Quantitative analysis of malondialdehyde-guanine adducts in genomic DNA samples by liquid chromatography/tandem mass spectrometry.

RATIONALE: The lipid peroxidation product malondialdehyde forms M1 dG adducts with guanine bases in genomic DNA. The analysis of these adducts is important as a biomarker of lipid peroxidation, oxidative stress and inflammation which may be linked to disease risk or exposure to a range of chemicals. METHODS: Genomic DNA samples were subjected to acid hydrolysis to release the adducts in the base form (M1 G) alongside the other purines. A liquid chromatography/mass spectrometry method was optimised for the quantitation of the M1 G adducts in genomic DNA samples using product ion and multiple reaction monitoring (MRM) scans. RESULTS: Product ion scans revealed four product ions from the precursor ion; m/z 188 â†’ 160, 133, 106 and 79. The two smallest ions have not been observed previously and optimisation of the method revealed that these gave better sensitivity (LOQ m/z 79: 162 adducts per 107 nucleotides; m/z 106: 147 adducts per 107 nucleotides) than the other two ions. An MRM method gave similar sensitivity but the two smallest product ions gave better accuracy (94-95%). Genomic DNA treated with malondialdehyde showed a linear dose-response relationship. CONCLUSIONS: A fast reliable sample preparation method was used to release adducts in the base form rather than the nucleoside. The methods were optimised to selectively analyse the adducts in the presence of other DNA bases without the need for further sample clean-up. Analysis of genomic DNA gave results consistent with previous work and was applied to new samples. Thus, the method is suitable for the analysis of M1 (d)G adducts in biological samples. Copyright © 2017 John Wiley & Sons, Ltd.

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour.

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods.

Defining the Properties of an Array of -NH2-Modified Substrates for the Induction of a Mature Osteoblast/Osteocyte Phenotype from a Primary Human Osteoblast Population Using Controlled Nanotopography and Surface Chemistry.

Accelerating the integration of a joint replacement or the healing of a bone fracture, particularly a complicated non-union fracture, would improve patient welfare and decrease healthcare costs. Currently, an autologous bone graft is the gold standard method for the treatment of complicated non-union fractures, but it is not always possible to harvest such a graft. A proactive highly inductive so-called smart material approach is pertinent in these cases. In this study, the surface chemistry of a previously approved material with desirable bulk material properties was modified to investigate its potential as an economical and effective alternative. The objective was to create stable synthetic chemical coatings that could guide cells along the osteogenic lineage required to generate mineralised tissue that would induce and accelerate bone healing. Primary human osteoblast-like cells were cultured in vitro for 7, 14 and 28 days on amine-terminated (chain length in the range 3-11) silane-modified glass surfaces with controlled nanotopography, to determine how surface chemistry and nanotopography change osteoblast function. The materials were characterised using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA) and a novel ninhydrin assay. The cells were analysed using qRT-PCR, von Kossa tinctural staining for mineralisation, and visualised using both transmitted white light and electron microscopy. Bone-like nodules, quantified using microscopy, only formed on the short-chain (chain length 3 and 4) amines after 7 days, as did the up-regulation of sclerostin, suggestive of a more mature osteoblast phenotype. In this paper, we report more rapid nodule formation than has previously been observed, without the addition of exogenous factors in the culture medium. This suggests that the coating would improve the integration of implants with bone or be the basis of a smart biomaterial that would accelerate the bone regeneration process.

The effects of acoustic vibration on fibroblast cell migration.

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (µm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration.

Acoustic vibration can enhance bacterial biofilm formation.

This paper explores the use of low-frequency-low-amplitude acoustic vibration on biofilm formation. Biofilm development is thought to be governed by a diverse range of environmental signals and much effort has gone into researching the effects of environmental factors including; nutrient availability, pH and temperature on the growth of biofilms. Many biofilm-forming organisms have evolved to thrive in mechanically challenging environments, for example soil yet, the effects of the physical environment on biofilm formation has been largely ignored. Exposure of Pseudomonas aeruginosa to vibration at 100, 800 and 1600 Hz for 48 h, resulted in a significant increase in biofilm formation compared with the control, with the greatest growth seen at 800 Hz vibration. The results also show that this increase in biofilm formation is accompanied with an increase in P. aeruginosa cell number. Acoustic vibration was also found to regulate the spatial distribution of biofilm formation in a frequency-dependent manner. Exposure of Staphylococcus aureus to acoustic vibration also resulted in enhanced biofilm formation with the greatest level of biofilm being formed following 48 h exposure at 1600 Hz. These results show that acoustic vibration can be used to control biofilm formation and therefore presents a novel and potentially cost effective means to manipulate the development and yield of biofilms in a range of important industrial and medical processes.

Analysis of dynamic cantilever behavior in tapping mode atomic force microscopy.

