Additional file 4: Figure S4. of Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress

Inhibition of dehydroascorbate reductase activity catalyzed by rCsGSTo1 and 2 with praziquantel (PZQ). Lineweaver-Burk plots showing inhibition of rCsGSTo1 and 2 activities (1/v) versus 1/[DHA] (mM-1) (a, b) or rCsGSTo1 and 2 activities (1/v) versus 1/[GSH] (mM-1) (c, d) in the absence (diamond) and presence of 10 μM (rectangle), 50 μM (triangle) and 100 μM (circle) of PZQ, with variable concentrations of DHA and GSH (0.01–100 mM). Data are plotted in double reciprocal form. Insets demonstrate determination of Ki values. All assays were independently done in triplicate (n = 3, mean ± standard deviation, SD) and representative figures are shown. (TIF 207 kb

Additional file 3: Figure S3. of Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress

Determination of optimal temperature and pH. The effects of temperature a and pH b on enzymatic activity were determined by the standard dehydroascorbate reductase assay. The reactions were initiated by adding DHA and recorded for 5 min with temperature ranges from 4 °C to 55 °C. Sodium phosphate buffer (100 mM, pH 6.2–7.8) and Tris-HCl buffer (100 mM, pH 8.0–9.4) were used to observe optimal pH. All enzyme assays were independently performed in triplicate (n = 3, mean ± standard deviation, SD). (TIF 102 kb

Calibration of radiochromic EBT3 film using laser-accelerated protons

We present a proof of principle for onsite calibration of a radiochromic film (EBT3) using CR-39 as an absolute proton-counting detector and laser-accelerated protons as a calibration source. A special detector assembly composed of aluminum range filters, an EBT3 film, and a CR-39 detector is used to expose the EBT3 film with protons in an energy range of 3.65 MeV-5.85 MeV. In our design, the proton beam is divided into small beamlets and their projection images are taken on the EBT3 film and the CR-39 detector by maintaining a certain distance between the two detectors. Owing to the geometrical factor of the configuration and scattering inside the EBT3, the areal number density of protons was kept below the saturation level of the CR-39 detector. We also present a method to relate the number of protons detected on the CR-39 in a narrow energy range to protons with a broad energy spectrum that contribute to the dose deposited in the EBT3 film. The energy spectrum of protons emitted along the target normal direction is simultaneously measured using another CR-39 detector installed in a Thomson parabola spectrometer. The calibration curves for the EBT3 film were obtained in the optical density range of 0.01-0.25 for low dose values of 0.1 Gy-3.0 Gy. Our results are in good agreement with the calibrations of the EBT3 film that are traditionally carried out using conventional accelerators. The method presented here can be further extended for onsite calibration of radiochromic films of other types and for a higher range of dose values.11Nsciescopu

Additional file 5: Table S1. of Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress

Inhibitory mode of rCsGSTo1 and 2 by inhibitors. (DOCX 18 kb

Additional file 2: Figure S2. of Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress

Expression and purification of rCsGSTo1 and 2. a The full-length ORFs of CsGSTo1 and 2 were transformed into E. coli BL21. The recombinant proteins were induced with 0.1 mM IPTG for 4 h at 37 °C. The cells were sonicated and cleared by centrifugation. Soluble fractions were subjected to Ni-NTA affinity chromatography. His-tag was further removed by thrombin cleavage. Proteins were separated by 12 % reducing SDS-PAGE and stained with Coomassie brilliant G-250. Abbreviations: U, uninduced cell lysates; I, soluble fractions of the induced cells; W, washing fractions; E, purified rCsGSTo1 and 2; P, thrombin-cleaved rCsGSTo1 and 2. b His-tag removed rCsGSTo1 and 2 were separated by 12 % reducing SDS-PAGE, electroblotted to nitrocellulose membrane and probed with anti-rCsGSTo1 or anti-rCsGSTo2. The blots were developed with ECL. (TIF 531 kb