Prevalence of the prothrombin G20210A mutation in the Irish populations:use of a novel polymerase chain reaction approach

The prothrombin G20210A polymorphism is associated with a threefold-increased risk of venous thrombosis. There is considerable variation in the reported prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determined. A restriction fragment length polymorphism (RFLP)-based assay is commonly used for the detection of the prothrombin 20210A allele. This assay does not include a control restriction digest fragment and, consequently, failure of the enzyme activity or lack of addition of enzyme to the sample cannot be distinguished from wild-type prothrombin. We developed a RFLP-based assay, which incorporates an invariant digest site, resulting in the generation of a control digest fragment. Furthermore, we developed a nested polymerase chain reaction (PCR) method for the amplification and digestion of poor-quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the prothrombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Anglo-Saxon populations. The nested PCR method successfully amplified and digested 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method. In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant control digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative results.

Gait period estimation algorithm based on the angles of extremities

Gait period estimation is an important step in the gait recognition framework. In this paper, we propose a new gait cycle detection method based on the angles of extreme points of both legs. In addition to that, to further improve the estimation of the gait period, the proposed algorithm divides the gait sequence into sections before identifying the maximum values. The proposed algorithm is scale invariant and less dependent on the silhouette shape. The performance of the proposed method was evaluated using the OU-ISIR speed variation gait database. The experimental results show that the proposed method achieved 90.2% gait recognition accuracy and outperforms previous methods found in the literature with the second best only achieved 67.65% accuracy.

CCL2 is transcriptionally controlled by the lysosomal protease cathepsin S in a CD74-dependent manner

Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA depletion of CatS resulted in reduced CCL2 expression. This regulation is transcriptionally mediated, as evident from RT-PCR analysis and CCL2 promoter studies. We revealed that CatS regulation of CCL2 is modulated through CD74 (also known as the invariant chain), a known substrate of CatS and a mediator of NFkB activity. Furthermore, CatS and CCL2 show a strong clinical correlation in brain, breast and colon tumours. In summary, these results highlight a novel mechanism by which CatS controls CCL2, which may present a useful pharmacodynamic marker for CatS inhibition.