Data-driven modelling makes quantitative predictions regarding bacteria surface motility.

In this work, we quantitatively compare computer simulations and existing cell tracking data of P. aeruginosa surface motility in order to analyse the underlying motility mechanism. We present a three dimensional twitching motility model, that simulates the extension, retraction and surface association of individual Type IV Pili (TFP), and is informed by recent experimental observations of TFP. Sensitivity analysis is implemented to minimise the number of model parameters, and quantitative estimates for the remaining parameters are inferred from tracking data by approximate Bayesian computation. We argue that the motility mechanism is highly sensitive to experimental conditions. We predict a TFP retraction speed for the tracking data we study that is in a good agreement with experimental results obtained under very similar conditions. Furthermore, we examine whether estimates for biologically important parameters, whose direct experimental determination is challenging, can be inferred directly from tracking data. One example is the width of the distribution of TFP on the bacteria body. We predict that the TFP are broadly distributed over the bacteria pole in both walking and crawling motility types. Moreover, we identified specific configurations of TFP that lead to transitions between walking and crawling states.

Single-detector double-beam modulation for high-sensitivity infrared spectroscopy.

Balanced detection based on double beams is widely used to reduce common-mode noises, such as laser intensity fluctuation and irregular wavelength scanning, in absorption spectroscopy. However, employing an additional detector can increase the total system noise due to added non-negligible thermal noise of the detector, particularly with mid-infrared (IR) detectors. Herein, we demonstrate a new optical method based on double-beam modulation (DBM) that uses a single-element detector but keeps the advantage of double-beam balanced detection. The sample and reference path beams were modulated out-of-phase with each other at a high frequency, and their average and difference signals were measured by two lock-in amplifiers and converted into absorbance. DBM was coupled with our previously reported solvent absorption compensation (SAC) method to eliminate the IR absorption contribution of water in aqueous solutions. The DBM-SAC method enabled us to acquire IR absorption spectra of bovine serum albumin solutions down to 0.02 mg/mL. We investigated the noise characteristics of DBM measurements when the wavelength was either fixed or scanned. The results demonstrate that DBM can lower the limit of detection by ten times compared to the non-modulation method.

History-dependent attachment ofPseudomonas aeruginosato solid-liquid interfaces and the dependence of the bacterial surface density on the residence time distribution.

This study investigates how the recent history of bacteria affects their attachment to a solid-liquid interface. We compare the attachment from a flowing suspension of the bacterium,Pseudomonas aeruginosaPAO1, after one of two histories: (a) passage through a tube packed with glass beads or (b) passage through an empty tube. The glass beads were designed to increase the rate of bacterial interactions with solid-liquid surfaces prior to observation in a flow cell. Analysis of time-lapse microscopy of the bacteria in the flow cells shows that the residence time distribution and surface density of bacteria differ for these two histories. In particular, bacteria exiting the bead-filled tube, in contrast to those bacteria exiting the empty tube, are less likely to attach to the subsequent flow cell window and begin surface growth. In contrast, when we compared two histories defined by different lengths of tubing, there was no difference in either the mean residence time or the surface density. In order to provide a framework for understanding these results, we present a phenomenological model in which the rate of bacterial surface density growth,dN(t)/dt, depends on two terms. One term models the initial attachment of bacteria to a surface, and is proportional to the nonprocessive cumulative residence time distribution for bacteria that attach and detach from the surface without cell division. The second term for the rate is proportional to the bacterial surface density and models surface cell division. The model is in surprisingly good agreement with the data even though the surface growth process is a complex interplay between attachment/detachment at the solid-liquid interface and cell division on the surface.

Changing the Wound: Covalent Immobilization of the Epidermal Growth Factor.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.

Effect of Topographical Steps on the Surface Motility of the Bacterium Pseudomonas aeruginosa.

Bacteria traverse surfaces as part of colonizing solids, and it is of interest to hinder this motion to potentially thwart infections in humans. Here, we demonstrate that topographical steps hinder the ability of Pseudomonas aeruginosa PAO1 (P. aeruginosa) to traverse a solid-liquid interface. Using time-lapse fluorescence microscopy and image analysis, we analyzed the motion of P. aeruginosa that were challenged with steps ranging in height from 0.4 to 9.0 µm. Bacterial trajectories are sensitive to the height of the step, the curvature of the step face, and the direction of their motion relative to gravity. When the step height is ≥0.9 µm, which is similar to the cell diameter, there is a reduced probability of the cell crossing the step. For those bacteria that do cross a step, there is a time penalty for crossing steps of height 2-3 µm; this height is similar to the length of the bacterium. For higher steps, the bacteria reorient their cell body while traversing the step riser. Our findings elucidate how topography influences the motion of bacteria and informs strategies for hindering bacterial motion at surfaces.

