Rapid and effective processing of blood specimens for diagnostic PCR using filter paper and Chelex-100.

AIM: To provide a more efficient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis. METHODS: The use of blood impregnated filter paper and Chelex-100 in DNA isolation was evaluated and compared with standard DNA isolation techniques. RESULTS: In polymerase chain reaction (PCR) based assays of five point mutations, identical results were obtained with DNA isolated routinely from peripheral blood and isolated using the filter paper and Chelex-100 method. CONCLUSION: In the clinical setting, this method provides a useful alternative to conventional DNA isolation. It is easily implemented and inexpensive, and provides sufficient, stable DNA for multiple assays. The potential for specimen contamination is reduced because most of the steps are performed in a single microcentrifuge tube. In addition, this method provides for easy storage and transport of samples from the point of acquisition.     

Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis

Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis. P. gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity. P. gingivalis grew well at all three temperatures. An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01). A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0. 01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased, P. gingivalis adopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.     

The effect of automated landmark identification on morphometric analyses

Morphometric analysis of anatomical landmarks allows researchers to identify specific morphological differences between natural populations or experimental groups, but manually identifying landmarks is time‐consuming. We compare manually and automatically generated adult mouse skull landmarks and subsequent morphometric analyses to elucidate how switching from manual to automated landmarking will impact morphometric analysis results for large mouse (Mus musculus) samples (n = 1205) that represent a wide range of ‘normal’ phenotypic variation (62 genotypes). Other studies have suggested that the use of automated landmarking methods is feasible, but this study is the first to compare the utility of current automated approaches to manual landmarking for a large dataset that allows the quantification of intra‐ and inter‐strain variation. With this unique sample, we investigated how switching to a non‐linear image registration‐based automated landmarking method impacts estimated differences in genotype mean shape and shape variance‐covariance structure. In addition, we tested whether an initial registration of specimen images to genotype‐specific averages improves automatic landmark identification accuracy. Our results indicated that automated landmark placement was significantly different than manual landmark placement but that estimated skull shape covariation was correlated across methods. The addition of a preliminary genotype‐specific registration step as part of a two‐level procedure did not substantially improve on the accuracy of one‐level automatic landmark placement. The landmarks with the lowest automatic landmark accuracy are found in locations with poor image registration alignment. The most serious outliers within morphometric analysis of automated landmarks displayed instances of stochastic image registration error that are likely representative of errors common when applying image registration methods to micro‐computed tomography datasets that were initially collected with manual landmarking in mind. Additional efforts during specimen preparation and image acquisition can help reduce the number of registration errors and improve registration results. A reduction in skull shape variance estimates were noted for automated landmarking methods compared with manual landmarking. This partially reflects an underestimation of more extreme genotype shapes and loss of biological signal, but largely represents the fact that automated methods do not suffer from intra‐observer landmarking error. For appropriate samples and research questions, our image registration‐based automated landmarking method can eliminate the time required for manual landmarking and have a similar power to identify shape differences between inbred mouse genotypes.     

Extending psychosocial assessment of patients with psoriasis in the UK,using a self‐rated,web‐based survey

Background. Quality of life self‐rating using a web‐based survey has not previously been evaluated for psoriasis in the UK. Aim. To use an open‐access web‐based survey to assess the effect of psoriasis on patients’ daily life. Methods. The survey was conducted using a dedicated website endorsed by a UK psoriasis patient charity. Results. In total, 1760 patients (1102 women, 658 men; median age range 40–44 years) assessed their psoriasis using the website. Psoriasis was ‘very’ or ‘extremely’ active in 52%, and 71% had been diagnosed > 10 years previously. Psoriasis had negatively affected the working life of 59% of patients, and the educational performance of 31%. Conclusions. The use of an open‐access web‐based survey may address potential bias in previous studies, but may itself introduce a bias towards younger patients. This is the first report of a web‐based survey of UK patients with psoriasis, providing further recent evidence of how psoriasis affects patients’ lives.     

Comparison of five elastin histochemical stains to identify pulmonary small vasculature*

Optimization of an elastic tissue histochemical stain to enable clear, crisp visualization and quantification of pulmonary small vasculature is central to the histomorphologic quantitation of pulmonary vasculature wall thickness. To accomplish elastic tissue histochemical stain optimization, five histochemical elastin stains were compared to identify the internal and external elastic laminae of small arteries (50–100 μm in external diameter) to very small intra-acinar vessels (10–50 μm in external diameter) in rat lung tissue sections. The five elastin stains included: a modified Verhoeff’s elastin stain, Miller’s elastic van Gieson, and three modifications of the Miller’s stain. The Miller elastin stain is a progressive procedure that does not require a differentiation step, thus enabling consistency and reliability of staining from slide to slide. A modified Miller’s histochemical staining methodology successfully highlighted the pulmonary small caliber vasculature wall thickness. The modified method was technically easier and less time consuming to perform than regressive methods. To improve elastin-to-background contrast, modifications to the Miller’s stain included bypassing the nuclear staining and using a neutral red counterstain in place of the van Gieson counterstain, both of which greatly facilitated observer-assisted pulmonary vascular structure identification for histomorphometric quantitation.