm dispersal. These results provide a new insight into the role of membrane potentials in the bacterial stress response and could be used in the development of 405 nm light based biofilm treatments.

Microrheology and Spatial Heterogeneity of Staphylococcus aureus Biofilms Modulated by Hydrodynamic Shear and Biofilm-Degrading Enzymes.

Particle tracking microrheology was used to investigate the viscoelasticity of Staphylococcus aureus biofilms grown in microfluidic cells at various flow rates and when subjected to biofilm-degrading enzymes. Biofilm viscoelasticity was found to harden as a function of shear rate but soften with increasing height away from the attachment surface in good agreement with previous bulk results. Ripley's K-function was used to quantify the spatial distribution of the bacteria within the biofilm. For all conditions, biofilms would cluster as a function of height during growth. The effects of proteinase K and DNase-1 on the viscoelasticity of biofilms were also investigated. Proteinase K caused an order of magnitude change in the compliances, softening the biofilms. However, DNase-1 was found to have no significant effects over the first 6 h of development, indicating that DNA is less important in biofilm maintenance during the initial stages of growth. Our results demonstrate that during the preliminary stages of Staphylococcus aureus biofilm development, column-like structures with a vertical gradient of viscoelasticity are established and modulated by the hydrodynamic shear caused by fluid flow in the surrounding environment. An understanding of these mechanical properties will provide more accurate insights for removal strategies of early-stage biofilms.

Optical coherence tomography velocimetry of colloidal suspensions.

Optical coherence tomography velocimetry combined with a rheometer and optical modulation techniques provides increased sensitivity to the low shear rate motion of complex fluid systems. Optical modulation coupled with a new interferometer design yields improved signal to noise ratios and is demonstrated with optically opaque colloidal suspensions. Thus the measurable range of shear velocities with complex fluids can be as low as ∼40 µm s(-1), more than an order of magnitude improvement on the previous lower limit of ∼700 µm s(-1). Furthermore the apparatus demonstrates improved sensitivity to the measurement of velocity. The instrument was used to study two hard sphere colloidal systems, sterically stabilized PVP spheres of 1 µm radius and sterically stabilized polystyrene spheres of 600 nm radius, which display shear banding behavior due to shear induced concentration gradients. OCT velocimetry also allows the velocity fluctuations of the system to be quantified as a function of the distance across the rheometer gap to help classify underlying unsteady or turbulent phenomena.

Electronics of peptide- and protein-based biomaterials.

Biologically inspired peptide- and protein-based materials are at the forefront of organic bioelectronics research due to their inherent conduction properties and excellent biocompatibility. Peptides have the advantages of structural simplicity and ease of synthesis providing credible prospects for mass production, whereas naturally expressed proteins offer inspiration with many examples of high performance evolutionary optimised bioelectronics properties. We review recent advances in the fundamental conduction mechanisms, experimental techniques and exemplar applications for the bioelectronics of self-assembling peptides and proteins. Diverse charge transfer processes, such as tunnelling, hopping and coupled transfer, are found in naturally occurring biological systems with peptides and proteins as the predominant building blocks to enable conduction in biology. Both theory and experiments allow detailed investigation of bioelectronic properties in order to design functionalized peptide- and protein-based biomaterials, e.g. to create biocompatible aqueous electrodes. We also highlight the design of bioelectronics devices based on peptides/proteins including field-effect transistors, piezoelectric energy harvesters and optoelectronics.

Spatial propagation of electrical signals in circular biofilms: A combined experimental and agent-based fire-diffuse-fire study.

Bacterial biofilms are a risk to human health, playing critical roles in persistent infections. Recent studies have observed electrical signaling in biofilms and thus biofilms represent a new class of active excitable matter in which cell division is the active process and the spiking of the individual bacterial cells is the excitable process. Electrophysiological models have predominantly been developed to describe eukaryotic systems, but we demonstrate their use in understanding bacterial biofilms. Our agent-based fire-diffuse-fire (ABFDF) model successfully simulates the propagation of both centrifugal (away from the center) and centripetal (toward the center) electrical signals through biofilms of Bacillus subtilis. Furthermore, the ABFDF model allows realistic spatial positioning of the bacteria in two dimensions to be included in the fire-diffuse-fire model and this is the crucial factor that improves agreement with experiments. The speed of propagation is not constant and depends on the radius of the propagating electrical wave front. Centripetal waves are observed to move faster than centrifugal waves, which is a curvature driven effect and is correctly captured by our simulations.

