As indicated in Fig. 8, the 15 min incubations with high drug concentrations (1 mM diamide, 50 mM H2O2, or 50 ��M MB) led to changes in the JQ1 Epigenetic Reader Do inhibitor total thiol status. All conditions �C including the 24 h incubations at pharmacologically meaningful drug concentrations (4��IC50 of methylene blue or artemisinin) �C led to a pronounced drop in glutathione concentrations. Figure 8 Total thiols and total glutathione concentrations in P. falciparum after drug treatment. Interestingly, only the most stressful condition (1 mM diamide), which led to partial loss of protein in short-term experiments and was only survived by a part of the cells at longer incubations, induced changes in the ratio of reduced to oxidized thioredoxin (Fig. S8).

Discussion hGrx1-roGFP2 as a dynamic biosensor in Plasmodium falciparum Due to the great importance of cellular redox reactions in malaria parasites and the mechanism of action of antimalarial drugs, it would be of great value to be able to follow the intracellular glutathione redox potential in living cells. We therefore tested the applicability of the ratiometric hGrx1-roGFP2 redox sensor in Plasmodium falciparum. The parasites could be transfected with the probe, and a stable expression of the protein without decomposition products was determined under (patho)physiologically and pharmacologically meaningful conditions. Furthermore, a disturbing influence of host cell hemoglobin autofluorescence could be ruled out. In accordance with previous studies [31], we observed a rapid, dynamic, and ratiometric response of hGrx1-roGFP2 upon oxidation with diamide and reduction with DTT, which indicates its applicability as a live cell EGSH biosensor in P.

falciparum. Interestingly, when comparing the basal hGrx1-roGFP2 redox ratios of the CQ-sensitive P. falciparum 3D7 strain and the CQ-resistant Dd2 strain, the latter constantly showed slightly lower values (0.59��0.09 in 3D7 vs. 0.29��0.08 in Dd2). This observation might at least partially be explained by higher concentrations of total glutathione in Dd2, which have been previously reported [21] and might induce a stronger basal reduction of hGrx1-roGFP2 in Dd2. The basal redox ratio of the hGrx1-roGFP2 sensor primarily depends on the GSH/GSSG ratio. However, as shown by Meyer et al. [33], the total glutathione concentration can also influence the fluorescence ratio of the sensor.

This is not unexpected since the glutathione redox potential also depends on both the GSH/GSSG ratio and the glutathione concentration. Therefore, when employing the hGrx1-roGFP sensor to Batimastat monitor drug effects in living Plasmodium falciparum parasites, it is important to thoroughly monitor the basal redox ratio and then follow the changes in direct comparison with control cells. When interpreting the results, one must keep in mind that the observed changes can be due to a change in the GSH/GSSG ratio and/or to changes in the total glutathione concentration [24], [33], which can occur, e.g.