α-氨基酸酯酰基转移酶表达纯化、酶学性质及其催化应用

α-氨基酸酯酰基转移酶(α-amino acid ester acyltransferase,AET)能够催化底物L-丙氨酸甲酯盐酸盐、L-谷氨酰胺合成L-丙氨酰-L-谷氨酰胺(L-alanyl-L-glutamine,丙谷二肽)。利用重组大肠杆菌saet-QC01表达α-氨基酸酯酰基转移酶,对其表达条件进行了优化,通过Ni-NTA亲和层析法分离纯化重组蛋白,并对其酶学性质、催化应用进行了研究。适合酶表达的诱导条件:温度20℃,诱导阶段(OD_(600)=2.0-2.5),IPTG浓度0.6 mmol/L,诱导时间12 h。α-氨基酸酯酰基转移酶的最适反应温度27℃,最适pH 8.5,在pH 7.0-8.0很稳定,在酸性条件下相对稳定,低浓度的Co~(2+)、低浓度的EDTA对酶活有促进作用。在底物浓度丙氨酸甲酯盐酸盐600 mmol/L、谷氨酰胺480 mmol/L,丙谷二肽的产量达到78.2 g/L,生产速率达到1.955 g/(L·min),转化率达到75.0%。α-氨基酸酯酰基转移酶具有良好的酸碱耐受性,催化效率高的优良特性,在工业生产中具有较好的应用潜力。     

Molecular Insight into the Steric Shielding Effect of PEG on the Conjugated Staphylokinase: Biochemical Characterization and Molecular Dynamics Simulation

PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.     

水生态文明城市建设对城市水生态承载力的影响——以武汉市为例

城市水生态承载力能较好反映水资源-水环境-水生态-经济社会复合系统间多要素互馈关系,水生态文明城市建设对城市水生态系统具有多方面影响,对城市水生态承载力在水生态文明城市建设前后的变化进行定量研究具有重要意义。以城市"社会-经济-自然"复合生态理论为指导,充分考虑人类活动与城市水生态系统的响应关系,基于DPSIR框架构建城市水生态承载力评价指标体系,以水生态文明建设试点城市武汉市为例,综合运用熵权法、TOPSIS和障碍度诊断模型等方法,分析2008-2019年武汉市水生态承载力的变化趋势和影响因素。结果表明,水生态文明城市试点建设期间（2015-2017年）的水生态承载力平均水平（0.552）明显高于建设前（0.361）和建设后（0.438）,建设成果主要体现在压力和影响指标的改善以及响应指标的大幅度提升上。阻碍武汉市水生态承载力提升的主要指标为生态环境状况指数、建成区绿化覆盖率、农田灌溉亩均用水量、酸雨量和饮用水源地水质达标率。根据评价结果提出水城共生共荣发展模式、引导产业结构转型等提升武汉市水生态承载力的建议。     

Developmental toxicity of triclocarban in zebrafish (Danio rerio) embryos

Triclocarban (TCC), which is used as an antimicrobial agent in personal care products, has been widely detected in aquatic ecosystems. However, the consequence of TCC exposure on embryo development is still elusive. Here, by using zebrafish embryos, we aimed to understand the developmental defects caused by TCC exposure. After exposure to 0.3, 30, and 300 μg/L TCC from 4‐hour postfertilization (hpf) to 120 hpf, we observed that TCC exposure significantly increased the mortality and malformation, delayed hatching, and reduced body length. Exposure to TCC also affected the heart rate and expressions of cardiac development–related genes in zebrafish embryos. In addition, TCC exposure altered the expressions of the genes involved in hormonal pathways, indicating its endocrine disrupting effects. In sum, our data highlight the impact of TCC on embryo development and its interference with the hormone system of zebrafish.     

Monitoring <i>Thosea sinensis</i> Walker in Tea Plantations Based on UAV Multi-Spectral Image

Thosea sinensis Walker (TSW) rapidly spreads and severely damages the tea plants. Therefore, finding a reliable operational method for identifying the TSW-damaged areas via remote sensing has been a focus of a research community. Such methods also enable us to calculate the precise application of pesticides and prevent the subsequent spread of the pests. In this work, based on the unmanned aerial vehicle (UAV) platform, five band images of multispectral red-edge camera were obtained and used for monitoring the TSW in tea plantations. By combining the minimum redundancy maximum relevance (mRMR) with the selected spectral features, a comprehensive spectral selection strategy was proposed. Then, based on the selected spectral features, three classic machine learning algorithms, including random forest (RF), support vector machine (SVM), and k-nearest neighbors (KNN) were used to construct the pest monitoring model and were evaluated and compared. The results showed that the strategy proposed in this work obtained ideal monitoring accuracy by only using the combination of a few optimized features (2 or 4). In order to differentiate the healthy and TSW-damaged areas (2-class model), the monitoring accuracies of all the three models were computed, which were above 96%. The RF model used the least number of features, including only SAVI and Band_red. In order to further discriminate the pest incidence levels (3-class model), the monitoring accuracies of all the three models were computed, which were above 80%, among which the RF algorithm based on SAVI, Band_red, VARI_green, and Band_{red_edge} features achieve the highest accuracy (OAA of 87%, and Kappa of 0.79). Considering the computational cost and model accuracy, this work recommends the RF model based on a few optimal feature combinations to monitor and distinguish the severity of TSW in tea plantations. According to the UAV remote sensing mapping results, the TSW infestation exhibited an aggregated distribution pattern. The spatial information of occurrence and severity can offer effective guidance for precise control of the pest. In addition, the relevant methods provide a reference for monitoring other leaf-eating pests, effectively improving the management level of plant protection in tea plantations, and guaranting the yield and quality of tea plantations.     

H7亚型禽流感病毒一步法RT-PCR检测方法的建立

通过分析流感数据库45个H7亚型禽流感病毒的HA序列,在保守区内设计并合成引物,建立了一步法RT-PCR检测方法,扩增片段大小为501bp。通过对H7亚型禽流感病毒尿囊液和棉拭子浸出液不同滴度检测,证实病毒尿囊液最低检出量为105.5EID50/mL;阳性棉拭子最低检出量为103EID50/mL。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原进行检测,仅有H7亚型AIV有特异性目的条带,与其他均无交叉反应。从脏器及咽喉、泄殖腔棉拭子样品的病毒分离和RT-PCR方法比较,表明在10-1的样品浓度下,两者可以达到相同的检出量。表明该一步法RT-PCR方法具有特异性强、敏感性高和准确率高的特点。     

河南郑州汪沟遗址出土的植硅体

汪沟遗址是豫中地区仰韶文化晚期一处高等级的中心性聚落遗址。在2014-2016年三个季度的发掘中我们系统采集了植硅体土样并进行了分析。研究结果显示：汪沟聚落仰韶文化晚期的农作物有粟、黍和水稻;黍粟比例较高,水稻的比例较低;稻作农业较旱作农业规模小,种植少,但水稻和粟黍的出土概率相差很小,说明水稻和粟黍一样都是汪沟先民日常食用的作物,是汪沟先民植物性食物的重要组成部分。我们根据炭化植物遗存分析的结果推测,汪沟遗址的粟黍和水稻在不同的季节以连杆带穗的方式收割,然后在壕沟南部区域对谷物进行集中脱粒,脱粒后的粟黍和稻被共同储藏在房址周围,个体家庭需要食用的时候在房屋内或周围进行脱壳。大规模的谷物收割和在特定场所集中进行的脱粒加工活动说明,汪沟聚落有着较强的劳动组织能力,有较大型社会生产组织的存在,大家庭或家族公社是聚落生产与生活中的基本组成单位。