Tapping mode atomic force microscopy (AFM) provides phase images in addition to height and amplitude images. Although the behavior of tapping mode AFM has been investigated using mathematical modeling, comprehensive understanding of the behavior of tapping mode AFM still poses a significant challenge to the AFM community, involving issues such as the correct interpretation of the phase images. In this paper, the cantilever's dynamic behavior in tapping mode AFM is studied through a three dimensional finite element method. The cantilever's dynamic displacement responses are firstly obtained via simulation under different tip-sample separations, and for different tip-sample interaction forces, such as elastic force, adhesion force, viscosity force, and the van der Waals force, which correspond to the cantilever's action upon various different representative computer-generated test samples. Simulated results show that the dynamic cantilever displacement response can be divided into three zones: a free vibration zone, a transition zone, and a contact vibration zone. Phase trajectory, phase shift, transition time, pseudo stable amplitude, and frequency changes are then analyzed from the dynamic displacement responses that are obtained. Finally, experiments are carried out on a real AFM system to support the findings of the simulations.

The crystal engineering of salbutamol sulphate via simulated pulmonary surfactant monolayers.

PURPOSE: This study aims to crystallise salbutamol sulphate beneath simulated pulmonary surfactant monolayers. Such ensembles serve as heterogeneous nucleating sites to direct crystallisation. This contribution builds upon previous work to confirm the suitability of Langmuir monolayers in supporting the rational generation of respirable therapeutic material. METHODS: Langmuir monolayers (i.e. DPPC or a 'mixed' system) were supported on a subphase containing the extremely water soluble model drug (2.5 g/ml) and compressed to 5 mN m(-1) or 35 mN m(-1) whilst experiencing a temperature reduction and positioned within a humid environment. Control samples were produced via batch crystallisation. Analysis involved scanning electron microscopy (SEM), atomic force microscopy (AFM), powder X-ray diffraction (PXRD) and inverse gas chromatography (IGC). RESULTS: Expanded Langmuir isotherms confirmed drug-surfactant interaction; crystal growth was inhibited at high surface pressure. Resultant crystals exhibited a range of morphologies, dependent upon the crystallisation route. AFM analysis highlighted nanoscale surface undulations. IGC data confirmed sample surface energy profiles were variable and influenced by crystallisation route. CONCLUSIONS: Principal modes of drug-surfactant interaction are proposed as hydrogen bond and ion-dipole associations. A range of pharmaceutical approaches have been applied to understand drug-surfactant complementarity. The results strengthen the argument for the use of Langmuir monolayers in drug particle engineering.

Actin bundling and polymerisation properties of eukaryotic elongation factor 1 alpha (eEF1A), histone H2A-H2B and lysozyme in vitro.

Elongation factor 1 alpha (eEF1A) is a positively charged protein which has been shown to interact with the actin cytoskeleton. However, to date, a specific actin binding site within the eEF1A sequence has not been identified and the mechanism by which eEF1A interacts with actin remains unresolved. Many protein-protein interactions occur as a consequence of their physicochemical properties and actin bundle formation has been shown to result from non-specific electrostatic interaction with basic proteins. This study investigated interactions between actin, eEF1A and two other positively charged proteins which are not regarded as classic actin binding proteins (namely lysozyme and H2A-H2B) in order to compare their actin organising effects in vitro. For the first time using atomic force microscopy (AFM) we have been able to image the interaction of eEF1A with actin and the subsequent bundling of actin in vitro. Interestingly, we found that eEF1A dramatically increases the rate of polymerisation (45-fold above control levels). We also show for the first time that H2A-H2B has remarkably similar effects upon actin bundling (relative bundle size/number) and polymerisation (35-fold increase above control levels) as eEF1a. The presence of lysozyme resulted in bundles which were distinct from those formed due to eEF1A and H2A-H2B. Lysozyme also increased the rate of actin polymerisation above the control level (by 10-fold). Given the striking similarities between the actin bundling and polymerisation properties of eEF1A and H2A-H2B, our results hint that dimerisation and electrostatic binding may provide clues to the mechanism through which eEF1A-actin bundling occurs.

Towards crystal engineering via simulated pulmonary surfactant monolayers to optimise inhaled drug delivery.

PURPOSE: To generate theophylline monohydrate crystals underneath Langmuir monolayers composed of material expressed at the alveolar air-liquid interface. Such monolayers can act as nucleation sites to direct crystallisation. The approach offers a novel route to rationally engineer therapeutic crystals and thereby optimise inhaled drug delivery. METHODS: Langmuir monolayers consisting of either dipalmitoylphosphatidylcholine (DPPC) or a surfactant mix reflecting pulmonary surfactant were supported on an aqueous theophylline (5.7 mg/ml) subphase. The monolayers were compressed to surface pressures reflecting inhalation and exhalation (i.e. 5 mNm(-1) or 55 mNm(-1)) with a period of 16 h to allow crystallisation. Analysis involved scanning electron microscopy (SEM), atomic force microscopy (AFM) and powder X-ray diffraction (PXRD). RESULTS: Condensed isotherms were acquired, which signified surfactant-theophylline interaction. Theophylline monohydrate crystals were obtained and exhibited needle-like morphology. SEM and AFM data highlighted regions of roughened growth along with smooth, stepwise growth on the same crystal face. The surfactant monolayers appeared to influence crystal morphology over time. CONCLUSIONS: The data indicate a favourable interaction between each species. The principal mechanism of interaction is thought to be an ion-dipole association. This approach may be applied to generate material with improved complementarity with pulmonary surfactant thus enhancing the interaction between inhaled drug particles and internal lung surfaces.

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy.

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.