Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR

A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed. The target genes used were: the aerolysin (aero) gene of Aeromonas hydrophila, the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, the attachment invasion locus (ail) gene of Yersinia enterocolitca, the invasion plasmid antigen B (ipaB) gene of Salmonella typhimurium, the enterotoxin extracellular secretion protein (epsM) gene of Vibrio cholerae and a species-specific region of the 16S-23S rDNA (Vpara) gene of Vibrio parahaemolyticus were used as the gene targets. Multiplex PCR using the six pairs of primers produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in seawater and from pure cultures. The m-PCR assay was specific and rapid, with a turnaround time of <12h. The detection limit of the assay for the bacterial targets was estimated at 100-102cfu. Multiplex PCR analysis was performed on 19 seawater samples collected around Hong Kong and the results indicated significant levels of four bacterial pathogens at several sites where primary sewage wastes are discharged, and the levels of which showed no correlation with E. coli counts. Overall, both laboratory and field validation results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of water quality. © 2002 Elsevier Science Ltd. All rights reserved.link_to_subscribed_fulltex

Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences

Vibrios are widespread in the marine environment and a few pathogenic species are known to be commonly associated with outbreaks of diarrheal diseases in humans due to the consumption of raw or improperly cooked seafood. However, there are also many Vibrio species which are potentially pathogenic to vertebrate and invertebrate aquatic animals, and of which little is known. In an attempt to develop rapid PCR detection methods for these latter class of vibrios, we have examined the 16S-23S intergenic spacers (IGSs) of 10 lesser-known Vibrio species and successfully developed species-specific primers for eight of them - Vibrio costicola, V. diazotrophicus, V. fluvialis, V. nigripulchritudo, V. proteolyticus, V. salmonicida, V. splendidus and V. tubiashii. The IGS amplicons were amplified using primers complementary to conserved regions of the 16S and 23S rRNA genes, and cloned into plasmid vectors and sequenced. Analysis of the IGS sequences showed that 37 ribosomal RNA (rrn) operons representing seven different IGS types have been cloned from the 10 vibrios. The three IGS types - IGS0, IGSIA and IGSGlu - were the most prevalent forms detected. Multiple alignment of representative sequences of these three IGS types from different Vibrio species revealed several domains of high sequence variability, which were used to design species-specific primers for PCR. The specificity of the primers were evaluated using total DNA prepared from different Vibrio species and bacterial genera. The results showed that the PCR method can be used to reliably detect eight of the 10 Vibrio species in marine waters in this study. © 2002 Elsevier Science Ltd. All rights reserved.link_to_subscribed_fulltex

The first transmembrane region of complement component-9 acts as a brake on its self-assembly

The Complement component 9 (C9) is the pore-forming component of the Membrane Attack Complex which targets pathogens. Here authors use structural biology to compare monomeric C9 to C9 within the polymeric assembly and identify the element which inhibits C9 self-assembly in the absence of the target membrane

Constitutional Conflict in Hong Kong Under Chinese Sovereignty

© 2016, T.M.C. Asser Press.The protracted constitutional conflicts between the Chinese state and Hong Kong society over the pace and form of democratization have become a source of political instability. This article sheds new light on these dynamics by analyzing the two sides as locked in a coordination dilemma: both have important common interests to coordinate with each other, but both rank in divergent ways their preferences for and against introducing competitive electoral arrangements into Hong Kong. It is shown how the unwillingness of either side to compromise has caused the gradual breakdown of coordination as focal points like the Basic Law have eroded, culminating in the Umbrella Revolution of 2014, the largest and longest-lasting popular movement in Hong Kong history. The bloodless ending of the Movement is chronicled, along with the persistence of political stalemate in this highly anomalous polity, known throughout the world for its combination of genuine civil liberties with authoritarian rule.Link_to_subscribed_fulltex