LabCaS: Labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields

The calpain family of Ca²⁺‐dependent cysteine proteases plays a vital role in many important biological processes which is closely related with a variety of pathological states. Activated calpains selectively cleave relevant substrates at specific cleavage sites, yielding multiple fragments that can have different functions from the intact substrate protein. Until now, our knowledge about the calpain functions and their substrate cleavage mechanisms are limited because the experimental determination and validation on calpain binding are usually laborious and expensive. In this work, we aim to develop a new computational approach (LabCaS) for accurate prediction of the calpain substrate cleavage sites from amino acid sequences. To overcome the imbalance of negative and positive samples in the machine‐learning training which have been suffered by most of the former approaches when splitting sequences into short peptides, we designed a conditional random field algorithm that can label the potential cleavage sites directly from the entire sequences. By integrating the multiple amino acid features and those derived from sequences, LabCaS achieves an accurate recognition of the cleave sites for most calpain proteins. In a jackknife test on a set of 129 benchmark proteins, LabCaS generates an AUC score 0.862. The LabCaS program is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/LabCaS . Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Preparative Separation of Enantiomers Based on Functional Nucleic Acids Modified Gold Nanoparticles

The preparative‐scale separation of chiral compounds is vitally important for the pharmaceutical industry and related fields. Herein we report a simple approach for rapid preparative separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs). The separation of _DL‐tryptophan (_DL‐Trp) is demonstrated as an example to show the feasibility of the approach. AuNPs modified with enantioselective aptamers were added into a racemic mixture of _DL‐Trp. The aptamer‐specific enantiomer (_L‐Trp) binds to the AuNPs surface through aptamer‐_L‐Trp interaction. The separation of _DL‐Trp is then simply accomplished by centrifugation: the precipitate containing _L‐Trp bounded AuNPs is separated from the solution, while the _D‐Trp remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured under 95 °C and a second centrifugation is then performed, resulting in the separation of AuNPs and _L‐Trp. The supernatant is finally collected to obtain pure _L‐Trp in water. The results show that the racemic mixture of _DL‐Trp is completely separated into _D‐Trp and _L‐Trp, respectively, after 5 rounds of repeated addition of fresh aptamer‐modified AuNPs to the _DL‐Trp mixture solution. Additionally, the aptamer‐modified AuNPs can be repeatedly used for at least eight times without significant loss of its binding ability because the aptamer can be easily denatured and renatured in relatively mild conditions. The proposed approach could be scaled up and extended to the separation of other enantiomers by the adoption of other enantioselective aptamers. Chirality 25:751–756, 2013. © 2013 Wiley Periodicals, Inc.     

MAIGO2 is involved in abscisic acid‐mediated response to abiotic stresses and Golgi‐to‐ER retrograde transport

The central role of multisubunit tethering complexes in intracellular trafficking has been established in yeast and mammalian systems. However, little is known about their roles in the stress responses and the early secretory pathway in Arabidopsis. In this study, Maigo2 (MAG2), which is equivalent to the yeast Tip20p and mammalian Rad50‐interacting protein, is found to be required for the responses to salt stress, osmotic stress and abscisic acid in seed germination and vegetative growth, and MAG2‐like (MAG2L) is partially redundant with MAG2 in response to environmental stresses. MAG2 strongly interacts with the central region of ZW10, and both proteins are important as plant endoplasmic reticulum (ER)‐stress regulators. ER morphology and vacuolar protein trafficking are unaffected in the mag2, mag2l and zw10 mutants, and the secretory marker to the apoplast is correctly transported in mag2 plants, which indicate that MAG2 functions as a complex with ZW10, and is potentially involved in Golgi‐to‐ER retrograde trafficking. Therefore, a new role for ER–Golgi membrane trafficking in abiotic‐stress and ER‐stress responses is discovered.     