The self-assembly, elasticity, and dynamics of cardiac thin filaments.

Solutions of intact cardiac thin filaments were examined with transmission electron microscopy, dynamic light scattering (DLS), and particle-tracking microrheology. The filaments self-assembled in solution with a bell-shaped distribution of contour lengths that contained a population of filaments of much greater length than the in vivo sarcomere size ( approximately 1 mum) due to a one-dimensional annealing process. Dynamic semiflexible modes were found in DLS measurements at fast timescales (12.5 ns-0.0001 s). The bending modulus of the fibers is found to be in the range 4.5-16 x 10(-27) Jm and is weakly dependent on calcium concentration (with Ca2+ > or = without Ca2+). Good quantitative agreement was found for the values of the fiber diameter calculated from transmission electron microscopy and from the initial decay of DLS correlation functions: 9.9 nm and 9.7 nm with and without Ca2+, respectively. In contrast, at slower timescales and high polymer concentrations, microrheology indicates that the cardiac filaments act as short rods in solution according to the predictions of the Doi-Edwards chopsticks model (viscosity, eta approximately c(3), where c is the polymer concentration). This differs from the semiflexible behavior of long synthetic actin filaments at comparable polymer concentrations and timescales (elastic shear modulus, G' approximately c(1.4), tightly entangled) and is due to the relative ratio of the contour lengths ( approximately 30). The scaling dependence of the elastic shear modulus on the frequency (omega) for cardiac thin filaments is G' approximately omega(3/4 +/- 0.03), which is thought to arise from flexural modes of the filaments.

Elastic turbulence in entangled semi-dilute DNA solutions measured with optical coherence tomography velocimetry.

The flow instabilities of solutions of high molecular weight DNA in the entangled semi-dilute concentration regime were investigated using optical coherence tomography velocimetry, a technique that provides high spatial (probe volumes of 3.4 pL) and temporal resolution (sub µs) information on the flow behaviour of complex fluids in a rheometer. The velocity profiles of the opaque DNA solutions (high and low salt) were measured as a function of the distance across the gap of a parallel plate rheometer, and their evolution over time was measured. At lower DNA concentrations and low shear rates, the velocity fluctuations were well described by Gaussian functions and the velocity gradient was uniform across the rheometer gap, which is expected for Newtonian flows. As the DNA concentration and shear rate were increased there was a stable wall slip regime followed by an evolving wall slip regime, which is finally followed by the onset of elastic turbulence. Strain localization (shear banding) is observed on the boundaries of the flows at intermediate shear rates, but decreases in the high shear elastic turbulence regime, where bulk strain localization occurs. A dynamic phase diagram for non-linear flow was created to describe the different behaviours.

Microrheology of polymeric solutions using x-ray photon correlation spectroscopy.

We demonstrate the technique of XPCS microrheology on opaque polymeric solutions (1-20% w/w) using colloidal silica probes. The short time decay of the intensity correlation function provides the mean square displacement (MSD) of the colloidal probes. The MSDs of the probes are subsequently transformed using the generalized Stokes-Einstein equation, allowing the linear viscoelastic spectra of a biopolymer (gellan) and a synthetic polyelectrolyte (polystyrene sulfonate, PSS) to be calculated over two decades of frequency. MSDs can be measured that are two orders of magnitude smaller than those possible with video particle tracking microrheology, with a sensitivity of ∼10 nm s(-1) for displacements of ∼nms. The XPCS data for water, glycerol and PSS combs are in agreement with video particle tracking microrheology experiments performed at lower polymer concentrations.

The phase transformations in starch during gelatinisation: a liquid crystalline approach.

