BackgroundIn T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective Kv1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases.MethodsWe combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin.ResultsThe peptide completely blocked Kv1.3 channels currents without impairing TEM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-γ and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of Kv1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery.ConclusionsThe Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of Kv1.3 channels in regulating TEM lymphocyte function.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0257-7) contains supplementary material, which is available to authorized users.
Two 1,4-benzoquinone derivatives, found in the venom of the scorpionDiplocentrus melicifollowing exposure to air, have been isolated, characterized, synthesized, and assessed for antimicrobial activities. Initially a white, viscous liquid, the extracted venom colors within minutes under ambient conditions. From this colored mixture, two compounds, one red, the other blue, were isolated and purified using chromatography. After a variety of NMR and mass spectrometry experiments, the red compound was determined to be 3,5- dimethoxy-2-(methylthio)cyclohexa-2,5-diene-1,4-dione, and the blue compound was determined to be 5-methoxy-2,3- bis(methylthio)cyclohexa-2,5-diene-1,4-dione. Because extremely small amounts of these compounds were isolated from the scorpion venom, we developed laboratory syntheses from commercially available precursors, allowing us to produce sufficient quantities for crystallization and biological assays. The red benzoquinone is effective againstStaphylococcus aureus[minimum inhibitory concentration (MIC) = 4 µg/mL], while the blue benzoquinone is active againstMycobacterium tuberculosis(MIC = 4 µg/mL) and even against a multidrug-resistant (MDR) strain with nearly equal effectiveness. The bactericidal effects of both benzoquinones show comparable activity to commercially available antibiotics used against these pathogens and were cytotoxic to neoplastic cell lines, suggesting their potential as lead compounds for the development of novel antimicrobial and anticancer drugs. Importantly, the blue benzoquinone was also effective in vivo with mouse models of MDR tuberculosis infection. After treatment for 2 mo, four mice with late-stage active MDR tuberculosis had a significant decrease in pulmonary bacillary loads and tissue damage. Healthy mice served as negative controls and tolerated treatment well, without adverse side effects.
Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus —a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics—via growth in the presence of distinct azido (-N 3 ) analogues of 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N 3 -Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy- d -arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N 3 -Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide–alkyne cycloaddition (SPAAC) “click” chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N 3 -Kdo and 7-N 3 -Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N 3 -Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N 3 -Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N 3 -Kdo is indeed a substrate of the enzyme, 7-N 3 -Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N 3 -Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N 3 -Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.
The aim of this study was to establish and validate an alternative high‐performance liquid chromatography method for simultaneous quantification of pyrazinamide, isoniazid, acetyl‐isoniazid and rifampicin in plasma of patients under treatment for tuberculosis. The performed method was lineal (r2 > 0.99) in the range of 2.00–50.00 μg/mL for pyrazinamide, 0.50–20.00 μg/mL for both acetyl‐isoniazid and isoniazid, and 1.20–25.00 μg/mL for rifampicin. Precision and trueness were demonstrated with coefficient of variation < 15% and deviations < 15%, respectively, for quality controls samples. The lower limits of quantification were 2.00, 0.50, 0.50, and 1.20 μg/mL for pyrazinamide, isoniazid, acetyl‐isoniazid and rifampicin, respectively. The method was applied for the analysis of plasma from patients with tuberculosis. This method allowed ensuring reliable quantification of the target compounds and their pharmacokinetics parameters. In general, the mean values of maximum concentration of each antituberculosis drug were located within their respective reference therapeutic ranges. However, patients with sub‐therapeutic plasma concentrations of isoniazid and rifampicin were detected. This is the first analytical technique that simultaneously quantifies isoniazid, acetyl‐isoniazid, rifampicin, and pyrazinamide concentrations from plasma samples by high‐performance liquid chromatography with ultraviolet/visible. The proposed method could be applied for therapeutic drug monitoring and pharmacokinetics studies of the four compounds throughout the treatment of tuberculosis patients.
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