噬菌体展示HIV-1 Tat38-61碱性区51和55位随机突变体文库的构建

为了构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库,进一步研究HIV-1 Tat38-61表位的分子进化筛选,采用含随机核苷酸序列的引物,通过Overlap PCR的方法获得51和55位氨基酸随机突变的全长Tat编码序列,再以此为模板PCR扩增出两端含有Xba I识别序列的Tat38-61突变体片段HIV-1 Tat38-61(51N/55N),克隆至噬菌体展示载体pCANTAB5S上,转化大肠杆菌TG1,经M13K07辅助噬菌体拯救,构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库。结果显示文库的库容量为5.0×106,滴度为2.65×1012 TU/mL,阳性克隆率为56.50％;序列分析显示文库中51、55位核苷酸与氨基酸均呈随机性分布,达到了对文库进行分子进化筛选的要求,为获得可用作疫苗候选物的新型Tat突变体奠定基础。     

Identification of curcumin derivatives as human glyoxalase I inhibitors: A combination of biological evaluation, molecular docking, 3D-QSAR and molecular dynamics simulation studies

Several recent developments suggest that the human glyoxalase I (GLO I) is a potential target for anti-tumor drug development. In present study, a series of curcumin derivatives with high inhibitory activity against human GLO I were discovered. Inhibition constant (K(i)) values of compounds 8, 9, 10, 11 and 13 to GLO I are 4.600μM, 2.600μM, 3.200μM, 3.600μM and 3.600μM, respectively. To elucidate the structural features of potent inhibitors, docking-based three-dimensional structure-activity relationship (3D-QSAR) analyses were performed. Satisfactory agreement between experiment and theory suggests that comparative molecular similarity index analysis (CoMSIA) modeling exhibit much better correlation and predictive power. The cross-validated q(2) value is 0.638 while no-validation r(2) value is 0.930. Integrated with docking-based 3D-QSAR CoMSIA modeling, molecular surface property (electrostatic and steric) mapping and molecular dynamics simulation, a set of receptor-ligand binding models and bio-affinity predictive models for rational design of more potent inhibitors of GLO I are established.     

Prediction of N-myristoylation modification of proteins by SVM

Attachment of a myristoyl group to NH(2)-terminus of a nascent protein among protein post-translational modification (PTM) is called myristoylation. The myristate moiety of proteins plays an important role for their biological functions, such as regulation of membrane binding (HIV-1 Gag) and enzyme activity (AMPK). Several predictors based on protein sequences alone are hitherto proposed. However, they produce a great number of false positive and false negative predictions; or they cannot be used for general purpose (i.e., taxon-specific); or threshold values of the decision rule of predictors need to be selected with cautiousness. Here, we present novel and taxon-free predictors based on protein primary structure. To identify myristoylated proteins accurately, we employ a widely used machinelearning algorithm, support vector machine (SVM). A series of SVM predictors are developed in the present study where various scales representing physicochemical and biological properties of amino acids (from the AAindex database) are used for numerical transformation of protein sequences. Of the predictors, the top ten achieve accuracies of >98% (the average value is 98.34%), and also the area under the ROC curve (AUC) values of >0.98. Compared with those of previous studies, the prediction accuracies are improved by about 3 to 4%.     

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.     

人类免疫缺陷病毒1型Tat蛋白N末端缺失突变体融合蛋白的表达及其免疫原性分析

本研究采用PCR方法从人类免疫缺陷病毒1型（Human immunodeficiency virus 1,HIV-1）HXB2株tat基因中扩增编码Tat蛋白N末端1-21位氨基酸缺失的突变体Tat22-101基因片段,构建其原核表达质粒pET32a-Tat22-101,经双酶切及测序验证后,转化大肠埃希菌BL21（DE3）,进行IPTG诱导表达及Ni²⁺-NTA柱亲和层析纯化。纯化后的突变体融合蛋白PET32a-Tat22-101经SDS-PAGE及Western blotting鉴定,其相对分子质量约为26.9kD。该融合蛋白免疫BALB/c小鼠,经ELISA检测结果表明,pET32a-Tat22-101融合蛋白不仅较好地保留其免疫原性,而且能诱导产生高滴度的针对Tat N末端区之外的Tat其他功能区表位的抗体,为进一步研究Tat生物学功能和研制新型HIV Tat疫苗奠定试验基础。     

