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.

Identification of Biomarker IL2Rß in Ankylosing Spondylitis via Multi-Chip Integration Analysis of Gene Differential Expression.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease that affects axial joints such as the spine. Early diagnosis is essential to improve treatment outcomes. The purpose of this study is to uncover underlying genetic diagnostic features of AS. METHODS: We downloaded gene expression data from the Gene Expression Omnibus (GEO) database for three studies of groups of healthy and AS samples. After preprocessing and normalizing the data, we employed linear models to identify significant differentially expressed genes (DEGs) and further integrated the differential genes to acquire reliable differential transcriptional markers. Gene functional enrichment analysis was conducted to obtain enriched pathways and regulatory gene interactions were extracted from pathways to further elucidate pathway networks. Seventy-three reliably differentially expressed genes (DEGs) were integrated by differential analysis. Utilizing the regulatory relationships of the 21 Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway genes that were enriched in the analysis, a regulatory network of 622 genes was constructed and its topological properties were further analyzed. RESULTS: Functional enrichment analysis found 73 DEGs that were strongly associated with immune pathways like Th17, Th1 and Th2 cell differentiation. Using KEGG combined with DEGs, six hub genes (KLRD1, HLA-DRB3, HLA-DRB5, IL2Rß, CD247, and CXCL10) were suggested from the network. Of these, the IL2Rß gene was significantly differentially expressed compared with the normal control. CONCLUSION: IL2Rß (Interleukin-2 receptor beta) is strongly associated with the onset and progression of autoimmune joint diseases, and may be used as a potential biomarker of AS. This study offers new characteristics that can help in the diagnosis and individualized therapy of AS.

Production of a Natural Dihydropteroate Synthase and Development of a Signal-Amplified Pseudo-Immunoassay for the Determination of Sulfonamides in Pork.

In this study, a type of magnetic photoaffinity-labeled activity-based protein profiling probe for sulfonamide drugs was first synthesized for the purpose of capturing the natural dihydropteroate synthase of Escherichia coli by using simple incubation and magnetic separation. After characterization of its identity with LC-ESI-MS/MS, this enzyme was used as a recognition reagent to develop a direct competitive pseudo-ELISA for the determination of the residues of 40 sulfonamides in pork. Because of the use of streptavidin-horseradish peroxidase and biotinylated horseradish peroxidase as a signal-amplified system, the limits of detection for the 40 drugs were in the range of 0.001-0.016 ng/mL. Compared to the steps in a conventional assay formation, the operation steps were the same, but the sensitivities increased 32-88-fold. Furthermore, the assay performances were better than the previously reported immunoassays performances for sulfonamides. Therefore, this method could be used as a practical tool for multiscreening the trace levels of sulfonamides residues in food samples.

Molecularly imprinted microspheres based multiplexed fluorescence method for simultaneous detection of benzimidazoles and pyrethroids in meat samples.

Two molecularly imprinted microspheres and two fluorescent tracers for benzimidazoles and pyrethroids were synthesized respectively. The two types of microspheres were coated in the wells of conventional microplate simultaneously. Then the sample extracts and the two traces were added for differential competition. The fluorescence intensities at two different emission wavelengths were excited and recorded for quantification of the two classes of drugs respectively. The optimized multiplexed fluorescence method could be used to determine 8 benzimidazoles and 10 pyrethroids in mutton and beef samples simultaneously. The limits of detection of the method for the 18 drugs were in the range of 5.2-17 ng/mL, and the recoveries from the standards fortified blank samples were in the range of 67.7%-109%. From the analysis of 60 real mutton and beef samples, this method could be used for multi-screening the residues of benzimidazoles and pyrethroids in meat samples.

Production of generic monoclonal antibody and development of chemiluminescence immunoassay for determination of 32 sulfonamides in chicken muscle.

