Systematic random sampling of the comet assay

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory ‘tail’ DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the ‘randomness’ of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Shell scarring in Calliostoma zizyphinum (Prosobranch: Trochidae) from Strangford Lough, Northern Ireland.

Pink and white shells of Calliostoma zizyphinum show both undamaged and damaged scars. White shelled individuals predominate in Strangford Lough, Northern Ireland where Calliostoma densities and scarring levels are high. No white shells were found intertidally, outside the entrance channel to the Lough. Significant differences occur in the size of Calliostoma shells between sampling sites but not between different colour morphs at each site. However in pooled samples, pink individuals are significantly larger than white individuals. When sites and morphs are considered separately, pink Calliostoma density is negatively correlated with water movement. At sites where pink Calliostoma occur, the percentage of pink shelled individuals is negatively correlated with total Calliostoma density. Damaged and undamaged scarring values per unit area of shell, show highly significant differences between sites and damaged scars are significantly higher in white individuals. Undamaged scars are not correlated with any of the environmental parameters recorded, but are positively correlated with damaged scars suggesting a common causative factor. The level of damaged scarring is positively correlated with crab/total Calliostoma ratio at all sites and where each colour morph was considered separately. Multiple regression analyses reveal that crab/Calliostoma ratios account for 42% of the between site variation in damaged scars. Significantly higher levels of damaged scars are found at sites with high crab densities and significantly larger individuals are found at sites where crab densities are intermediate in value. The largest and most highly scarred individuals occur at sites with most coarse substrata where Calliostoma are present in their lowest densities. The higher scarring levels and smaller size of white individuals reflect either higher mortality or reduced growth in white shelled Calliostoma.

Monitoring and repair of an impact damaged prestressed bridge

This paper details the monitoring and repair of an impact damaged prestressed concrete bridge. The repair was required following an impact from a low-loader carrying an excavator while passing underneath the bridge. The repair was carried out by preloading the bridge in the vicinity of the damage to relieve some prestressing. This preload was removed following the hardening and considerable strength gain of the repair material. The true behaviour of damaged prestressed concrete bridges during repair is difficult to estimate theoretically due to lack of benchmarking and inadequacy of assumed damage models. A network of strain gauges at locations of interest was thus installed during the entire period of repair. Effects of various activities were qualitatively and quantitatively observed. The interaction and rapid, model-free calibration of damaged and undamaged beams, including identification of damaged gauges were also probed. This full scale experiment is expected to be of interest and benefit to the practising engineer and the researcher alike.

Stiffener debonding mechanisms in post-buckled CFRP aerospace panels

This paper describes the fractographic analysis of five CFRP post-buckled skin/stringer panels that were tested to failure in compression. The detailed damage mechanisms for skin/stiffener detachment in an undamaged panel were characterised and related to the stress conditions during post-buckling; in particular the sites of peak twist (at buckling nodes) and peak bending moments (at buckling anti-nodes). The initial event was intralaminar splitting of the +45 degrees plies adjacent to the skin/stiffener interface, induced by high twist at a nodeline. This was followed by mode II delamination, parallel to +/- 45 degrees plies and then lengthwise (0 degrees) shear along the stiffener centreline. The presence of defects or damage was found to influence this failure process, leading to a reduction in strength. This research provides an insight into the processes that control post-buckled performance of stiffened panels and suggests that 2D models and element tests do not capture the true physics of skin/stiffener detachment: a full 3D approach is required.