The analogy between starch and a chiral side-chain polymeric liquid crystal is examined in relation to the processes involved during gelatinisation. There are three important parameters for characterisation of the molecular phase behaviour of the amylopectin: the lamellar order parameter (psi), the orientational order parameter of the amylopectin double helices (phi), and the helicity of the sample (h, the helix/coil ratio, a measure of the helix-coil transition of the double helices). The coupling between the double helices and the backbone through the flexible spacers is affected dramatically by the water content and it is this factor which dictates the particular phase adopted by the amylopectin inside the starch granule as a function of temperature. SAXS, WAXS and 13C CP/MAS NMR are used to examine these phenomena in excess water. Furthermore, previous experimental evidence pertaining to the limiting water case is reviewed with respect to this new theoretical framework.

Optical coherence tomography velocimetry in controlled shear flow.

Doppler-shift optical coherence tomography with infrared light was used to probe the velocity profiles of concentrated solutions of complex fluids with samples experiencing steady-state shear flow. The apparatus is sensitive to a velocity range of 0.7-330 mm/s probing very small volumes of material (quasicylindrical volume elements of 9-µm length and 11-µm radius with 3.4-picoliter volumes) inside a plate-plate rheometer with a total sample volume of ~100-1000 µL. The technique can scan the flow in the plane perpendicular to the shear direction, building up a two-dimensional map of the velocity flow field. The use of a coherence gate with a broad-band infrared source (9-µm coherence length, 1300-nm wavelength) allows opaque specimens, such as concentrated colloidal suspensions (2% w/w) and margarine, to be probed. We observe the phenomena of wall slip (margarine) and shear banding (polyacrylamide, a linear flexible polyelectrolyte) using this technique.

The viscoelasticity of self-assembled proteoglycan combs.

Particle tracking microrheology with a fast digital camera allowed the slow and intermediate time regimes (10(-4)-10(1) s) of the linear viscoelasticity of giant aggrecan proteoglycans to be mapped. Combined with diffusing wave spectroscopy experiments this enabled us to probe the linear viscoelasticity of aggrecan over seven orders of magnitude in time (10(-6)-10(1) s) [Palmer et al., Biophys. J., 1999, 76, 1063; Papagiannopoulos et al., Biomacromolecules, 2007, 7, 2162]. When the comb side-groups self-assemble on the hyaluronic acid backbones they cause a dramatic increase in the relaxation time of the solutions and consequently the viscosity of the sample, but leave the elasticity of the solutions relatively unchanged. The experiments illustrate the modular nature of aggrecan's viscosity and clearly demonstrate the role of this molecule in vivo in cartilaginous composites, where it dissipates energy. Both one- and two-particle tracking microrheology were used to investigate the length-scale dependent viscoelasticity of the comb superstructures [Lui et al., Phys. Rev. Lett., 2006, 96, 118104] and the errors inherent in the two techniques were quantified [Savin and Doyle, Biophys. J., 2005, 88, 623; Waigh, Rep. Prog. Phys., 2005, 68, 685]. The behaviour of the viscoelasticity is compared with the predictions of dynamic scaling theory, indicating a significant contribution of the side-chain dynamics to the reptative motion of both the aggrecan aggregate and the monomers. The results have important implications for a molecular understanding of tissue function and pathology in osteoarthritis.

Microrheology of bacterial biofilms in vitro: Staphylococcus aureus and Pseudomonas aeruginosa.

The rheology of bacterial biofilms at the micron scale is an important step to understanding the communal lifecycles of bacteria that adhere to solid surfaces, as it measures how they mutually adhere and desorb. Improvements in particle-tracking software and imaging hardware have allowed us to successfully employ particle-tracking microrheology to measuring single-species bacterial biofilms, based on Staphlococcus aureus and Pseudomonas aeruginosa. By tracking displacements of the cells at a range of timescales, we separate active and thermal contributions to the cell motion. The S. aureus biofilms in particular show power-law rheology, in common with other dense colloidal suspensions. By calculating the mean compliance of S. aureus biofilms, we observe them becoming less compliant during growth, and more compliant during starvation. The biofilms are rheologically inhomogeneous on the micron scale, as a result of the strength of initial adhesion to the flow cell surface, the arrangement of individual bacteria, and larger-scale structures such as flocs of P. aeruginosa. Our S. aureus biofilms became homogeneous as a function of height as they matured: the rheological environment experienced by a bacterium became independent of how far it lived from the flow cell surface. Particle-tracking microrheology provides a quantitative measure of the "strength" of a biofilm. It may therefore prove useful in identifying drug targets and characterizing the effect of specific molecular changes on the micron-scale rheology of biofilms.

