Molecular methods which can identify extensive mutations associated with multiple anti-tuberculous drugs are in urgent need so that the research on drug resistant mutations should be extended.

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BackgroundThe uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity.MethodRecombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study.ResultsWe revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity.ConclusionThis is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.

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According to the updates in the review, it can be concluded artemisinin resistance does not disseminate to India and African countries within short period whereas regular tracking of these mutants is necessary.

This study revealed that V.cholerae in Sabah were genetically diverse and were atypical El Tor strains. Fingerprint patterns of these isolates will be useful in tracing the origin of this pathogen in the future.

Plasmodium ovale is widely distributed in tropical countries, whereas it has not been reported in the Americas. It is not a problem globally because it is rarely detected by microscopy owing to low parasite density, which is a feature of clinical ovale malaria. P.o. curtisi and P.o. wallikeri are widespread in both Africa and Asia, and were known to be sympatric in many African countries and in southeast Asian countries. Small subunit ribosomal RNA (SSUrRNA) gene, cytochrome b (cytb) gene, and merozoite surface protein-1 (msp-1) gene were initially studied for molecular discrimination of P.o. curtisi and P.o. wallikeri using polymerase chain reaction (PCR) and DNA sequencing. DNA sequences of other genes from P. ovale in Southeast Asia and the southwestern Pacific regions were also targeted to differentiate the two sympatric types. In terms of clinical manifestations, P.o. wallikeri tended to produce higher parasitemia levels and more severe symptoms. To date, there have been a few studies that used the quantitative PCR method for discrimination of the two distinct P. ovale types. Conventional PCR with consequent DNA sequencing is the common method used to differentiate these two types. It is necessary to identify these two types because relapse periodicity, drug susceptibility, and mosquito species preference need to be studied to reduce ovale malaria. In this article, an easier method of molecular-level discrimination of P.o. curtisi and P.o. wallikeri is proposed.

Xpert has improved the bacteriological confirmation of PTB among hospitalised children in Myanmar.