An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.

PIM protein family, short-lived serine/threonine kinases (PIM1, PIM2 and PIM3), are weak oncogenes but contribute to tumorigenesis as cancer targets. Thus, design of a novel pan-PIM inhibitor is still a challenge for current cancer drug discovery. Herein, we used a Naïve Bayesian model to construct the PIM network and identified Bad and Hsp90 to interact with PIMs. Then, we screened a series of candidate small-molecule compounds targeting PIMs, and subsequently synthesized a novel small-molecule compound PI003 with remarkable anti-proliferative activities in cervical cancer cells. Moreover, we found that PI003 induced apoptosis via the death-receptor and mitochondrial pathways by targeting PIMs and affecting Bad and Hsp90. Combined with microRNA microarray analyses, we demonstrated that some microRNAs such as miR-1296 and miR-1299 could affect PIM1-STAT3 pathway in PI003-induced apoptosis. Finally, we reported that PI003 had remarkable anti-tumor activity and apoptosis-inducing effect in in vivo mouse model. In conclusion, these results demonstrate that PI003, as a novel synthesized pan-PIM inhibitor, induces the death-receptor and mitochondrial apoptosis involved in microRNA regulation, and also possessed remarkable anti-tumor activity and apoptosis-inducing effect in vivo. Thus, these findings would shed light on discovering more potential new small-molecule pan-PIM inhibitors in future cervical cancer therapy.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (∆Ψ m), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ∆Ψ m. RSV treatment could increase the rate of high mitochondrial ∆Ψ m of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor-and mitochondria-mediated apoptotic pathways.

Deoxynivalenol is one the of most common mycotoxins in cereals and grains and causes a serious health threat to poultry and farm animals. Our previous study found that DON decreased the production performance of laying hens. It has been reported that DON could exert significant toxic effects on the intestinal barrier and microbiota. However, whether the decline of laying performance is related to intestinal barrier damage, and the underlying mechanisms of DON induced intestine function injury remain largely unclear in laying hens. In this study, 80 Hy-line brown laying hens at 26 weeks were randomly divided into 0, 1, 5 and 10 mg/kg.bw (body weight) DON daily for 6 weeks. The morphology of the duodenum, the expression of inflammation factors and tight junction proteins, and the diversity and abundance of microbiota were analyzed in different levels of DON treated to laying hens. The results demonstrated that the mucosal detachment and reduction of the villi number were presented in different DON treated groups with a dose-effect manner. Additionally, the genes expression of pro-inflammatory factors IL-1β, IL-8, TNF-α and anti-inflammatory factors IL-10 were increased or decreased at 5 and 10 mg/kg.bw DON groups, respectively. The levels of ZO-1 and claudin-1 expression were significantly decreased in 5 and 10 mg/kg.bw DON groups. Moreover, the alpha diversity including Chao, ACE and Shannon indices were all reduced in DON treated groups. At the phylum level, Firmicutes and Actinobacteria and Bacteroidetes, Proteobacteria, and Spirochaetes were decreased and increased in 10 mg/kg.bw DON group, respectively. At the genus levels, the relative abundance of Clostridium and Lactobacillus in 5 and 10 mg/kg.bw DON groups, and Alkanindiges and Spirochaeta in the 10 mg/kg.bw DON were significantly decreased and increased, respectively. Moreover, there were significant correlation between the expression of tight junction proteins and the relative abundance of Lactobacillus and Succinispira. These results indicated that DON exposure to the laying hens can induce the inflammation and disrupt intestinal tight junctions, suggesting that DON can directly damage barrier function, which may be closely related to the dysbiosis of intestinal microbiota.

No abstract