Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

Modification in Silver Staining Procedure for Enhanced Protein Staining.

Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein silver staining technology has higher sensitivity and is suitable for the detection of low-concentration proteins compared to other staining techniques including the Coomassie brilliant blue detection method. The present study was conducted to enhance the detection ability of the protein staining method. Herein, we modified the recipe of silver staining, a very reproducible method, by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. Furthermore, the particle size and potentiometer were used to detect the particle size and potential difference of the silver ions in the prepared dyeing materials, and then, the morphology, transparency, and size of the dyed silver particles in different dyeing solutions were studied using a transmission electron microscopy (TEM). The obtained results revealed that the use of 0.5% of AMP, PVP, Tween-80, and xylene improved the staining ability of protein silver staining, compared with the original method. Furthermore, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. These results suggested a new idea for further improving the detection ability of protein silver staining.

Application value of real-time shear wave elastography in differential diagnosis of testicular torsion.

AIM: To evaluate the clinical value of real-time shear wave elastography (SWE) in differential diagnosis of testicular torsion and acute orchiditis. MATERIAL AND METHODS: During a 3-year period, 14 cases of testicular torsion and 16 cases of acute orchiditis met the inclusion criteria. Young's modulus maximum hardness (Emax) of testicular capsule region, middle testicular parenchyma, warped spermatic segment or inferior spermatic segment was measured in each group. SWE "stiff ring sign" of testis refers to the appearance of a red ring in the testicular capsule area and "stiff knot sign" of spermatic cord refers to the appearance of a red knot in the lower segment of the spermatic cord. RESULTS: Emax value of the testicular capsule in the torsion group was higher than in the acute inflammation group (138.76±58.27 vs 16.40±4.71 kPa, p=0.0001). Emax value in the middle parenchyma of the testis showed no statistically significant difference between groups (p=0.053). Emax value in the twisted spermatic segment was higher than that in the lower spermatic segment with acute inflammation (166.61±60.07 vs 14.14±4.93, p=0.0001). In the torsion group, 12 testicular capsule areas showed "stiff ring sign" and all twisted segments of spermatic cord showed "stiff knot sign" but no signs were found in the inflammatory group. CONCLUSIONS: "Stiff ring sign" of testis, "stiff knot sign" of spermatic cord, high stiffness of the testicular capsule and in the twisted spermatic segment are the typical SWE findings of testicular torsion, with important clinical value in the differential diagnosis of testicular torsion and acute orchiditis.