Differences in capacities of in vitro organ regeneration between two Arabidopsis ecotypes Wassilewskija and Columbia

In vitro organogenesis is well-controlled and thus provides an ideal system to study mechanisms of plant organ development. Although it has been well investigated for a long time that exogenous hormones play important roles in determining the types of organs regenerated in vitro, there is currently limited information available for other key factors that mediate de novo organ regeneration. Here, we reported simple and efficient one-step processes for evaluating capacities of inflorescence stem-derived in vitro organogenesis between two different ecotypes in Arabidopsis. Different types of organs, including shoots and roots were initiated from inflorescence stem explants cultured on the media containing 216 combinations of exogenous auxin and cytokinin. Further, we showed that Wassilewskija ecotype had the much higher shoot regeneration capacity than Columbia with different combinations of hormones, indicating that the ecotype is an essential factor determining de novo organogenesis. Our results also suggested that the defined expression patterns of genes involved in auxin and cytokinin biosynthesis were correlated with the variations in organogenesis capacities between the two ecotypes. Thus, in vitro organogenesis is likely regulated by ecotypes through mediating endogenous hormonal biosynthesis.     

Abscisic acid is required for somatic embryo initiation through mediating spatial auxin response in Arabidopsis

Abscisic acid (ABA) regulates many aspects of plant development, including somatic embryo (SE) initiation. However, mechanisms of ABA functions on SE initiation have remained to be investigated. In this study, we examined the endogenous ABA contents of calli in Arabidopsis during the SE inductive process. We further found that the capacity for SE initiation was strongly impaired by treatment of fluridone, a potent inhibitor of ABA biosynthesis, as well as by mutation of ABA biosynthetic gene ABA2, suggesting that ABA is required for SE initiation. Furthermore, treatment of fluridone inhibited local auxin biosynthesis and auxin polar transport in the embryonic calli, resulting in the disturbance of auxin response pattern and the decreased regeneration frequency of SEs. However, application of exogenous ABA in the medium almost recovered patterns of auxin response and SE initiation. Thus, the results suggest that ABA functions on SE initiation through mediating both auxin biosynthesis and polar transport for establishment of auxin response pattern in callus. Our study provides new information for understanding mechanisms of SE initiation.     

Defense against Pieris rapae in cabbage plants induced by Bemisia tabaci biotype B

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and Pieris rapae L. (Lepidoptera: Pieridae) are serious pests of vegetables, that can occur simultaneously on a single cabbage plant [Brassica oleracea var. capitata L. (Brassicaceae)]. We determined whether pre‐feeding or infestation by B. tabaci on cabbage could induce physiological and biochemical responses of the plant against P. rapae. Developmental time, length, and weight of immature P. rapae, and defense‐related plant compounds (SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; APX, ascorbate peroxidase) were measured. Development of P. rapae larvae was 21% slower on B. tabaci‐pre‐infested plants than on plants without B. tabaci infestation. When feeding on B. tabaci‐pre‐infested plants, 22% of P. rapae larvae pupated as compared with 83% on B. tabaci‐free plants. Weights of P. rapae from first to fourth instars that fed on B. tabaci‐pre‐infested plants were also reduced, whereas those of fifth instars and pupae were not. Similarly, body length of P. rapae from first to fourth instars was affected by B. tabaci pre‐infestation, whereas that of the fifth instars was not. Peroxidase and APX activities of the B. tabaci‐pre‐infested plants increased more than SOD and CAT. Peroxidase and SOD activities of B. tabaci and P. rapae co‐infested plants increased as compared with those of P. rapae‐infested plants; however, CAT and APX activities were not different between B. tabaci‐ and P. rapae‐infested plants. These results showed that B. tabaci infestation had a negative effect on P. rapae when they occurred simultaneously on the same host plant. The implications of the induced plant changes on the herbivore are discussed.     

