Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

2011年、2013年和2016年医院内获得性血流感染常见病原菌分布及其耐药性分析

动态监测2011年、2013年和2016年我国不同地区医院内获得性血流感染病原菌分布及耐药进展趋势。从全国10个城市回顾性收集血流感染病原菌非重复性株,采用琼脂稀释法或微量肉汤稀释法进行药物敏感性试验,采用Whonet 5.6软件对药敏试验结果进行分析。收集的2 248株血流感染病原菌中革兰阴性杆菌为1 657株 (占73.7%),革兰阳性球菌为591株 (占26.3%)。分离率排名前五的病原菌依次为大肠埃希菌 (32.6%,733株/2 248株)、肺炎克雷伯菌 (14.5%,327株/2 248株)、金黄色葡萄球菌 (10.0%,225株/2 248株)、鲍曼不动杆菌 (8.7%,196株/ 2 248株) 和铜绿假单胞菌 (6.2%,140株/2 248株)。血流感染分离的革兰阴性杆菌对抗菌药物体外敏感率较高的抗菌药物依次为粘菌素 (96.5%,1 525株/1 581株,不包括天然耐药菌株)、替加环素 (95.6%,1 375株/1 438株,不包括天然耐药菌株)、头孢他啶/克拉维酸 (89.2%,1 112株/1 246株)、阿米卡星 (86.4%,1 382株/1 599株) 和美罗培南 (85.7%,1 376株/1 605株);革兰阳性球菌对抗菌药物体外敏感率较高的抗菌药物依次为替加环素、替考拉宁和达托霉素 (敏感率均为100.0%)、万古霉素和利奈唑胺 (敏感率均为99.7%)。2011年、2013年和2016年产超广谱β-内酰胺酶肠杆菌科细菌分离率分别为50.6% (206株/407株)、49.8% (136株/273株) 和38.9% (167株/429株);碳青霉烯不敏感肠杆菌科细菌分离率分别为2.2% (9株/408株)、4.0% (16株/402株) 和3.9% (17株/ 439株);多重耐药鲍曼不动杆菌分离率分别为76.4% (55株/72株)、82.7% (43株/52株) 和87.5% (63株/72株),多重耐药铜绿假单胞菌分离率分别为9.8% (5株/51株)、20.0% (7株/35株) 和13.0% (7株/54株);甲氧西林耐药金黄色葡萄球菌的分离率分别为51.9% (41株/79株)、29.7% (19株/64株) 和31.7% (26株/82株)。屎肠球菌和粪肠球菌中高水平庆大霉素耐药株分离率分别为43.2% (48株/111株) 和40.9% (27株/66株)。碳青霉烯不敏感肠杆菌科细菌中肺炎克雷伯菌居首位,占57.1% (24株/42株) 。肠杆菌科细菌中分离出30株替加环素不敏感株,其中肺炎克雷伯菌占76.7% (23株/30株);分离出粘菌素耐药肠杆菌科细菌39株,其中大肠埃希菌、阴沟肠杆菌和肺炎克雷伯菌分别占43.6% (17株/39株)、35.9% (14株/39株) 和15.4% (6株/39株)。医院获得性血流感染病原菌主要为革兰阴性杆菌 (以大肠埃希菌和肺炎克雷伯菌为主),其对替加环素、粘菌素和碳青霉烯类药物的敏感率较高;革兰阳性球菌中分离率最高的为金黄色葡萄球菌,其次为屎肠球菌,这两种细菌对替加环素、达托霉素、利奈唑胺、万古霉素和替考拉宁的敏感率较高。粘菌素耐药肠杆菌科细菌、替加环素不敏感肠杆菌科细菌、利奈唑胺或万古霉素不敏感革兰阳性球菌的分离,警示临床高度关注,仍需动态监测耐药进展趋势。     

非嗜食植物乙醇提取物对小菜蛾种群控制作用评价

通过建立实验种群生命表和自然种群生命表,应用种群趋势指数(indexofpopulationtrend,I)和干扰作用控制指数(interferenceindexofpopulationcontrol,IIPC),评价花椒(Zanthoxylumbungeanum)、细叶桉(Eucalyptustereticornis)、烟草(Nicotianatabacum)、构树(Broussonetiapapyrifera)、羊蹄甲(Bauhiniavariegata)、假莲翘(Durantarepens)、飞扬草(Euphorbiahirta)、茶枯(Camelliaoleifera)8种非嗜食植物乙醇提取物对小菜蛾实验种群的控制作用,以及细叶桉、烟草、茶枯3种非嗜食植物乙醇提取物及其混合液对小菜蛾自然种群的控制作用.室内试验结果表明,在各种植物乙醇提取物作用下,I值从小到大的顺序为4.4842(细叶桉)、5.3702(花椒)、5.5199(飞扬草)、6.1609(假莲翘)、6.8937(羊蹄甲)8.0945(烟草)、9.8052(茶枯)、11.1382(构树),对照的I值为69.8964;IIPC值从小到大的顺序为0.0642(细叶桉)、0.0768(花椒)、0.0790(飞扬草)、0.0881(假莲翘)、0.0986(羊蹄甲)、0.1158(烟草)、0.1403(茶枯)、0.1594(构树),说明供试植物提取物对小菜蛾实验种群增长都有一定的抑制和干扰作用.小菜蛾自然种群生命表研究结果表明,在各种植物乙醇提取物作用下,I值从小到大的顺序为5.1997(细叶桉)、7.4160(烟草)、7.3644(茶枯)和3.1399(混合液),对照的I值为21.6232;IIPC值从小到大的顺序为混合液(0.1608)、细叶桉(0.2405)、茶枯(0.3549)、烟草(0.3695),说明供试植物提取物都能明显降低种群趋势指数,在一定程度上抑制和干扰小菜蛾自然种群增长,在生产中有一定的应用前景.     

