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Zygote injection of CRISPR/Cas9 system is an efficient method for generation of gene modified animals. Efficiency of zygote collection, embryo survival rate after micro-injection and pregnancy rate of surrogate are the key influence factors for the generation of gene modified animals using this strategy. Here, we investigated the influence of CIDR treatment at different time during the estrus cycle to the superovulation, and analyzed the influence of the estrus terminal time between embryo donor and embryo receptor to the pregnancy rate in Ziwuling black goats. We designed three sgRNAs targeting myostatin ( MSTN ) and also optimized the micro-injected parameter of the CRISPR/Cas9 system. The results showed that embryo donor goats effectively increase the corpus luteum amount (15.25±3.69) and zygote amount (13.875±4.015) when they were treated with CIDR at the fourth day of estrus cycle. The mixture including Cas9 mRNA (60 ng/μL) and sgRNAs (60 ng/μL) targeting MSTN was injected into zygote cytoplasm using a micromanipulator at the optimized injection parameters (Pi, Ti and Pc was 300, 0.5 and 60, respectively). Embryo cleavage rate was 76.79 % after 24 h of embryo injection. Pregnancy rate of embryo receptors whose terminal estrus time was delayed 12–16 h compared with that of embryo donor was the highest (38.46%). A total of 19 living lambs were obtained, and indels were detected in all the lambs using TA cloning sequencing. Overall, we established the technological system based on the zygote injection of CRISPR/Cas9 in Ziwuling black goats. The established technological system is the basic for generation of gene modified goats.

The aim of this study was to develop an analytical method for the simultaneous determination of ultra-trace levels of 35 organophosphorus pesticide residues in Sanjie Zhentong capsules, a traditional Chinese medicine prescription. A method based on multiclass and multiresidue sample preparation was developed. First, samples were hydrated with water at 4°C. A ratio of 1:3 sample/water was used for each of the sample amounts. Then, different extraction solvents were screened. This step was followed by a dispersive solid-phase extraction clean-up procedure using both primary secondary amine and polyamide. A comprehensive sensitive multiresidue liquid chromatography tandem mass spectrometry method was investigated and validated. Good linearity was achieved in the range of 10-500 μg/kg for each analyte. The average recovery ranged from 70 to 120%, except for methamidophos, fonophos, diazinon, and chlorpyriphos-ethyl, the recoveries of which ranged from 60-70% at the lower concentration level. The precision values were lower than 10% for all the compounds in three concentration levels. The limits of detection and limits of quantification values were 0.01-2.1 and 0.05-3.4 μg/kg, respectively. The matrix effects were determined, and most of the compounds showed signal suppression. Finally, this optimized procedure was applied for the analysis of real samples.

To analyze and identify the chemical constituents from Yinyanghuo Zonghuangtong capsule by using UPLC/Q-TOF-MS/MS. Chromatographic separation was carried out on an Agilent SB-C₁₈ column (2.1 mm×100 mm, 1.8 μm) at the temperature of 30 ℃. The mobile phase was 0.1% formic acid and acetonitrile by gradient elution, with a flow rate at 0.30 mL•min⁻¹, and the injection volume of 2 μL. The MS spectrum was acquired in both negative and positive ion modes using ESI ion source. Based on relative accurate molecular weight, secondary mass spectrometry pyrolysis fragments and chromatographic peak retention time, as well as fragmentation regularity summarized from reference substance and the literature, we could effectively identify the chemical structure of the components under test. As a result, a total of 46 compounds, including flavonoid glycosides, phenolic acids and alkaloids, were successfully identified or tentatively predicted. The results provide technical support for quality control and late-stage clinical application of Yinyanghuo Zonghuangtong capsule, and reference for further expound the pharmacodynamic material basis.

Titanium alloy TC11 rods with Φ25 mm, were rolled to that with Φ17 mm in four passes by a triangle-round-triangle-round sequence. The rod temperature drop was measured at entry temperature 950 °C during continuous rolling, and the rolling forces and the roll torques were measured in the case of different area reductions at 750, 850, 950, and 1050 °C. Then, the geometry shapes of deformation zone were analyzed for different passes. The mathematic model of the rolling force for Y-type mill was modified based on empirical modifications of two-roll theories. A number of experiments were also carried out to check the validity of the proposed mathematic model. The result shows that the difference between the calculated value and the experimental value is relatively minor. Therefore, this mathematic model can provide the foundation for theoretical research and engineering application of Y-type rolling process.

RATIONALE The Direct Analysis in Real Time (DART) ionization source coupled with a quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) system has the capability to desorb analytes directly from samples from complex Chinese herbal preparations without sample cleanup or chromatographic separation. METHODS In this work, a method based on DART/Q-TOF MS/MS has been developed for rapid determination of geniposide present in 'Re Du Ning Injections', a Chinese herbal preparation. The method has been evaluated for both qualitative and quantitative analysis of geniposide in Re Du Ning Injections. RESULTS Variables including polarity for ion detection, DART gas heater temperature, matrix effect and sample presentation speed were investigated. The quantitative method was validated with respect to linearity, sensitivity, repeatability, precision and accuracy by using both internal and external standards. A comparison of the results obtained using the DART-based method was made with those obtained using a conventional High-Performance Liquid Chromatography/Diode-Array Detector (HPLC/DAD) by analyzing geniposide in four batches of Re Du Ning Injections. CONCLUSIONS The DART/Q-TOF MS/MS-based method provides a rapid, efficient and powerful method to analyze compounds from complex Traditional Chinese Medicines with limited sample preparation thus reducing time and complexity of quality control for those materials. Copyright © 2012 John Wiley & Sons, Ltd.

OBJECTIVE To investigate pharmacokinetic parameters of peoniflorin, albiflorin and amygdaloside after administration of Guizhi Fuling capsule in beagle dogs. METHOD Plasma was collected from forelimb vein of Beagle dogs after oral administration of Guizhi Fuling capsule. HPLC-MS/MS method was used to determine the concentrations of constituents in plasma. The pharmacokinetic parameters were analyzed by program DAS 2.0. RESULT The limit of quantitation of peoniflorin, albiflorin and amygdaloside were 0.25, 2.64, 0.04 microg x L(-1), respectively. After administrated with different doses, half-life of peoniflorin in dogs were 4.33, 3.62 h, albiflorin were 6.16, 5.91 h, amygdaloside were 2.43, 1.32 h. The AUC(0-t) of all components were related to dose. CONCLUSION The pharmacokinetic course of peoniflorin, albiflorin and amygdaloside can be described by two-compartment model, and these components have high expose.