EBV裂解复制周期调控机制研究新进展

EBV与许多恶性疾病包括霍奇金病、伯基特淋巴瘤、鼻咽癌等恶性肿瘤发病有关人类B淋巴细胞是EBV天然宿主,其在宿主细胞中的生活周期分为潜伏感染和裂解感染。EBV潜伏感染时,为逃避宿主细胞的免疫杀伤,仅表达少量基因产物。而在外界条件如化学、物理或宿主细胞分化的刺激下,EBV可由潜伏感染进入到裂解复制(Lytic Replication)感染周期,促进病毒在宿主细胞中播散。根据EBV裂解复制产物出现的时间顺序可将裂解复制周期分为裂解复制立即早期、早期和晚期。1 EBV裂解复制不同时期产物的调控作用     

秸秆还田条件下灌水模式对冬小麦产量和水肥利用效率的影响

于2008-2010年,在山西省临汾市尧都区半干旱、半湿润季风气候区,通过大田试验研究了玉米秸秆连续还田条件下灌水模式对冬小麦籽粒产量、干物质转移及水肥利用效率的影响.结果表明:浇越冬水可促进小麦分蘖;浇拔节水可提高分蘖成穗率,增加成穗数;浇孕穗水可促进穗部干物质积累,提高千粒重.浇2水时,推迟第2次浇水时期使叶片干物质转移量和穗粒数增加;浇2水比浇l水的肥料表观利用率高,可促进穗部干物质积累.越冬水灌水量和总灌水量对分蘖、穗部干物质积累的影响较小;拔节期或孕穗期增加灌水量则更有利于养分吸收及干物质积累与转移,提高籽粒水分利用效率,产量构成因素协调,增产效果明显.因此,确保越冬水可实现稳产,在越冬水基础上,拔节期增量灌水(900 m3·hm-2)可满足冬小麦中后期生长发育的需要,提高籽粒水分利用效率,实现节水高产栽培.     

基于过程的小麦茎鞘夹角动态模拟

基于不同株型品种和不同密度处理的小麦田间试验,连续观察并记录各处理不同叶位叶鞘与主茎之间的夹角,进一步利用系统分析方法和动态建模技术,构建基于过程的小麦叶片茎鞘夹角的动态模拟模型.结果表明:小麦茎鞘夹角随叶片生育进程不断加大,并随密度的增大而减小;从第2叶开始,最大茎鞘夹角随叶位的增加而减小.所建模型利用Logistic方程描述叶片茎鞘夹角随生育进程的动态变化过程,使用分段函数描述最大茎鞘夹角随叶位的动态变化,引入品种参数(第2叶茎鞘夹角的最大值)量化了茎鞘夹角在不同品种之间的差异,并利用基本苗量化了密度对茎鞘夹角的影响.基于独立的田间试验资料对所建模型进行测试与检验,结果显示茎鞘夹角模拟值与观测值之间的均方根差为1.7°.表明模型对小麦主茎叶片茎鞘夹角的动态变化过程具有较好的预测性,为小麦生长过程的可视化表达奠定了技术基础.     

Effects of different irrigation modes on winter wheat grain yield and water- and nitrogen use efficiency

Taking the widely planted winter wheat cultivar Tainong 18 as test material, a field experiment was conducted to study the effects of different irrigation modes on the winter wheat grain yield and water- and nitrogen use efficiency in drier year (2009-2010) in Tai' an City of Shandong Province, China. Five treatments were installed, i. e., irrigation before sowing (CK), irrigation before sowing and at jointing stage (W1), irrigation before sowing and at jointing stages and at over-wintering stage with alternative irrigation at milking stage (W2), irrigation before sowing and at jointing and flowering stages (optimized traditional irrigation mode, W3), and irrigation before sowing and at over-wintering, jointing, and milking stages (traditional irrigation mode, W4). The irrigation amount was 600 m3 hm(-2) one time. Under the condition of 119.7 mm precipitation in the winter wheat growth season, no significant difference was observed in the grain yield between treatments W2 and W4, but the water use efficiency was significantly higher in W2 than in W4. Comparing with treatment W3, treatments W2 and W4 had obviously higher grain yield, but the water use efficiency had no significant difference. The partial factor productivity from N fertilization was the highest in W2 and W4, and the NO3(-)-N accumulation amount in 0-100 cm soil layer at harvest was significantly higher in W2 than in W3 and W4, suggesting that W2 could reduce NO3(-)-N leaching loss. Under the conditions of our experiment, irrigation before sowing and jointing stages and at over-wintering stage with alternative irrigation at milking stage was the optimal irrigation mode in considering both the grain yield and the water- and nitrogen use efficiency.     

大型医疗设备成本绩效考核数学模型的建立

目的探讨医疗设备绩效管理的新模式,提高医疗设备的经济效益。方法研制医疗器械成本效益分析评价模型,对医疗设备使用中影响成本的元素进行分析,并提出合理的改善方法。结果通过开展医疗设备使用效益分析,为决策提供可靠的数字依据,对医疗设备的投资及使用进行监督核算。