Environmental DNA phylogeography: Successful reconstruction of phylogeographic patterns of multiple fish species from cups of water

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionize phylogeography.     

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7α-hydroxy bile acids, are detected by an enzymatic method using 7α-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7α-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by β-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7α-hydroxy bile acids are detected with 7α-HSD in the presence of β-NAD⁺, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7α-hydroxy bile acids may be useful for clinical diagnosis.     

Temperature shift-up leads to simultaneous and continuous plasmid DNA relaxation and induction of DnaK and GroEL proteins in anaerobically growing Escherichia coli cells

Abstract We previously reported that plasmid DNA in Escherichia coli cells growing under aerobic conditions relaxed immediately and transiently after heat shock (Mizushima, T., Natori, S. and Sekimizu, K., Mol. Gen. Genet. (1993) 238, 1–5). We next examined DNA relaxation and induction of heat shock proteins after heat shock in cells growing under anaerobic conditions. DNA in these cells relaxed rapidly (2 min) after heat shock (42°C), as was the case with aerobically growing cells, but full superhelicity was not recovered. The relaxed state of DNA topology was maintained for 60 min after heat shock. Induction of DnaK and GroEL proteins, which was transient in aerobically growing cells, was continuous in anaerobically growing cells. Therefore, induction of heat shock proteins correlated with DNA relaxation in both aerobic and anaerobic conditions.     

The extension of α-d-1,3-branch linkages by 1,3-α-d-glucan synthase from Streptococcus sobrinus

In the presence of an acceptor, 1,3-alpha-D-glucan synthase of Streptococcus sobrinus synthesizes water-insoluble glucans from sucrose. Under such conditions, 1,3-alpha-D-glucoside linkages were extended without any change in the glucose-residue number between the 1,3,6-branch points on the acceptor. From these results, the mechanisms of water-insoluble-glucan formation were proposed as follows: (i) the attachment of an acceptor to the glucan binding sites of 1,3-alpha-D-glucan synthase occurs during the initiation of the reaction, and concurrently determines the positions of the branched portions of 1,3,6 on the acceptor, and (ii) the 1,3-alpha-D-glucoside linkage extends from these positions.