Development of a receptor based signal amplified fluorescence polarization assay for multi-detection of 35 sulfonamides in pork.

With the increasing focus on food security, a screening method with high-throughput, ultra-sensitivity, and user-friendly operation is urgently needed for monitoring of sulfonamides residues in animal-derived foods. In this study, the sulfonamides' receptor dihydropteroate synthase of Staphylococcus aureus was subjected to saturate mutation, and a mutant with higher affinities for sulfonamides was obtained. The mutant was then used as recognition material to establish a fluorescence polarization assay for determination of 35 sulfonamides in pork. Due to the use of an enhanced fluorescent tracer containing two fluorophore molecules, the sensitivities for the 35 sulfonamides were improved for 2.8-8.6 folds (LODs 0.03-1.16 ng/mL) in comparison with using conventional fluorescent tracer. The present method outperformed all previous fluorescence polarization (immuno)assays for sulfonamides due to its broader spectrum, higher sensitivity, and shorter assay time. Furthermore, this is the first study reporting an enhanced fluorescence polarization assay for determination of small molecule substance.

Function and regulation mechanism of Chk1 during meiotic maturation in porcine oocytes.

Checkpoint 1 (Chk1), as an important member of DNA replication checkpoint and DNA damage response, has an important role during the G2/M stage of mitosis. In this study, we used porcine oocyte as a model to investigate the function of Chk1 during porcine oocyte maturation. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages, mainly localized in the cytoplasm at GV stage and moved to the spindle after germinal vesicle breakdown (GVBD). Chk1 depletion not only induced oocytes to be arrested at MI stage with abnormal chromosomes arrangement, but also inhibited the degradation of Cyclin B1 and decreased the expression of Mitotic Arrest Deficient 2-Like 1 (Mad2L1), one of spindle assembly checkpoint (SAC) proteins, and cadherin 1 (Cdh1), one of coactivation for anaphase-promoting complex/cyclosome (APC/C). Moreover, Chk1 overexpression delayed GVBD. These results demonstrated that Chk1 facilitated the timely degradation of Cyclin B1 at anaphase I (AI) and maintained the expression of Mad2L1 and Cdh1, which ensured that all chromosomes were accurately located in a line, and then oocytes passed metaphase I (MI) and AI and exited from the first meiotic division successfully. In addition, we proved that Chk1 had not function on GVBD of porcine oocytes, which suggested that maturation of porcine oocytes did not need the DNA damage checkpoint, which was different from the mouse oocyte maturation.