Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells In Vitro and In Vivo

Background

Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro.

Methods & Findings

Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production

Conclusions

This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.     

Arachidonic acid effect on the allosteric gating mechanism of BK (Slo1) channels associated with the β1 subunit

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary β1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with β1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca²⁺ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the β1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary β1 subunit.     

How important is it to consider lineage diversification heterogeneity in macroevolutionary studies? Lessons from the lizard family Liolaemidae

Macroevolutionary and biogeographical studies commonly apply multiple models to test state-dependent diversification. These models track the association between states of interest along a phylogeny, although many of them do not consider whether different clades might be evolving under different evolutionary drivers. Yet, they are still commonly applied to empirical studies without careful consideration of possible lineage diversification heterogeneity along the phylogenetic tree. A recent biogeographic study has suggested that orogenic uplift of the southern Andes has acted as a species pump, driving diversification of the lizard family Liolaemidae (307 described species), native to temperate southern South America. Here, we argue against the Andean uplift as main driver of evolution in this group. We show that there is a clear pattern of heterogeneous diversification in the Liolaemidae, which biases state- and environment-dependent analyses in, respectively, the GeoSSE and RPANDA programs. We show here that there are two shifts to accelerated speciation rates involving two clades that have both been classified as having “Andean” distributions. We incorporated the Geographic Hidden-State Speciation and Extinction model (GeoHiSSE) to accommodate unrelated diversification shifts, and also re-analyzed the data in RPANDA program after splitting biologically distinct clades for separate analyses, as well as including a more appropriate set of models. We demonstrate that the “Andean uplift” hypothesis is not supported when the heterogeneous diversification histories among these lizards is considered. We use the Liolaemidae as an ideal system to demonstrate potential risks of ignoring clade-specific differences in diversification patterns in macroevolutionary studies. We also implemented simulations to show that, in agreement with previous findings, the HiSSE approach can effectively and substantially reduce the level of distribution-dependent models receiving the highest AIC weights in such scenarios. However, we still find a relatively high rate (15%) of distribution-dependent models receiving the highest AIC weights, and provide recommendations related to the set of models included in the analyses that reduce these rates by half. Finally, we demonstrate that trees including clades following different dependent-drivers affect RPANDA analyses by producing different outcomes, ranging from partially correct models to completely misleading results. We provide recommendations for the implementation of both programs.     

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: separation and identification of contact sequences of neighboring proteins after CNBr fragmentation

Mammalian ribosomal proteins were cross-linked in situ with the primarily cysteine-selective heterobifunctional reagents N-succinimidyl 2-(4-hydroxy-2-maleimidophenylazo)benzoate (reagent A, maximum range approx. 8 A) and N-succinimidyl 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoate (reagent B, maximum range approx. 12 A). With reagent B the secondarily attached (N-aryolated) protein becomes labelled specifically at the receptor amino group (lysine). The cross-linked proteins were fragmented with CNBr in attempts to isolate and identify sequences involved in the next-neighbor contacts. Two experimental schemes were adopted. Heavy complexes containing the large protein L4 cross-linked to protein L14 and/or L18 were isolated and treated with CNBr. The split products were submitted to diagonal electrophoresis for separation and identification of the two pairs of contact fragments. Proteins cross-linked with the radiolabelled reagent B were submitted to diagonal electrophoresis. The labelled receptor proteins were excised and treated with CNBr. Fragments carrying the contact sequences were separated by gradient gel electrophoresis and identified by autoradiography. By use of these methods CNBr fragments were isolated containing one or the dual contact sites of the following binary protein complexes: L4-L14, L4-L18, L4-L13a/L18a, L6'-L23, L6-L29, L7-L29, L14-L13a, L21-L18a, and L27-L30 (asterisks indicate the labelled receptor proteins). By varying the site of labelling of the heterobifunctional reagents and the methods of protein fragmentation a complete analysis of the contact sequences of these proteins should be possible.     

An analyzer and monitor for rapid microscale peptide separations

An automatic peptide analyzer is described, which can be used to monitor quantitatively preparative microscale separations of protein hydrolysates, using microbore ion exchange columns in combination with volatile buffers. Complex protein mixtures can be effectively resolved within 5 h without deterioration of the elution profiles. By use of a novel system of split valves well-defined aliquots of the separated protein digest (5–25 nmol) are diverted into the monitor at regular intervals, while the rest of the eluate is collected intact for further characterization. Using low splitting ratios the peptide peaks become nearly indistinguishable from those obtained in analytical separations. At higher splitting ratios (1:5, 1:10) the sampling of early, rapidly eluting fractions becomes somewhat inaccurate. This is outweighed by the small amounts of material (less than 1 nmol) consumed by the analyzer under these conditions.