A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry

We present the demonstration of a rapid “middle-up” liquid chromatography mass spectrometry (LC–MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody–drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison—a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug–antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.     

Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly‐isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral‐associated Symbiodinium had higher protein content than did cultured and sea anemone‐associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone‐associated Symbiodinium had a distinct pattern compared coral‐associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral‐associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts.     

藜异型性种子后代植株盐响应生理机制

种子异型性是植物适应异质生境的重要策略,异型性对后代植株的影响值得深入研究。以具有种子异型性的荒漠植物藜为材料,测定了异型种子后代植株在不同浓度(0,50,300 mmol/L)Na Cl胁迫下的表型及与耐盐相关的生理指标变化,探讨了异型种子后代植株之间对盐胁迫生理响应机制的差异。结果显示,褐色种子后代植株在各浓度盐胁迫下的表型(株高、分枝数)均优于黑色种子。对其生理指标测定结果显示,褐色种子植株除电导率显著低于黑色种子植株外,氧化损伤指标(氧自由基、丙二醛含量)及抗氧化酶(SOD、CAT、POX)活性、抗氧化剂(Car、As A)含量、渗调物质(可溶性糖、脯氨酸、甜菜碱)含量等在两者之间无明显差异。研究结果暗示,藜异型性种子后代植株的早期生长表型在盐胁迫下所产生的差异可能是由种子胚的大小及萌发快慢差异所导致,而这种差异并未引起盐胁迫下两种植株抗氧化系统响应的差异。     

江西武夷山国家级自然保护区蛇类物种资源调查初报

目的了解江西武夷山国家级自然保护区蛇类资源现状。方法 2014年7月22日~8月4日采用样线法对江西武夷山国家级自然保护区两栖爬行动物资源展开调查。结果本次调查记录蛇类动物14种,隶属1目3科12属,以游蛇科为主,占遇见蛇类物种总数的78.6%。大眼斜鳞蛇(Pseudoxenodon macrops)为江西省蛇类新纪录。调查过程中蛇的遇见率为0.88条/km,数量较多的福建竹叶青蛇、山溪后棱蛇和黄链蛇,遇见率分别为0.31条/km、0.24条/km和0.11条/km。结论江西武夷山国家级自然保护区福建竹叶青蛇、山溪后棱蛇和黄链蛇等常见种的数量较为丰富。     

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.     

西藏独一味属一新变型——白花独一味

描述了唇形科独一味属植物独一味[Lamiophlomis rotato.(Benth.ex Hook.f.)Kudo]的1个新变型--白花独一味[Lamiophlomis rotata(Benth.ex Hook.f.)Kudo f.dlbiflora J.Luo et S.L.Wang].原变型的花冠为淡紫色、红紫色或粉红褐色,而新变型花瓣为白色,花萼为绿色.     

中微量元素和有益元素对水稻生长和吸收镉的影响

采用盆栽试验,研究了中微量元素和有益元素对水稻生长和吸收镉的影响。结果表明,在所有测试的元素和施用方法中,硅酸钠叶面喷施显著增加稻谷产量,而碳酸钙、硼酸、硅酸钠土施和亚硒酸钠显著降低了稻谷产量。镁、锌、铁的盐酸盐形态对水稻籽粒的增产效果优于硫酸盐形态,而钙、铜的硫酸盐形态增产效果略高于盐酸盐形态。在钙、镁、硫三种中量元素中,钙增加了水稻籽粒中的Cd浓度和吸收量,而镁和硫则降低了籽粒中的Cd浓度和吸收量,以硫磺粉处理为最低。稻草中的Cd浓度和总量均以氯化镁处理为最高,硫磺粉处理最低。镁能有效抑制Cd从秸秆向籽粒的转移,其盐酸盐优于硫酸盐。在微量元素中,锌对水稻Cd的吸收抑制作用最为显著,其次是铜,而有益元素肥料硅酸钠叶面喷施则显著增加了稻谷中的Cd浓度和吸收量。硫酸亚铁、氯化锰、氯化铜、硼酸和硼砂处理都能有效地抑制Cd从秸秆向籽粒的转移,而硅酸钠叶面喷施和锌处理则促进了Cd的转移,表明硅酸钠抑制水稻吸收Cd的机制很可能发生在土壤中,而非在植株体内或地上部分。在Cd污染土壤上选用适宜的中微量和有益元素肥料及其施用方法,能有效降低水稻对镉的吸收和稻米中的Cd含量。     

