Report from the EPAA workshop: In vitro ADME in safety testing used by EPAA industry sectors

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritisation, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.     

欧猥迭宫绦虫膜联蛋白E1基因的生物信息学分析

目的:从欧猥迭宫绦虫成虫cDNA文库中识别出膜联蛋白E1(Annexin E1)基因,并对其进行生物信息学分析和功能预测。方法:从欧猥迭宫绦虫cDNA文库中获取Annexin E1基因的核酸序列,应用NCBI、ExPASy等多种生物信息学在线分析工具结合Vector NTI Advance10、Geneious Pro等软件包,对所获基因及其编码蛋白的基本理化特征、亚细胞定位、保守功能域、抗原表位、二级结构及拓扑结构等进行预测,建立蛋白质三级空间结构模型及构建其分子进化树。结果:Annexin E1编码354个氨基酸残基,理论分子量为40168.0Da,具有4个完整的保守功能域。位于细胞内,无信号肽及跨膜结构,存在6个潜在抗原表位。二级结构主要以α螺旋为主,结构和功能有关的位点高度保守,具有多个磷酸化位点。在进化的过程中与绦虫类亲缘较近,而与脊椎类亲缘较远。结论:Annexin E1编码蛋白及潜在的抗原表位与宿主同源性低,可能作为研发新型免疫诊断方法的理想分子靶标。     

新形势下医学生核心能力探析

医学生核心能力是医学专业学生在完成医疗卫生活动中所必备的个性心理特征的综合,主要由语言交流能力、自我学习能力、问题解决能力和医学职业人格四部分构成,在医学生能力体系中居于核心地位,其发展受到学校教育、实践活动、非智力因素的影响。     

棘突根部与椎板连接复合体椎间植骨结合椎弓根钉棒系统内固定治疗腰椎不稳的临床研究

目的 研究棘突根部与椎板连接复合体椎间植骨结合后路椎弓根钉棒系统进行腰椎融合治疗腰椎不稳及退行性疾病的手术方法,以期为临床提供一种较为理想的椎間植骨选择.方法 A组41例腰椎不稳及退行性疾病,为对照组,行椎间自体髂骨块植骨;B组46例腰椎不稳及退行性疾病,为实验组,行椎间自体棘突根部与椎板连接复合体植骨,对两组患者的手术时间、失血量、住院时间、疼痛改善、融合率以及融合所需时间和影像学测量结果均进行记录比较.结果 两组在手术时间、失血量、住院时间、疼痛缓解评分程度、融合率以及融合所需时间等均无明显差异,但在维持椎间隙高度及减少腰椎滑移危险方面有显著性差异.结论 棘突根部与椎板连接复合体椎间植骨结合椎弓根钉棒系统内固定治疗腰椎不稳及退行性疾病手术方法可行有效,在维持椎间隙高度与减少腰椎滑移方面有较好效果,是一种较为理想的腰椎融合方法.     

股骨头坏死的关节软骨病变研究进展

股骨头坏死治疗方式的选择,应充分考虑到关节软骨的状况。软骨、软骨下皮质骨及软骨下小梁骨在结构和功能上被看成是一个功能单元体。软骨下小梁骨坏死,既改变了关节软骨的载荷传导与分布途径,使相邻的软骨下皮质骨与软骨易于遭受机械性损伤,也破坏了软骨下区的微循环,影响软骨营养的获得及代谢产物的排出,使相邻的软骨下皮质骨与软骨易于遭受代谢性损伤。股骨头骨坏死的临床研究和实验研究显示,股骨头坏死的骨缺损对覆盖其上的邻近关节软骨的结构和代谢有损害作用。     

