Development of a Efficient and Sensitive Dispersive Liquid–Liquid Microextraction Technique for Extraction and Preconcentration of 10 β2-Agonists in Animal Urine

Dispersive liquid–liquid microextraction (DLLME) coupled with ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was developed for the extraction and determination of 10 β₂-agonists in animal urine. Some experimental parameters, such as the type and volume of the extraction solvent, the concentration of the dispersant, the salt concentration, the pH value of the sample solution, the extraction time and the speed of centrifugation, were investigated and optimized. Under the optimized conditions, a good enrichment factors (4.8 to 32.3) were obtained for the extraction. The enrichment factor show that the concentration rate of DLLME is significantly higher than other pretreatment methods, and the detection sensitivity has been greatly improved. The calibration curves were linear, the correlation coefficient ranged from 0.9928 to 0.9999 for the concentration range of 0.05 to 50 ngmL^-1 and 0.1 to 50 ngmL^-1, and the relative standard deviations (RSDs, n = 15, intra and inter-day precision) at a concentration of 5 ngmL^-1 were in the range of 1.8 to 14.6%. The limits of detection (LODs) for the 10 β₂-agonists, based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.01 to 0.03 ngmL^-1. The proposed method was used to identify β₂-agonists in three types of animal urine (swine, cattle, sheep), and the relative recoveries from each matrix were in the range of 89.2 to 106.8%, 90.0 to 109.8% and 89.2 to 107.2%, respectively.     

Phosphorylation of Cdc42 Effector Protein-4 (CEP4) by Protein Kinase C Promotes Motility of Human Breast Cells

Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser¹⁸, Ser⁷⁷, Ser⁸⁰, and Ser⁸⁶) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser¹⁸ and Ser⁸⁰ were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser¹⁸ and Ser⁸⁰ causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.     

The model and algorithm of distributing cooperation profits among operators of urban rail transit under PPP pattern

The financing channels, investors and operators of urban rail transit are becoming more and more diversified, and public private partnership pattern has been increasingly suggested in financing and investment field of urban rail transit in China. The diversification of investors of urban rail transit will no doubt lead to the diversification of operators of urban rail transit network. To legitimately distribute the cooperation profits among operators, a model is developed based on passenger’s path choice behavior by considering travel period, travel time, transfer convenience and the comprehensive proportion of different service types provided by operators. In accordance with the features of urban rail transit network and origin-destination (OD) pairs of transferring among lines of different operators, a scheme of improved rail transit network is proposed. On the basis of the algorithm of breadth-first search and depth-first search, an algorithm of searching effective paths based on backtracking and traversing along the shortest path is established by considering the factor of transfer. Taking the example of Shenzhen’s rail transit network, three typical OD pairs are selected to measure and calculate, compare and analyze by six different conditions. The result shows that travel period, travel time, transfer convenience, and service types provided by operators exert great influence on the distribution of cooperation profits. Therefore, it is advisable to comprehensively consider all of these factors to improve the accuracy of cooperation profits distribution. Moreover, the proposed algorithm can search effective paths efficiently.     

Synthesis,Molecular Modeling,and Biological Evaluation of Novel Chiral Thiosemicarbazone Derivatives as Potent Anticancer Agents

A series of new chiral thiosemicarbazones derived from homochiral amines in both enantiomeric forms were synthesized and evaluated for their in vitro antiproliferative activity against A549 (human alveolar adenocarcinoma), MCF‐7 (human breast adenocarcinoma), HeLa (human cervical adenocarcinoma), and HGC‐27 (human stomach carcinoma) cell lines. Some of compounds showed inhibitory activities on the growth of cancer cell lines. Especially, compound 17b exhibited the most potent activity (IC₅₀ 4.6 μM) against HGC‐27 as compared with the reference compound, sindaxel (IC₅₀ 10.3 μM), and could be used as a lead compound to search new chiral thiosemicarbazone derivatives as antiproliferative agents. Chirality 27:177–188, 2015. © 2014 Wiley Periodicals, Inc.