The regulatory roles of DDIT4 in TDCIPP-induced autophagy and apoptosis in PC12 cells.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is a commonly used organophosphate-based flame retardant and can bio-accumulate in human tissues and organs. As its structure is similar to that of neurotoxic organophosphate pesticides, the neurotoxicity of TDCIPP has raised widespread concerns. TDCIPP can increase neuronal apoptosis and induce autophagy. However, its regulatory mechanism remains unclear. In this study, we found that the expression upregulation of the DNA Damage-Inducible Transcript 4 (DDIT4) protein, which might play essential roles in TDCIPP-induced neuronal autophagy and apoptosis, was observed in TDCIPP-treated differentiated rat PC12 cells. Furthermore, we determined the protective effect of the DDIT4 suppression on the autophagy and apoptosis induced by TDCIPP using Western blot (WB) and Flow cytometry (FACS) analysis. We observed that TDCIPP treatment increased the DDIT4, the autophagy marker Beclin-1, and the microtubule-associated protein light chain 3-II (LC3II) expressions and decreased the mTOR phosphorylation levels. Conversely, the suppression of DDIT4 expression increased the p-mTOR expression and decreased cell autophagy and apoptosis. Collectively, our results revealed the function of DDIT4 in cell death mechanisms triggered by TDCIPP through the mTOR signaling axis in differentiated PC12 cells. Thus, this study provided vital evidence necessary to explain the mechanism of TDCIPP-induced neurotoxicity in differentiated PC12 cells.

Artificial clickase-triggered fluorescence "turn on" based on a click bio-conjugation strategy for the immunoassay of food allergenic protein.

Herein, based on an artificial clickase-catalyzed bio-conjugation strategy, we established a sensitive fluorescent clickase-linked immunosorbent assay (FCLISA) platform using an oligonucleotide-molecular beacon (Oligo-MB) hairpin structure as a fluorescence switch for detection of food allergenic protein. Firstly, a highly stable Cu(I)-containing nanocube was prepared for usage as an artificial clickase, which could catalyze the bio-conjugation of two short oligonucleotides (i.e., Oligo-A and Oligo-B labeled by a 5'-alkyne and a 3'-azide group, respectively) through clickase-catalyzed azide/alkyne cycloaddition reaction. Subsequently, the formed long-chain oligonucleotide (Oligo-A-B) could hybridize with Oligo-MB hairpin to open hairpin structure, leading to its fluorescence turn on. By using clickase as an alternative enzymatic label in conventional ELISAs, the established FCLISA showed high sensitivity and accuracy in detection of casein, achieving a limit of detection as low as 1.5 × 10-8 g/mL. Additionally, FCLISA has been challenged by detecting the casein in real samples, indicating a great potential in food safety assay.

An aptamer-assisted biological nanopore biosensor for ultra-sensitive detection of ochratoxin A with a portable single-molecule measuring instrument.

Biological nanopore-based single-molecule detection technology has shown ultrahigh sensitivity to various target analyte. But the detection scope of interesting targets is limited due to the lack of effective signal conversion strategies. In addition, conventional nanopore detection instruments are cumbersome, resulting nanopore detection can only be performed in laboratory. Herein, a customizable nanopore current amplifier is constructed to lower the cost and increase the portability of the nanopore instrument, and then an immobilized aptamer-based signal conversion strategy is proposed for α-hemolysin (α-HL) nanopore to detect small molecules (ochratoxin A, OTA). The presence of OTA in sample would trigger the release of probe single-strand DNA (ssDNA) from magnetic beads, which could subsequently cause current blockage in nanopore. The results show that the signal frequency of probe ssDNA has a linear relationship with the OTA concentration in the range of 2 × 101～2 × 103 pmol/L. Compared to other methods, our sensing system has achieved an ultra-sensitive detection of OTA with the detection limit as low as 1.697 pmol/L. This strategy could broaden the scope of nanopore detection and have the potential for rapid and in-situ detection of other food contaminants in the future.