Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

Efficient loading and entrapment of tamoxifen in human serum albumin based nanoparticulate delivery system by a modified desolvation technique

Herein, the poorly water-soluble drug, Tamoxifen (Tmx), was loaded in the amphipathic matrix of human serum albumin (HSA) nanoparticles by a modified desolvation method. In order to enhance the drug loading (DL) and drug entrapment efficiency (DEE) (<2% and 10%, respectively), ultrasonication of Tmx-HSA mixture was performed prior to desolvation process. Tmx loading and entrapment efficiency were optimized by employment of the response surface methodology (RSM)-central composite design (CCD) of experiments. Under the optimum conditions of 1.59 mg Tmx/ml concentration, 7.76 pH and 5 h incubation of HSA-Tmx, the DL of 6.7% and DEE of 74% are achievable. Particles with the average size of 195 nm, zeta potential of −21 mV and polydispersity index of 0.09 were produced under these conditions. A more sustained Tmx release behavior was observed from polyethylene glycol (PEG) conjugated nanoparticles in comparison to the non-PEGylated ones. The short-term stability investigation showed no alteration in physicochemical properties of nanoparticles at 4 and 37 °C, but small increase in nanoparticles size was observed after three months of storage at room temperature. This is the first report for efficient production of a Tmx delivery system based on HSA nanoparticles.     

美藤果油水包油型乳液的制备及其稳定性研究

应用高压匀质法制备美藤果油水包油型乳液。以乳液的平均粒径和分散系数(PDI)为指标,探究Tween乳化剂类型、油质量分数、乳化剂添加量、剪切速率及均质压力对乳液的平均粒径以及PDI的影响。通过测量储藏期内乳液的粒径变化、油相氧化程度(过氧化值和硫代巴比妥酸含量)、液滴的自扩散系数以及游离脂肪酸含量变化,进一步探究储藏期内乳液的物理、氧化以及水解稳定性。结果表明:制备乳液的优化工艺条件为Tween60添加量1%、油质量分数10%、剪切速率16 000 r/min、均质压力40 MPa,此条件下制备的乳液平均粒径为(218. 27±2. 21) nm,PDI为0. 118±0. 002;该工艺条件下制备的美藤果油乳液在14 d的储藏期间内具有较好的耐受pH、盐离子的物理稳定性和较高的氧化稳定性,并且未发生明显的水解作用。     

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay/Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26 °C and micro-injected with Seleno-L-Methionine to reach 8 µg g⁻¹ selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P < 0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65 kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline.     

基于Edgecam的异型螺纹自动编程技术研究

针对异型螺纹的加工问题,研究数控程序编制的自动化。基于Edgecam软件提出了螺纹加工的智能化编程方法;论述了工艺设计、软件设计和程序编制等关键技术,利用PCI和PDI等二次开发语言编制了程序;以某零件的螺纹圆弧加工实例测试了方法的有效性,实现了螺纹制造的智能化和快速化,为其它智能编程提供了借鉴。     

电脑刺绣机自动注油系统

随着电脑刺绣机的不断发展,刺绣机的主轴转速越来越高[1].高速运转会加剧机械的磨损,特别是机头和旋梭部分.有效降低机械磨损的方法是关键机械部分定期地进行清洁和注油.本文针对目前电脑刺绣机的应用情况设计了一种自动注油控制系统,可以实现容积式和阻尼式润滑系统,满足了电脑刺绣机的润滑要求.目前本系统已广泛使用于高速电脑刺绣机...     

Step-scheme perylenediimide supramolecular nanosheet and TiO2 nanoparticle composites for boosted water splitting performance

Adjusting the band gap of organic-inorganic composites by chemical bonding can effectively construct Step-scheme (S-scheme) heterojunctions, featuring properties of fast photogenerated charge migration and excellent photocatalytic performance. In this work, a novel perylene-3, 4, 9, 10-tetracarboxylicdiimide (PDI)-titanium dioxide (TiO₂) heterojunction is elaborately synthesized through simple solvent compounding method. The monodispersed spherical TiO₂ nanoparticles was prepared with the capping agents of oleylamine and oleic acid, and suffered by a ligand exchange process with nitrosonium tetrafluoroborate (NOBF₄) to remove oleylamine and oleic acid. The NOBF₄ ligands were further replaced by PDI super molecular nanosheets to obtain two dimensional (2D)-zero dimensional (0D) PDI-TiO₂ composites. TiO₂ nanoparticles are evenly anchored on the surface of PDI nanosheets with intimate contact. The PDI-TiO₂ composites has emerged considerably superior activity in hydrogen evolution. The highest hydrogen evolution rate for PDI-TiO₂composites with the PDI weight percentage of 2.4% was 9766 μmol h^?1 g^?1 under solar light irradiation, which is 2.56 times of TiO₂-NOBF₄ catalyst. Moreover, PDI-TiO₂ composites possess stoichiometric overall water splitting performance with H₂ and O₂ release rates of 238.20 and 114.18 μmol h^?1 g^?1. The superior photocatalytic performance of PDI-TiO₂ composites can be attributed to the dramatic increase in visible and NIR light absorption caused by π-π stacking structure of PDI, the prevented charge recombination by the S-scheme heterojunction, and the enhanced oxygen evolution by the stronger oxidation capability of PDI. PDI supramolecular nanosheets may work as a novel functional support for many types of semiconductor nanomaterials as graphene, which will display a wide range of application prospects in the energy and environmental fields.     

基于EtherCAT从站的风电机组扩展I/O系统设计

针对目前在风电机组中功能日益丰富,以及硬件I/O点的需求量也越来越大的问题,本文提出了基于EtherCAT从站模式的扩展I/O系统的设计方法。系统的I/O硬件接口采用了FSMC数据线复用方式的PDI接口;所开发的系统软件和XML文件配点在搭建的EtherCAT从站平台上进行了测试。测试结果表明,主控扩展I/O系统能够实现模拟量输入输出、数字量输入输出等功能,可以满足风电机组的功能需要。     

不锈钢轧制中模型参数的优化

描述了不锈钢分公司1780mm热轧厂在不锈钢生产过程中产生的一系列与模型相关的问题,对模型重要设定控制参数进行了优化,总结了所取得效果和存在问题,为不锈钢正常生产提供了有力保证.     

基于Edgecam的液压阀块快速编程技术研究

为了解决液压阀块的数控铣削加工问题,提出了自动化数控编程的解决方案。论述了数控加工工艺规划、制造特征构建和编程操作定义,基于VB.NET和PCI、PDI等Edgecam的二次开发工具开发了快速编程系统。最后通过实例证明了该方法的有效性,为其他机床的智能编程提供了参考。