谷子窄颖花突变体sins1的表型分析和突变基因定位

利用甲基磺酸乙酯(EMS,ethyl methyl sulfonate)诱变剂处理野生型Yugu1(豫谷1号),在后代中发现了一个可以稳定遗传的颖花明显变窄的突变体,将其命名为sins1。与Yugu1相比,突变体sins1的株高显著降低了3.89%,穗长和穗粗分别显著降低了17.42%和21.62%,旗叶叶长和叶宽分别显著降低了15.09%和25.78%,千粒重显著降低了40.96%,谷码数显著降低了25%,均达到显著水平(P0.05)。利用突变体sins1为母本、SSR41为父本构建F_2定位群体,F_2正常颖花与窄颖花植株数目的分离比例为3∶1,表明该突变性状由隐性单基因控制。利用F_2群体隐性单株,最终将突变基因定位在3号染色体上SSR标记3-2658与CAAS3031间约7.709 Mb的距离内,为下一步精细定位提供了基础,同时也为促进禾本科作物颖花的研究提供了方向。     

昆虫体内储存蛋白的研究进展

储存蛋白是昆虫体内普遍存在的一种特异性血淋巴蛋白 ,通常在幼虫的脂肪体内合成 ,释放进入血淋巴中。化蛹时 ,又被脂肪体选择性吸收 ,作为氨基酸的贮存库对成虫变态发育和雌性卵发育起着重要的作用。该文介绍了昆虫体内储存蛋白的特性、功能、及调节机制。     

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Mut^s) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.     

Screening of binding proteins that interact with human Salvador 1 in a human fetal liver cDNA library by the yeast two-hybrid system

Salvador promotes both cell cycle exit and apoptosis through the modulation of both cyclin E and Drosophila inhibitor of apoptosis protein in Drosophila. However, the cellular function of human Salvador (hSav1) is rarely reported. To screen for novel binding proteins that interact with hSav1, the cDNA of hSav1 was cloned into a bait protein plasmid, and positive clones were screened from a human fetal liver cDNA library by the yeast two-hybrid system. hSav1 mRNA was expressed in yeast and there was no self-activation and toxicity in the yeast strain AH109. Twenty proteins were found to interact with hSav1, including HS1 (haematopoietic cell specific protein1)-associated protein X-1 (HAX-1); neural precursor cell expressed, developmentally down-regulated 9, pyruvate kinase, liver and RBC, cytochrome c oxidase subunit Vb, enoyl coenzyme A hydratase short chain 1, and NADH dehydrogenase (ubiquinone) 1 beta subcomplex, demonstrating that the yeast two-hybrid system is an efficient method for investigating protein interactions. Among the identified proteins, there were many mitochondrial proteins, indicating that hSav1 may play a role in mitochondrial function. We also confirmed the interaction of HAX-1 and hSav1 in mammalian cells. This investigation provides functional clues for further exploration of potential apoptosis-related proteins in disease biotherapy.     

铜藻中β-丙氨酸的分离及结构鉴定

非蛋白质氨基酸在抗癌、抗菌、抗结核、抗坏血病等方面有着重要的作用。本文主要对铜藻中的游离氨基酸进行检测分析和部分分离纯化及结构鉴定方面的研究,为更好的开发利用这些天然产物提供技术支持。采用离子交换树脂层析法分离铜藻粗提液中的游离氨基酸,收集3 mol/L的氨水洗脱液,用PITC-HPLC柱前衍生反相高效液相色谱法对其进行检测分析,结果显示粗提液中除含有多种组成蛋白质的氨基酸外还有3种未知组分,且含量较高的常见蛋白质氨基酸为丙氨酸、脯氨酸、缬氨酸。采用半制备高效液相色谱系统制备分离了其中一种未知组分的衍生物MWZ2,经真空冷冻干燥后为略黄固体粉末,结合核磁共振波谱、高分辨质谱、红外光谱数据,最终鉴定MWZ2去掉已知取代基团PITC后的成分为β-丙氨酸,分子式为C3H7NO2,分子量为89.09。     

FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

Diallyl trisulfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

Lung cancer is the leading cause of cancer-related mortality all over the world. In recent years, pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries. Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers. Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as: organosulfer compounds, OSC). DATS can induce apoptosis and inhibit the growth of many cancer cell lines. Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3, -8, and -9. Eventually, DATS induced the apoptosis and inhibited the proliferation in a concentration- and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549 xenografts, we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.