Simple,sensitive, and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes,using loop mediated isothermal amplification combined with the electrochemical biosensing method

BackgroundWolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia-based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity and cost issues.Methodology/Principal findingsWe describe here the development of a LAMP assay combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers used a low-cost dye detecting system and 4 oligo nucleotide primers which can reduce the cost of analysis while the specificity is comparable to the previous methods. The detection capacity of our LAMP technique was 1.4 nM and the detection limit reduced to 2.2 fM when combined with the BIOSENSOR. Our study demonstrates that a BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification.Conclusions/SignificanceOur results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.     

Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways

Eukaryotic cells activate the unfolded-protein response (UPR) upon endoplasmic reticulum (ER) stress, where the stress is assumed to be the accumulation of unfolded proteins in the ER. Consistent with previous in vitro studies of the ER-luminal domain of the mutant UPR initiator Ire1, our study show its association with a model unfolded protein in yeast cells. An Ire1 luminal domain mutation that compromises Ire1's unfolded-protein-associating ability weakens its ability to respond to stress stimuli, likely resulting in the accumulation of unfolded proteins in the ER. In contrast, this mutant was activated like wild-type Ire1 by depletion of the membrane lipid component inositol or by deletion of genes involved in lipid homeostasis. Another Ire1 mutant lacking the authentic luminal domain was up-regulated by inositol depletion as strongly as wild-type Ire1. We therefore conclude that the cytosolic (or transmembrane) domain of Ire1 senses membrane aberrancy, while, as proposed previously, unfolded proteins accumulating in the ER interact with and activate Ire1.     

How does each substituent functional group of oseltamivir lose its activity against virulent H5N1 influenza mutants?

To reveal the source of oseltamivir-resistance in influenza (A/H5N1) mutants, the drug-target interactions at each functional group were investigated using MD/LIE simulations. Oseltamivir in the H274Y mutation primarily loses the electrostatic and the vdW interaction energies at the –NH₃⁺ and –OCHEt₂ moieties corresponding to the weakened hydrogen-bonds and changed distances to N1 residues. Differentially, the N294S mutation showed small changes of binding energies and intermolecular interactions. Interestingly, the presence of different conformations of E276 positioned between the –OCHEt₂ group and the mutated residue is likely to play an important role in oseltamivir-resistant identification. In the H274Y mutant, it moves towards the –OCHEt₂ group leading to a reduction in hydrophobicity and pocket size, whilst in the N294S mutant it acts as the hydrogen network center bridging with R224 and the mutated residue S294. The molecular details have answered a question of how the H274Y and N294S mutations confer the high- and medium-level of oseltamivir-resistance to H5N1.