Solution structure and dynamics of cartilage aggrecan.

We studied the structure and dynamics of porcine laryngeal aggrecan in solution using a range of noninvasive techniques: dynamic light scattering (DLS), small-angle neutron scattering (SANS), video particle tracking (VPT) microrheology, and diffusing wave spectroscopy (DWS). The data are analyzed within the framework of a combined static and dynamic scaling model, and evidence is found for reptation of the comb backbones with unentangled side-chain dynamics. Small-angle neutron scattering indicated standard polyelectrolyte scaling of the mesh size (xi) with concentration (c) in semidilute solutions for the whole aggrecan aggregate, xi = Ac(-0.47+/-0.04), with the prefactor (A) implying there is on average 60 nm between the aggrecan subunits along the backbone. VPT demonstrated large exponents for the power law dependence of the intrinsic viscosity (eta) on the polymer concentration in the semidilute concentration regime, eta approximately c(alpha); with alpha equal to 2.04 +/- 0.06 and 1.95 +/- 0.08 for the assembled and disassembled aggrecan aggregates, respectively. DWS at high frequencies (10(4)-10(5) Hz) gave evidence for internal Rouse modes of the aggrecan monomers, independent of the degree of self-assembly of the molecules.

The microrheology of polystyrene sulfonate combs in aqueous solution.

Video particle tracking (VPT) and diffusing wave spectroscopy were used to characterize the microrheology of polystyrene sulfonate combs in aqueous solutions. At low frequencies VPT demonstrated predominantly viscous behavior. The manner in which the viscosity scaled as a function of monomer concentration was a sensitive function of the comb architecture. Densely branched combs or combs with long side chains demonstrated entangled polyelectrolyte scaling above the overlap concentration, whereas sparsely branched combs had unentangled polyelectrolyte scaling. A dynamic scaling model was developed for the viscosity of unentangled semidilute solutions of comb polyelectrolytes. Diffusing wave spectroscopy demonstrated Rouse modes (G' approximately G" approximately omega12) for the high-frequency dynamics of the semidilute comb solutions. The form of the high-frequency viscoelasticity was independent of the chain architecture and the modulus scaled as expected for linear flexible polyelectrolytes.

The internal dynamic modes of charged self-assembled peptide fibrils.

Photon correlation spectroscopy is used to study the internal dynamics of self-assembled charged peptide fibrils. Short neutral and charged polymeric aggregates have diffusive modes due to whole macromolecular motion. For long semiflexible fibrils the logarithm of the intermediate scattering function follows a q(2)t(3/4) scaling at long times consistent with a Kratky-Porod free energy and preaveraged Oseen hydrodynamics. Persistence lengths on the order of micrometers are calculated for the peptide fibrils consistent with estimates from the liquid-crystalline phase behavior. Fibril diameters (5-35 nm) calculated from the initial decay of the correlation functions are in agreement with transmission electron microscopy measurements.

Dynamic light scattering study of the dynamics of a gelled polymeric micellar system.

The dynamics of the E(92)B(18)/water system are studied by dynamic light scattering (DLS) in the liquid, soft gel, and hard gel phases. Both the liquid and the soft gel phases are micellar phases, although the structural order is higher in the soft gel phase than in the liquid phase. The hard gel phase corresponds to a face-centered cubic arrangement of micelles. DLS results show that the dilute liquid phase is characterized by a single characteristic time tau(1) associated with the diffusion of the micelles. In addition, a second characteristic time tau(2) associated with the presence of micellar clusters in the system is identified in the concentrated liquid and in the soft gel phases. According to these results, DLS suggests that the structure of the soft gel phase comprises micellar clusters coexisting with micellar fluid, in good agreement with hypotheses from our previous work. The dynamics of the system slows down as the hard gel phase is approached and a plateau is observed in the DLS correlation function. The structure of the hard gel is "softened" upon increasing temperature and/or decreasing concentration.