Cnidocyst structure and the biomechanics of discharge

The cnidocyst is the defining organelle of the cnidarians, used for capture of prey and defense. It consists of a cylindrical capsule, which releases a long tubule upon triggering. Cnidocysts develop inside a giant post-Golgi vesicle by a sequential accumulation of proteins from the Golgi apparatus. Traditionally three types of cnidocysts are distinguished: nematocysts, spirocysts, and ptychocysts. Here we focus on nematocysts, the prototypic cnidocyst and by far most diverse group of cnidocysts in this phylum. The mature nematocyst capsule comprises a collagenous polymer with remarkable biophysical properties, able to withstand an osmotic pressure of 150 bar. Release of the capsule and discharge is probably initiated by classical exocytosis. High-speed studies revealed the kinetics of discharge to be as short as 700 ns, generating an acceleration of 5,400,000 × g and a pressure of 7.7 GPa at the site of impact of the spines onto the prey. Thus nematocysts comprise a powerful molecular spring mechanism releasing energy stored in the wall polymer in the nanosecond time range. During the last few years, genomic, biochemical and structural studies have helped to unravel the molecular composition of the nematocyst supra-structure. Here we summarize these findings and present an integrative view of mechanical and molecular aspects that have shaped the nematocyst during evolution.     

A new twist in cardiac muscle: dislocated and helicoid arrangements of myofibrillar z-disks in mammalian ventricular myocytes

Using deconvolved confocal microscopy of fluorescently labeled markers for z-disks, t-tubules and ryanodine receptors, we have examined sarcomere organization in cardiac myocytes from rat, rabbit and human. We show that sarcomeres exhibit dislocations in registration and occasionally more complex helicoidal topology. This organization was present at both slack (∼ 1.8 μm) and long sarcomere lengths (∼ 2.2 μm). Misregistrations in z-disks persisted over 15-20 sarcomere lengths and appeared to arise primarily from variations in fiber direction; particularly as myofibrils pass around nuclei. In addition, myofibrils twist along the cell length. T-tubules generally follow the sarcomere z-disks although additional elements bridging adjacent myofibrils and along the length of the myofibril are present to varying degrees in all cells. Ryanodine receptors (the sarcoplasmic reticulum Ca²⁺ release channel) are generally located within 250 nm of the local plane containing t-tubules and z-disks, but a small fraction (∼ 2%) is found on longitudinal elements of the t-system between z-disks. The results are discussed with respect to the possible role(s) of such complex z-disk organization and z-disk dislocations in the maintenance of cell structure and sarcomere assembly. In addition, the non-planar organization of z-disks may be important in the propagation of local Ca²⁺ waves which may have a useful role in helping maintain the uniformity of sarcomere activation in the presence of t-tubule remodeling.     

Isolation,structural determination,synthesis and quantitative determination of impurities in Intron-A,leached from a silicone tubing

Investigation of unexpected levels of impurities in Intron product has revealed the presence of low levels of impurities leached from the silicone tubing (Rehau RAU-SIK) on the Bosch filling line. In order to investigate the effect of these compounds (1a, 1b and 2) on humans, they were isolated identified and synthesized. They were extracted from the tubing by stirring in Intron placebo at room temperature for 72 h and were enriched on a reverse phase CHP-20P column, eluting with gradient aqueous ACN and were separated by HPLC. Structural elucidation of 1a, 1b and 2 by MS and NMR studies demonstrated them to be halogenated biphenyl carboxylic acids. The structures were confirmed by independent synthesis. Levels of extractable impurities in first filled vials of actual production are estimated to be in the range of 0.01–0.55 μg/vial for each leached impurity. Potential toxicity of these extractables does not represent a risk for patients under the conditions of clinical use.     

老年冠心病合并代谢综合征患者踝臂指数的临床研究

目的 研究老年冠心病合并代谢综合征(MS)患者踝臂指数(ABI)和冠状动脉病变的严重程度及心功能的关系.方法 经冠状动脉造影明确诊断为冠心病的老年患者140例,其中未合并MS者74例(无MS组),合并MS者66例(Ms组),评估两组冠状动脉病变情况,测定其ABI及心功能.结果 MS组ABI为0.90±0.32,无MS组为1.03±0.26,两组比较差异有统计学意义(P=0.015).MS组ABI≤0.9者比率高于无MS组[43.9%(29/66)比27.0%(20/74)],差异有统计学意义(P=0.038).在MS组中,ABI≤0.9者冠状动脉3支病变发生率显著高于ABI>0.9者[86.3%(25/29)比37.8%(14/37),P=0.000];ABI≤0.9者心功能降低更显著(P<0.05).结论 老年冠心病合并MS患者,其ABI较低,冠状动脉病变程度更加严重,心功能降低更加明显,应进行强化治疗.