嗜碱单胞菌的新型羧基转移酶α亚基基因Aa-accA及其抗盐碱性能

[目的]探讨解淀粉嗜碱单胞菌(Alkalimonas amylolytica)N10来源的羧基转移酶α亚基(Acetyl-coenzyme A carboxylase subunit alpha,AccA)基因Aa-accA对细菌及植物细胞耐盐碱性的作用.[方法]通过PCR方法从嗜碱菌N10基因组中扩增基因Aa-accA,并在大肠杆菌(Escherichia coli)K12中表达,通过测定工程菌及对照菌在不同盐浓度[0%,2%,4%,6%(W/V) NaCl]及不同碱性pH(8.0,8.5,9.0,9.5)的LB中生长12 h后的OD600值,以及二者在分别含6%(W/V) NaCl及pH 9的LB中的生长曲线,评价Aa-accA对大肠杆菌耐盐碱性的影响.同时以pPZP111为载体,构建了植物细胞重组表达载体,通过农杆菌介导方法将该基因转入烟草BY-2悬浮细胞表达,利用FDA染色方法测定经盐碱溶液处理后残存的活细胞数量评价该基因对植物细胞耐盐碱性的影响.[结果]PCR扩增得到基因Aa-accA,其ORF含957 bp,编码318个氨基酸的多肽,BLAST比对显示该基因为羧基转移酶α亚基(AccA)家族中的成员,其氨基酸序列与E.coli的AccA具有76%同源性;含有Aa-accA的E.coli K12相较于对照组在不同NaCl浓度及不同碱性pH的LB中表现出了明显的生长优势,特别是在6%(W/V) NaCl及pH 9的LB中培养12 h后,终OD600分别是对照菌的2.6倍和3.5倍;缺失体实验结果显示基因缺失的突变体E.coli K12△accA在6%(W/V) NaCl及pH 9的LB中不能正常生长,而含有Aa-accA基因的重组质粒使得E.coli K12△accA在同样条件下OD600值达到0.5和0.2;转入此基因的烟草BY-2细胞,经盐碱溶液处理后,其存活细胞比例高于野生型.[结论]本研究首次发现了Aa-accA基因与盐碱性的相关性,可提高大肠杆菌及烟草BY-2细胞的耐盐碱能力.     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

2012年在上海市发现中国大陆首例输入性D8基因型麻疹病例

本文对中国首例输入性D8基因型麻疹病毒基因特征进行分析。用ELISA法检测血清麻疹病毒IgM抗体;用Vero/Slam细胞对采集的咽拭子标本进行病毒分离,分离到的麻疹毒株用RT-PCR方法扩增其核蛋白基因3′端的部分序列,并对扩增产物进行核苷酸序列测定和分析,以3′端456个核苷酸为目的片段进行基因亲缘性关系分析。结果表明,上海市2012年共报告1 105疑似麻疹病例,其中实验室确诊590例,临床符合病例2例,排除513例,报告发病率为2.52/10万;共采集到984份疑似麻疹病例咽拭子标本,分离到247株麻疹病毒,病毒分离阳性率为25.3%;除Shanghai12-239为D8基因型外,其他均为H1a基因亚型。Shanghai12-239与世界卫生组织(World Health Organization,WHO)参考株(Manchester.UNK30.94(D8)AF280803)核苷酸序列同源性为97.8%,氨基酸序列同源性为98.6%。与WHO其他基因型参考株核苷酸序列同源性为89.6%~94.5%,氨基酸序列同源性为88.7%~95.3%。     

Protein Phosphatase 2A Catalytic Subunit α Plays a MyD88-Dependent,Central Role in the Gene-Specific Regulation of Endotoxin Tolerance

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Highlights? PP2Ac is constitutively activated and targets MyD88 in LPS-tolerized macrophages ? Constitutively active PP2Ac shifts a proinflammatory MyD88 to its prosurvival mode ? Constitutively active PP2Ac reprograms gene-specific chromatin modification landscape ? Constitutively active PP2Ac broadly defines ET at both signaling and epigenetic levels