Surface Topography Hinders Bacterial Surface Motility.

We demonstrate that the surface motility of the bacterium, Pseudomonas aeruginosa, is hindered by a crystalline hemispherical topography with wavelength in the range of 2-8 µm. The motility was determined by the analysis of time-lapse microscopy images of cells in a flowing growth medium maintained at 37 °C. The net displacement of bacteria over 5 min is much lower on surfaces containing 2-8 µm hemispheres than on flat topography, but displacement on the 1 µm hemispheres is not lower. That is, there is a threshold between 1 and 2 µm for response to the topography. Cells on the 4 µm hemispheres were more likely to travel parallel to the local crystal axis than in other directions. Cells on the 8 µm topography were less likely to travel across the crowns of the hemispheres and were also more likely to make 30°-50° turns than on flat surfaces. These results show that surface topography can act as a significant barrier to surface motility and may therefore hinder surface exploration by bacteria. Because surface exploration can be a part of the process whereby bacteria form colonies and seek nutrients, these results help to elucidate the mechanism by which surface topography hinders biofilm formation.

Effects of Colloidal Crystals, Antibiotics, and Surface-Bound Antimicrobials on Pseudomonas aeruginosa Surface Density.

We examined the effect of a crystalline layer of silica particles in the size range 0.5-4 µm on the adsorption and surface growth of Pseudomonas aeruginosa. Growth on these colloidal crystal monolayers (CCMs) was compared to growth on a flat plate of silica. All surfaces were coated with a thin film of silica to provide chemical uniformity of the different topographies. The results showed that the CCM reduces the density of colony forming units (CFU) on the solid by 99-99.9% when the suspension load was 103 CFU. We also examined the interaction between the CCM and either antibiotics or a chemically bound antimicrobial. The addition of 20 µg/mL tobramycin after an initial 24 h growth period caused a further decrease in CFU counts of about 99-99.9% for all topographies. The percentage reduction as a result of the antibiotics was similar for all topographies, which suggested that there was no particular synergy between the topography and antibiotics. On the other hand, the additive nature of the two effects suggested promise for clinical studies: the large percentage reduction in CFU density on addition of the antibiotic to a flat surface was maintained on the topography, even starting from a much lower CFU density. A similar result was obtained for the combination of CCM and a covalently bound layer of antimicrobial poly(allylamine hydrochloride) (PAH). The PAH reduced the CFU, and the CCM caused a further reduction; the two factors behaved approximately independently. Overall the CCM was found to be very effective at reducing the density of adsorbed P. aeruginosa both with and without the additional reductions caused by antibiotics or surface-bound antimicrobials.

Colloidal Crystals Delay Formation of Early Stage Bacterial Biofilms.

The objective of this work was to examine whether close-packed spheres of polystyrene (colloidal crystals) could be used to delay the development of biofilms. We examined early stage biofilm formation of Pseudomonas aeruginosa after 2 days on a flat sheet of polystyrene and on the same solid coated in polystyrene spheres of 450 or 1500 nm diameter. All materials were coated in fetal bovine serum to enable comparison of the effects of different surface curvature while maintaining constant surface chemistry. After 2 days, fluorescence imaging showed that the volume of bacterial colonies was much smaller on the 1500 nm colloidal crystals than on the flat film. In addition, electron microscopy showed that the area covered by structures containing more than one layer of bacteria was significantly reduced on both the 450 and 1500 nm colloidal crystals compared to the flat sheet. This provides proof of concept of biofilm inhibition of a pathogen by a simple nonchemical coating that may find future application in reducing the incidence of infections. Even though the density of adhered bacteria on 450 and 1500 nm was similar after 1 day, biofilm formation after 2 days was delayed more on the 1500 nm spheres than on the 450 nm spheres. We also observed that bacteria have preferred adsorption sites on the 1500 nm colloidal crystals and that cell bodies were often separated. This leads us to hypothesize that the greater spacing between favorable sites on the 1500 nm colloidal crystal hindered the early stage biofilm formation by separation of cell bodies.

The intrinsic stiffness of human trabecular meshwork cells increases with senescence.

Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases.

Wnt inhibition induces persistent increases in intrinsic stiffness of human trabecular meshwork cells.

