Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection.

The functional properties of the three most widely distributed α subunit isoforms of the Na,K‐ATPase are not well known, particularly concerning the voltage dependence of their activity and cation binding kinetics. We measured the electrogenic activity generated by Na,K‐ATPases resulting from co‐expression of the rat α1, α2* or α3* subunits with the rat β1 subunit in Xenopus oocytes; α2* and α3* are ouabain‐resistant mutants of the α2 and α3 isoform, which allowed selective inhibition of the endogenous Na+,K+‐pump of the oocyte. In oocytes expressing the three isoforms of the α subunit, K+ induced robust outward currents that were largely ouabain‐sensitive. In addition, ouabain‐sensitive inward currents were recorded for all three isoforms in sodium‐free and potassium‐free acid solutions. The very similar voltage dependence of the Na+,K+‐pump activity observed in the absence of extracellular Na+ indicated a similar stoichiometry of the transported cations by the three isoforms. The affinity for extracellular K+ was slightly lower for the α2* and α3* than for the α1 isoform. The α2* isoform was, however, more sensitive to voltage‐dependent inhibition by extracellular Na+, indicating a higher affinity of the extracellular Na+ site in this isoform. We measured and controlled [Na+]i using a co‐expressed amiloride‐sensitive Na+ channel. The intracellular affinity for Na+ was slightly higher in the α2* than in the α1 or α3* isoforms. These results suggest that the α2 isoform could have an activity that is strongly dependent upon [Na+]o and [K+]o. These concentrations could selectively modulate its activity when large variations are present, for instance in the narrow intercellular spaces of brain or muscle tissues.

BackgroundJapanese encephalitis virus (JEV) is a neurotropic flavivirus causing mortality and morbidity in humans. Severe Japanese encephalitis cases display strong inflammatory responses in the central nervous system and an accumulation of viral particles in specific brain regions. Microglia cells are the unique brain-resident immune cell population with potent migratory functions and have been proposed to act as a viral reservoir for JEV. Animal models suggest that the targeting of microglia by JEV is partially responsible for inflammatory reactions in the brain. Nevertheless, the interactions between human microglia and JEV are poorly documented.MethodsUsing human primary microglia and a new model of human blood monocyte-derived microglia, the present study explores the interaction between human microglia and JEV as well as the role of these cells in viral transmission to susceptible cells. To achieve this work, vaccine-containing inactivated JEV and two live JEV strains were applied on human microglia.ResultsLive JEV was non-cytopathogenic to human microglia but increased levels of CCL2, CXCL9 and CXCL10 in such cultures. Furthermore, human microglia up-regulated the expression of the fraktalkine receptor CX3CR1 upon exposure to both JEV vaccine and live JEV. Although JEV vaccine enhanced MHC class II on all microglia, live JEV enhanced MHC class II mainly on CX3CR1+ microglia cells. Importantly, human microglia supported JEV replication, but infectivity was only transmitted to neighbouring cells in a contact-dependent manner.ConclusionOur findings suggest that human microglia may be a source of neuronal infection and sustain JEV brain pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0675-3) contains supplementary material, which is available to authorized users.

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The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe