Quenched phosphorescence detection in cyclodextrin-based electrokinetic chromatography

Quenched phosphorescence detection is a sensitive detection method recently introduced in capillary zone electrophoresis. It is based on the dynamic quenching interaction of the analytes (quenchers) with a phosphorophore, 1-bromo-4-naphthalenesulfonate (BrNS), present in the separation buffer. In this study, it is shown that this detection method can also be used in cyclodextrin-based electrokinetic chromatography (CD-EKC) despite the presence in the buffer solution of cyclodextrins, which are known to reduce the luminescence quenching rate constants. Experiments indicate that BrNS mainly resides in the aqueous phase, while the analytes are distributed between both phases. In principle, the observed quenching might arise from the interaction of BrNS with uncomplexed as well as complexed analytes. However, from the dependence of the fractional quenching on the capacity factor (the normalized fractional quenching was found to be equal to the fraction of analyte in the aqueous phase), it was concluded that only aqueous-phase quenching contributes significantly to the observed quenching. Nevertheless, separation and detection can be regarded as fully compatible, because the capacity factors encountered in CD-EKC are generally low (in this study they ranged from about 0.1 to 2.5). Indeed, with nitroaromatic compounds as the target analytes, limits of detection in the 10(-8) M range were achieved.     

A statistical approach to high-throughput screening of predicted orthologs

Orthologs are genes in different species that have diverged from a common ancestral gene after speciation. In contrast, paralogs are genes that have diverged after a gene duplication event. For many comparative analyses, it is of interest to identify orthologs with similar functions. Such orthologs tend to support species divergence (ssd-orthologs) in the sense that they have diverged only due to speciation, to the same relative degree as their species. However, due to incomplete sequencing or gene loss in a species, predicted orthologs can sometimes be paralogs or other non-ssd-orthologs. To increase the specificity of ssd-ortholog prediction, Fulton et al. [Fulton, D., Li, Y., Laird, M., Horsman, B., Roche, F., Brinkman, F., 2006. Improving the specificity of high-throughput ortholog prediction. BMC Bioinformatics 7 (1), 270] developed Ortholuge, a bioinformatics tool that identifies predicted orthologs with atypical genetic divergence. However, when the initial list of putative orthologs contains a non-negligible number of non-ssd-orthologs, the cut-off values that Ortholuge generates for orthology classification are difficult to interpret and can be too high, leading to decreased specificity of ssd-ortholog prediction. Therefore, we propose a complementary statistical approach to determining cut-off values. A benefit of the proposed approach is that it gives the user an estimated conditional probability that a predicted ortholog pair is unusually diverged. This enables the interpretation and selection of cut-off values based on a direct measure of the relative composition of ssd-orthologs versus non-ssd-orthologs. In a simulation comparison of the two approaches, we find that the statistical approach provides more stable cut-off values and improves the specificity of ssd-ortholog prediction for low-quality data sets of predicted orthologs.     

数字工厂--数字制造教学与研究中的技术个案

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