Wnt antagonism has been linked to glaucoma and intraocular pressure regulation, as has increased stiffness of human trabecular meshwork (HTM) tissue. We have shown culturing HTM cells on substrates that mimic the elevated stiffness of glaucomatous tissue leads to elevated expression of the Wnt antagonist secreted frizzled related protein 1 (SFRP1), suggesting a linkage between SFRP1 and HTM mechanobiology. In this study, we document biomechanical consequences of Wnt antagonism on HTM cells. Cells were treated with the Wnt antagonists (SFRP1, KY02111, and LGK-974) for 8 days and allowed to recover for 4 days. After recovery, intrinsic cell stiffness and activation of the Wnt pathway via ß-catenin staining and blotting were assayed. Basal cell stiffness values were 3.71 ± 0.37, 4.33 ± 3.07, and 3.07 ± kPa (median ± S.D.) for cells derived from 3 donors. Cell stiffness increased after 0.25 µg/mL (4.32 ± 5.12, 8.86 ± 8.51, 4.84 ± 3.15 kPa) and 0.5 µg/mL (16.75 ± 5.59, 13.18 ± 7.99, and 8.54 ± 5.77 kPa) SFRP1 treatment. Stiffening was observed after 10 µM KY02111 (10.72 ± 5.63 and 6.57 ± 5.53 kPa) as well as LGK-974 (9.60 ± 7.41 and 11.40 ± 9.24 kPa) treatment compared with controls (3.79 ± 1.01 and 5.16 ± 2.14 kPa). Additionally, Wnt inhibition resulted in decreased ß-catenin staining and increased phosphorylation at threonine 41 after recovery. In conclusion, this work demonstrates a causal relationship between Wnt inhibition and cell stiffening. Additionally, these findings suggest transient Wnt inhibition resulted in durable modulation of the mechanical phenotype of HTM cells. When placed in context with previous results, these findings provide a causal link between Wnt antagonism and cell stiffness and suggest a feedback loop contributing to glaucoma progression.

Transforming Growth Factor Beta 3 Modifies Mechanics and Composition of Extracellular Matrix Deposited by Human Trabecular Meshwork Cells.

Pseudoexfoliation syndrome is a systemic disorder of the extracellular matrix (ECM) with ocular manifestations in the form of chronic open angle glaucoma. Elevated levels of TGFß3 in the aqueous humor of individuals with pseudoexfoliation glaucoma (PEX) have been reported. The influence of TGFß3 on the biochemical composition and biomechanics of ECM of human trabecular meshwork (HTM) cells was investigated. HTM cells from eye bank donor eyes were isolated, plated on aminosilane functionalized glass substrates and cultured in the presence or absence of 1 ng/mL TGFß3 for 4 weeks. After incubation, samples were decellularized and decellularization was verified by immunostaining. The mechanics of the remaining ECM that was deposited by the treated or the control cells were measured by atomic force microscopy (AFM). Imaged by AFM, the surface features of the ECM from both sets of samples had a similar roughness/topography (as determined by RMS values) suggesting surface features of the ECM were similar in both cases; however, the ECM from the HTM cells treated with TGFß3 was between 3- and 5-fold stiffer than that produced by the control HTM cells. Proteins present in the ECM were solubilized and analyzed using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Data indicate that multiple proteins previously reported to be altered in glaucoma were changed in the ECM as a result of the presence of TGFß3, including inhibitors of the BMP and Wnt signaling pathways. Gremlin1and 4, SERPINE1 and 2, periostin, secreted frizzled related protein (SFRP) 1 and 4, and ANGPTL4 were among those proteins that were overexpressed in the ECM after TGFß3 treatment.

Automated AFM force curve analysis for determining elastic modulus of biomaterials and biological samples.

The analysis of atomic force microscopy (AFM) force data requires the selection of a contact point (CP) and is often time consuming and subjective due to influence from intermolecular forces and low signal-to-noise ratios (SNR). In this report, we present an automated algorithm for the selection of CPs in AFM force data and the evaluation of elastic moduli. We propose that CP may be algorithmically easier to detect by identifying a linear elastic indentation region of data (high SNR) rather than the contact point itself (low SNR). Utilizing Hertzian mechanics, the data are fitted for the CP. We first detail the algorithm and then evaluate it on sample polymeric and biological materials. As a demonstration of automation, 64 × 64 force maps were analyzed to yield spatially varying topographical and mechanical information of cells. Finally, we compared manually selected CPs to automatically identified CPs and demonstrated that our automated approach is both accurate (< 10nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show that the algorithm is useful for analysis of both biomaterials and biological samples.

A cell culture substrate with biologically relevant size-scale topography and compliance of the basement membrane.

A growing body of literature broadly documents that a wide array of fundamental cell behaviors are modulated by the physical attributes of the cellular microenvironment, yet in vitro assays are typically carried out using tissue culture plastic or glass substrates that lack the 3-dimensional topography present in vivo and have stiffness values that far exceed that of cellular and stromal microenvironments. This work presents a method for the fabrication of thin hydrogel films that can replicate arbitrary topographies with a resolution of 400 nm that possess an elastic modulus of approximately 250 kPa. Material characterization including swelling behavior and mechanics were performed and reported. Cells cultured on these surfaces patterned with anisotropic ridges and grooves react to the biophysical cues present and show an alignment response.