Renewable bio ionic liquids‐water mixtures‐mediated selective removal of lignin from rice straw: Visualization of changes in composition and cell wall structure

Pretreatment of rice straw by using renewable cholinium amino acids ionic liquids ([Ch][AA] ILs)‐water mixtures and the subsequent enzymatic hydrolysis of the residues were conducted in the present work. Of the eight mixtures composed of ILs and water, most were found to be effective for rice straw pretreatment. After pretreatment with 50% ILs‐water mixtures, the enzymatic digestion of the lignocellulosic biomass was enhanced significantly, thus leading to satisfactory sugar yields of >80% for glucose and approximately 50% for xylose. To better understand the ILs pretreatment mechanism, confocal laser scanning microscopy combined with immunolabeling and transmission electron microscopy were used to visualize changes in the contents and distribution of two major components—lignin and xylan. The results coupled with changes in chemical structures (infrared spectra) of the substrates indicated occurrence of extensive delignification, especially in cell corner and compound middle lumen of cell walls, which made polysaccharides more accessible to enzymes. This pretreatment process is promising for large‐scale application because of the high sugar yields, easy handling, being environmentally benign and highly tolerant to moisture, and significantly reduced cost and energy consumption. Biotechnol. Bioeng. 2013; 110: 1895–1902. © 2013 Wiley Periodicals, Inc.     

家蝇溶菌酶MDLZM2基因的克隆、序列分析及原核表达

对家蝇溶菌酶(Musca domestica lysozyme,MDLZM2)基因进行克隆、序列分析,构建原核表达载体并在大肠杆菌中表达。从Gen Bank家蝇基因组中筛选获得MDLZM2基因。以该基因的序列设计引物,进行PCR扩增,测序分析获得该基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构和保守结构域等方面进行预测和分析。构建p EASY-E1-MDLZM2重组质粒,转化到大肠杆菌BL21(DE3)p Lys S Chemically Competent Cell中进行诱导表达及纯化。结果表明MDLZM2基因ORF全长552 bp,编码183个氨基酸,理论分子量21.2 k Da;等电点为6.13,具有Lysozyme家族的蛋白保守结构域。成功构建重组原核表达p EASY-E1-MDLZM2并诱导表达、纯化重组蛋白,为进一步研究该蛋白的生物学及免疫学活性奠定了基础。     

鸡肉粉完全替代鱼粉饲料中补充包被氨基酸对凡纳滨对虾生长、体成分及组织氨基酸含量的影响

为了探讨鸡肉粉完全替代鱼粉时饲料氨基酸的平衡性以及外源氨基酸的添加方式与凡纳滨对虾生长、体成分、血浆游离氨基酸及肌肉氨基酸含量的关系, 本试验采用26因子试验设计进行了为期56d的饲养试验。2个饲料蛋白质水平分别为40%和32%, 6个饲料处理分别为鱼粉组(对照组)、鸡肉粉组、鸡肉粉+晶体EAA组、鸡肉粉+晶体EAA+晶体NEAA组、鸡肉粉+包被EAA组、鸡肉粉+包被EAA+包被NEAA组, 配制12组饲料。将凡纳滨对虾(0.300.01) g随机分配到36个圆桶(150 L)中, 每桶30尾, 每3个桶为一个处理组, 饲喂一种饲料, 每天饱食投喂三次。在每一饲料蛋白质水平下, 无论是补充晶体氨基酸(CAA)组还是包被氨基酸组对虾的增重率均显著高于鸡肉粉组(P0.05), 且在32%蛋白质水平下, 包被EAA组对虾增重率达到了鱼粉组水平(P0.05); 补充晶体EAA+NEAA组对虾增重率与补充晶体EAA组无差异(P0.05), 但均显著低于补充包被氨基酸组(P0.05); 补充包被EAA组对虾增重率显著高于补充包被EAA+NEAA组(P0.05)。饲料系数的变化正好与增重率变化相反(P0.05)。饲喂高蛋白质水平饲料较之饲喂低蛋白质饲料明显提高对虾增重率、虾体蛋白含量(P0.05), 但降低虾体脂肪含量(P0.05)。包被氨基酸组凡纳滨对虾血浆游离氨基酸含量总体显著低于CAA组(P0.05)。除谷氨酸、甘氨酸以及脯氨酸外, 各组对虾肌肉氨基酸含量无显著差异(P0.05)。结果表明, 在32%饲料蛋白质水平下, 用鸡肉粉完全替代鱼粉时, 饲料中补充包被EAA可明显促进凡纳滨对虾的生长, 且达到了鱼粉组的饲喂效果。     

The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

Effects of tobacco–peanut relay intercropping on soil bacteria community structure

A reasonable cultivation pattern is beneficial to maintain soil microbial activity and optimize the structure of the soil microbial community. To determine the effect of tobacco−peanut (Nicotiana tabacum−Arachis hypogaea) relay intercropping on the microbial community structure in soil, we compared the effects of relay intercropping and continuous cropping on the soil bacteria community structure. We collected soil samples from three different cropping patterns and analyzed microbial community structure and diversity using high-throughput sequencing technology. The number of operational taxonomic units (OTU) for bacterial species in the soil was maximal under continuous peanut cropping. At the phylum level, the main bacteria identified in soil were Proteobacteria, Actinobacteria, and Acidobacteria, which accounted for approximately 70% of the total. The proportions of Actinobacteria and Firmicutes increased, whereas the proportion of Proteobacteria decreased in soil with tobacco–peanut relay intercropping. Moreover, the proportions of Firmicutes and Proteobacteria among the soil bacteria further shifted over time with tobacco–peanut relay intercropping. At the genus level, the proportions of Bacillus and Lactococcus increased in soil with tobacco–peanut relay intercropping. The community structure of soil bacteria differed considerably with tobacco–peanut relay intercropping from that detected under peanut continuous cropping, and the proportions of beneficial bacteria (the phyla Actinobacteria and Firmicutes, and the genera Bacillus and Lactococcus) increased while the proportion of potentially pathogenic bacteria (the genera Variibacter and Burkholderia) decreased. These results provide a basis for adopting tobacco–peanut relay intercropping to improve soil ecology and microorganisms, while making better use of limited cultivable land.