UV-B辐射胁迫对杨桐幼苗生长及光合生理的影响

通过对UV-B辐射胁迫下亚热带典型木本杨桐幼苗的生长及光合生理的研究,探讨植物对于UV-B辐射胁迫的生理响应及适应性机理,进而揭示UV-B辐射变化对亚热带森林树种的影响.实验设置UV-B辐射滤光组、自然光对照组以及辐射增强组,选择亚热带典型树种杨桐(Cleyera japonica Thunb.)幼苗为实验材料.研究结果表明:(1)增强UV-B辐射会降低杨桐幼苗的叶绿素含量,而降低辐射则会显著促进叶绿素的增加,且这种胁迫在时间上具有积累性.(2)增强或降低辐射强度都会抑制杨桐地径的生长,增强辐射会产生更显著的抑制;降低辐射强度会对杨桐幼苗的株高生长产生促进作用,反之,则会抑制其生长.3个测定期数据综合分析显示随着处理时间的加长,这种胁迫作用有减小的趋势.(3)对光响应曲线的分析表明相对于自然光条件下的UV-B辐射,降低其强度对杨桐幼苗光合作用有显著的促进作用,反之则会抑制,不过抑制作用并不显著;对于光合特征参数的分析表明增强或降低UV-B辐射会显著降低杨桐幼苗的光饱和点(LSP)和光补偿点(LcP),而对最大净光合速率(Amax)、表观光合量子效率(AQY)、暗呼吸速率(Rd)影响均不显著,表明辐射胁迫对杨桐幼苗利用光能的效率影响不大,从而也并未对杨桐的光合作用产生显著性的伤害,但是由于森林树种的多年生特性,这种影响将是积累性的或延迟的,UV-B所造成的光合作用或光能利用率的微小变化都可能会积累成长期影响.因此,对森林树种进行长期研究是必要的.     

乌拉山种子植物属的地理成分分析

运用植物区系地理学基本原理,对内蒙古阴山山脉乌拉山段种子植物属的组成及地理成分进行了统计分析.结果表明:乌拉山种子植物区系包含227属,约占内蒙古全区总属数的34.8%;属的分布区类型中,以温带成分为主,占非世界分布总属数的80.9%,其中,北温带分布占到了54.2%,旧大陆温带分布占18.0%,体现出乌拉山种子植物的分布与所处自然地理位置及其气候带相适应的结果.从生活型看,草本植物占绝对优势.     

MEKK3稳定表达细胞株的建立及其对A549细胞增殖的影响

目的 构建人MEKK3基因编码区序列（cDNA）的真核表达载体、建立其稳定表达细胞株并观察其对肺腺癌细胞增殖的影响.方法 从A549细胞中提取总RNA,应用RT-PCR扩增MEKK3 cDNA的全长序列后克隆入pcDNA3.1/hygro（＋）质粒中,构建成MEKK3基因的真核表达载体,然后转染入人肺腺癌A549细胞中,潮霉素筛选稳定转染克隆,通过MTT实验,研究转染MEKK3基因前后细胞增殖的变化.结果 重组载体经酶切鉴定和测序证实目的 基因正确无误,Western印迹检测结果显示MEKK3基因在A549细胞中具有良好的表达;荧光实时定量PCR结果表明MEKK3基因在其稳定转染的A549细胞克隆中表达上调,与空载体稳定转染及未转染细胞比较,差异具有统计学意义（P＜0.05）;MTT结果显示MEKK3表达上调的稳定克隆组,A549细胞的增殖活性显著增强（A570=0.876 1±0.074 5）,明显高于空载体稳定转染组（A570=0.582 8±0.070 3）及未转染亲代细胞组（A570=0.584 9±0.035 2）,差异具有统计学意义（P＜0.01）,而后两者之间差异无统计学意义（P＞0.05）.结论 MEKK3表达上调可导致肺腺癌细胞的增殖增强.