Sai Zhang,Ruixi Yang,Yaping Wang,Qinqin Gao,Langhong Wang,Guisheng Zhou,Qinglin Sheng
In recent years, food-borne pathogen Pseudomonas cocovenenans have caused many food safety incidents. Therefore, the detection of toxoflavin produced by the bacteria has aroused widespread attention. In this study, two fluorescent sensors were prepared using nitrogen-doped carbon dots (NCDs) materials as precursors: (1) Luminol-functionalized NCDs were synthesized by NHS-EDC reaction, and L-NCDs fluorescence sensor was constructed for the detection of toxoflavins in actual food samples. The linear range of the L-NCDs fluorescence sensor is 0.1–50 μM, the detection limit is 0.05 μM and the excitation wavelength is 375 nm. (2) The NCDs-CMC fluorescent film sensor was constructed by incorporating NCDs into sodium carboxymethyl cellulose. It was excited at a wavelength of 385 nm for visual detection of toxoflavin. The film sensor has a linear range of 1–60 μM and a detection limit of 0.45 μM. The results showed that the method has good sensitivity and reliability. This study extends the application of NCDs-based fluorescence sensing to detect toxoflavin. It enables simple, rapid, low-cost sensitive detection and visual monitoring of toxoflavin. Additionally, it offers a fast and convenient new technology for preventing and controlling Pseudomonas cocovenenans contamination in food.
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 IC 50 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 IC 50 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.
危险品化学品经营许可证（带存储）