Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development.

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 µg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 µg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

Effects of thiram exposure on liver metabolism of chickens.

Pesticides are widely used to control crop diseases, which have made an important contribution to the increase of global crop production. However, a considerable part of pesticides may remain in plants, posing a huge threat to animal safety. Thiram is a common pesticide and has been proven that its residues in the feed can affect the growth performance, bone formation, and intestinal health of chickens. However, there are few studies on the liver metabolism of chickens exposed to thiram. Here, the present study was conducted to investigate the effect of thiram exposure on liver metabolism of chickens. Metabolomics analysis shows that 62 metabolites were down-regulated (ginsenoside F5, arbekacin, coproporphyrinogen III, 3-keto Fusidic acid, marmesin, isofumonisin B1, 3-Hydroxyquinine, melleolide B, naphazoline, marmesin, dibenzyl ether, etc.) and 35 metabolites were up-regulated (tetrabromodiphenyl ethers, deoxycholic acid glycine conjugate, L-Palmitoylcarnitine, austalide K, hericene B, pentadecanoylcarnitine, glyceryl palmitostearate, quinestrol, 7-Ketocholesterol, tetrabromodiphenyl ethers, etc.) in thiram-induced chickens, mainly involved in the metabolic pathways including glycosylphosphatidylinositol (GPI)-anchor biosynthesis, porphyrin and chlorophyll metabolism, glycerophospholipid metabolism, primary bile acid biosynthesis and steroid hormone biosynthesis. Taken together, this research showed that thiram exposure significantly altered hepatic metabolism in chickens. Moreover, this study also provided a basis for regulating the use and disposal of thiram to ensure environmental quality and poultry health.

[Effects of Osthole on Apoptosis and Autophagy of Human Tongue Cancer Tca8113 Cells].

Objective To investigate the effects of osthole on the proliferation,apoptosis,and autophagy of human tongue cancer Tca8113 cells and explore its possible mechanism of action. Methods Tca8113 cells were cultured in vitro and divided into a control group without drugs and the experimental groups with 40,80,120,and 160 µmol/L osthole.The inhibitory effect of osthole on the proliferation of Tca8113 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay.Hoechst33342 staining method and annexin V-FITC/propidium iodide method were employed to detect the effect of osthole on the apoptosis of Tca8113 cells within 24 hours.Western blot was performed to detect the expression of apoptosis-related proteins(Bcl-2,Bax,and cleaved caspase-3)and autophagy-related proteins(LC3 and p62)in Tca8113 cells exposed to osthole. Results Osthole significantly inhibited the proliferation and induced the apoptosis of Tca8113 cells in a concentration-dependent manner,and it reduced the cell colony formation.Western blot results showed that osthole could up-regulate the expression of Bax and cleaved caspase-3 and down-regulate that of Bcl-2.At the same time,it increased the expression of LC3Ⅱ and P62 and reduced that of LC3Ⅰ. Conclusion Osthole may inhibit the proliferation of Tca8113 cells by promoting cell apoptosis and blocking autophagy flow to inhibit autophagy.

Immunoregulatory and Antimicrobial Activity of Bovine Neutrophil ß-Defensin-5-Loaded PLGA Nanoparticles against Mycobacterium bovis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex imposing a high zoonotic threat to human health. The limited efficacy of BCG (Bacillus Calmette-Guérin) and upsurges of drug-resistant tuberculosis require new effective vaccination approaches and anti-TB drugs. Poly (lactic-co-glycolic acid) (PLGA) is a preferential drug delivery system candidate. In this study, we formulated PLGA nanoparticles (NPs) encapsulating the recombinant protein bovine neutrophil ß-defensin-5 (B5), and investigated its role in immunomodulation and antimicrobial activity against M. bovis challenge. Using the classical water-oil-water solvent-evaporation method, B5-NPs were prepared, with encapsulation efficiency of 85.5% ± 2.5%. These spherical NPs were 206.6 ± 26.6 nm in diameter, with a negatively charged surface (ζ-potential -27.1 ± 1.5 mV). The encapsulated B5 protein from B5-NPs was released slowly under physiological conditions. B5 or B5-NPs efficiently enhanced the secretion of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-10 in J774A.1 macrophages. B5-NPs-immunized mice showed significant increases in the production of TNF-α and immunoglobulin A (IgA) in serum, and the proportion of CD4+ T cells in spleen compared with B5 alone. In immunoprotection studies, B5-NPs-immunized mice displayed significant reductions in pulmonary inflammatory area, bacterial burden in the lungs and spleen at 4-week after M. bovis challenge. In treatment studies, B5, but not B5-NPs, assisted rifampicin (RIF) with inhibition of bacterial replication in the lungs and spleen. Moreover, B5 alone also significantly reduced the bacterial load in the lungs and spleen. Altogether, our findings highlight the significance of the B5-PLGA NPs in terms of promoting the immune effect of BCG and the B5 in enhancing the therapeutic effect of RIF against M. bovis.