Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

Background

Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.

Methodology/Principal Findings

OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.

Conclusion/Significance

Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.     

Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with K(d) values of 2.50 and 4.00 microM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques.     

Phylogeography of Wild Musk Shrew (Suncus Murinus) Populations in Asia Based on Blood Protein/Enzyme Variation

The musk shrew (Suncus murinus) is an insectivore species that inhabits tropical and subtropical Asia widely. To clarify the genetic relationship among wild musk shrew populations, we examined the electrophoretic variants of biparentally inherited genetic markers at 10 loci coding for eight blood proteins/enzymes in a total of 639 animals and compared the results obtained from the mitochondrial DNA data. The principal-component analysis performed using the allele frequency data revealed that the 17 populations could be divided into two major groups, a South Asian group and a Southeast Asian group that includes several island populations bound by Myanmar. The degrees of genetic divergence among populations were higher within the Southeast Asian group than within the South Asian group. This finding was incongruent with the mtDNA diversity. Analysis conducted at the individual level showed that a shrew from the central region in Myanmar that carries a South Asian type of mtDNA showed the electrophoretic variants specific to the Southeast Asian group, suggesting that this region is a contact zone between the two major groups.     

The effects of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on isolated pea chloroplasts

The effects of a quinone analogue, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone(DBMIB), on the photochemical activities of isolated pea chloroplastshave been investigated. DBMIB completely inhibits pseudo-cyclicflow with methyl viologen (MV) and partially inhibits non-cyclicflow with ferricyanide (FeCN) in both coupled and uncoupledsystems. It is also shown that, under some circumstances, DBMIBacts like an uncoupler in that it stimulates electron flow andinhibits cyclic photophosphorylation. ¹ Present address: 32(1), 6 1/4 Mile, Prome Road, Kamayut P.O., Rangoon, Burma. (Received May 21, 1973; )     

Geographical distribution of Burkholderia pseudomallei in soil in Myanmar

BackgroundBurkholderia pseudomallei is a Gram-negative bacterium found in soil and water in many tropical countries. It causes melioidosis, a potentially fatal infection first described in 1911 in Myanmar. Melioidosis is a common cause of sepsis and death in South and South-east Asia, but it is rarely diagnosed in Myanmar. We conducted a nationwide soil study to identify areas where B. pseudomallei is present.Methodology/Principal findingsWe collected soil samples from 387 locations in all 15 states and regions of Myanmar between September 2017 and June 2019. At each site, three samples were taken at each of three different depths (30, 60 and 90 cm) and were cultured for B. pseudomallei separately, along with a pooled sample from each site (i.e. 10 cultures per site). We used a negative binomial regression model to assess associations between isolation of B. pseudomallei and environmental factors (season, soil depth, soil type, land use and climate zones). B. pseudomallei was isolated in 7 of 15 states and regions. Of the 387 sites, 31 (8%) had one or more positive samples and of the 3,870 samples cultured, 103 (2.7%) tested positive for B. pseudomallei. B. pseudomallei was isolated more frequently during the monsoon season [RR-2.28 (95% CI: 0.70–7.38)] and less in the hot dry season [RR-0.70 (95% CI: 0.19–2.56)] compared to the cool dry season, and in the tropical monsoon climate zone [RR-2.26; 95% CI (0.21–6.21)] compared to the tropical dry winter climate zone. However, these associations were not statistically significant. B. pseudomallei was detected at all three depths and from various soil types (clay, silt and sand). Isolation was higher in agricultural land (2.2%), pasture land (8.5%) and disused land (5.8%) than in residential land (0.4%), but these differences were also not significant.Conclusion/SignificanceThis study confirms a widespread distribution of B. pseudomallei in Myanmar. Clinical studies should follow to obtain a better picture of the burden of melioidosis in Myanmar.     

Estimating direct and spill-over impacts of political elections on COVID-19 transmission using synthetic control methods

Mass gathering events have been identified as high-risk environments for community transmission of coronavirus disease 2019 (COVID-19). Empirical estimates of their direct and spill-over effects however remain challenging to identify. In this study, we propose the use of a novel synthetic control framework to obtain causal estimates for direct and spill-over impacts of these events. The Sabah state elections in Malaysia were used as an example for our proposed methodology and we investigate the event’s spatial and temporal impacts on COVID-19 transmission. Results indicate an estimated (i) 70.0% of COVID-19 case counts within Sabah post-state election were attributable to the election’s direct effect; (ii) 64.4% of COVID-19 cases in the rest of Malaysia post-state election were attributable to the election’s spill-over effects. Sensitivity analysis was further conducted by examining epidemiological pre-trends, surveillance efforts, varying synthetic control matching characteristics and spill-over specifications. We demonstrate that our estimates are not due to pre-existing epidemiological trends, surveillance efforts, and/or preventive policies. These estimates highlight the potential of mass gatherings in one region to spill-over into an outbreak of national scale. Relaxations of mass gathering restrictions must therefore be carefully considered, even in the context of low community transmission and enforcement of safe distancing guidelines.     

Exploitation and community engagement: Can Community Advisory Boards successfully assume a role minimising exploitation in international research?

It has been suggested that community advisory boards (CABs) can play a role in minimising exploitation in international research. To get a better idea of what this requires and whether it might be achievable, the paper first describes core elements that we suggest must be in place for a CAB to reduce the potential for exploitation. The paper then examines a CAB established by the Shoklo Malaria Research Unit under conditions common in resource‐poor settings – namely, where individuals join with a very limited understanding of disease and medical research and where an existing organisational structure is not relied upon to serve as the CAB. Using the Tak Province Border Community Ethics Advisory Board (T‐CAB) as a case study, we assess the extent to which it might be able to take on a role minimising exploitation were it to decide to do so. We investigate whether, after two years in operation, T‐CAB is capable of assessing clinical trials for exploitative features and addressing those found to have them. The findings show that, although T‐CAB members have gained knowledge and developed capacities that are foundational for one‐day taking on a role to reduce exploitation, their ability to critically evaluate studies for the presence of exploitative elements has not yet been strongly demonstrated. In light of this example, we argue that CABs may not be able to perform such a role for a number of years after initial formation, making it an unsuitable responsibility for many short‐term CABs.     

Clinical diagnosis of malaria on the Thai-Myanmar border.

BACKGROUND: To evaluate the prevailing practice of presumptively diagnosing malaria in all cases of febrile illness in a clinic serving a refugee population on the Thai-Myanmar border METHODS: A retrospective review of 3,506 patient charts from December 1993 through June 1994 at the MaeSot medical clinic to compare clinical signs of malaria to blood smear findings. Patients presenting without fever were assumed not to have malaria; the remaining 2,111 patients presenting with fever had blood smears examined for malaria infection. RESULTS: Fever alone sufferedfrom poorpositive predictive value (54.7 percent) and specificity (59.3 percent). When fever was combined with hepatosplenomegaly and anemia, the positive predictive value and specificity improved (84.5 percent and 98.5 percent, respectively). However, this combination also resulted in an unacceptably poor sensitivity (16.5 percent) and false negative error rate (835/1,000). CONCLUSIONS. In this nonimmune refugee population, severe complications of falciparum malaria occur quickly and commonly; aggressive chemotherapy is necessary to reduce morbidity and mortality. Until laboratory facilities are made available, all cases offever should continue to be treated presumptively as malaria.