A hapten of sulfabenzamide was first synthesized to generate a monoclonal antibody that simultaneously recognized 32 sulfonamides. The computational simulation showed that the 3D conformation, molecular bend angle, molecular volume, electronic charge of core structure of these drugs all showed influences on the antibody binding. The antibody was combined with a heterologous enzyme-labeled hapten to develop a direct competitive chemiluminescence enzyme linked immunosorbent assay for determination of the 32 sulfonamides in chicken muscle sample. The CRs of the optimized method for these drugs were in the range of 7.3%-1778%, and the IC50 values were in the range of 0.038-11.2 ng/g. The limits of detection for detection of these drugs in chicken were in the range of 0.03-26 ng/g. Their recoveries from the standards fortified blank chicken samples were in the range of 60.8%-97.1%. Therefore, this method could be used as a useful tool for routine screening sulfonamides residues in meat.

Detection of chloramphenicol in meat with a chemiluminescence resonance energy transfer platform based on molecularly imprinted graphene.

In this study, a novel composite was synthesized by polymerizing the dummy-template molecularly imprinted microspheres on the surface of magnetic graphene. This composite was used as recognition reagent and energy acceptor to develop a platform for determination of chloramphenicol according to the principle of chemiluminescence resonance energy transfer. The light signal was induced with luminolH2O24-(imidazole-1-yl)phenol system, and the chemiluminescence intensity was positively correlated with the analyte concentration. The limit of detection for chloramphenicol in meat sample was 2.0 pg/g, and the recoveries from the standard fortified blank meat sample were in the range of 69.5%-97.3%. Furthermore, one single assay could be finished within 10 min, and the magnetic composite could be reused for at least thirty times. Therefore, this platform could be used as a rapid, simple, sensitive, accurate and recyclable tool for screening the residue of chloramphenicol in meat.

MiR-144 Inhibits Tumor Growth and Metastasis in Osteosarcoma via Dual-suppressing RhoA/ROCK1 Signaling Pathway.

Several microRNAs (miRNAs) have been found expressed differentially in osteosarcoma (OS), so they may function in the onset and progression of OS. In this study, we found that miR-144 significantly suppresses osteosarcoma cell proliferation, migration, and invasion ability in vitro and inhibited tumor growth and metastasis in vivo. Mechanically, we demonstrated that Ras homolog family member A (RhoA) and its pivotal downstream effector Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) were direct targets of miR-144. Moreover, the negative correlation between down-regulated miR-144 and up-regulated ROCK1/RhoA was verified in both OS cell lines and clinical patients' specimens. Functionally, RhoA with or without ROCK1 co-overexpression resulted a rescue phenotype on miR-144 inhibited cell growth, migration, and invasion abilities whereas individual overexpression of ROCK1 had no statistical significance compared with controls in miR-144-transfected SAOS2 and U2-OS cells. Taken together, this study demonstrates that miR-144 inhibited tumor growth and metastasis in OS via dual-suppressing of RhoA and ROCK1, which could be a new therapeutic approach for the treatment of OS.

Preparation of a chemiluminescence sensor for multi-detection of benzimidazoles in meat based on molecularly imprinted polymer.

In this study, a molecularly imprinted polymer capable of recognizing 8 benzimidazoles was first synthesized. The computation simulation showed that the shape and size of used template were the main factors influencing its recognition ability. Then the polymer was used as recognition reagent to prepare a chemiluminescence sensor on conventional 96-well microplate. The sample solution and a HRP-labeled hapten were added into the microplate wells to perform competitive binding, and the light signal was initiated with 4-(imidazol-1-yl)phenol enhanced luminol-H2O2 system. The optimized sensor was used to determine the residues of 8 benzimidazoles in mutton and beef. Result showed that the sensor achieved ultrahigh sensitivity (limits of detection of 1.5-21 pg/mL), rapid assay process (18 min) and satisfactory recovery (65.8%-91.2%). Furthermore, this sensor could be reused for 4 times. Therefore, this sensor could be used as a rapid, simple, sensitive and durable tool for screening the residual benzimidazoles in meat.