Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Current state and perspectives on erythropoietin production

Erythropoietin is a major regulator of erythropoiesis which maintains the body's red blood cell mass and tissue oxygenation at an optimum level. Recombinant human erythropoietin (rhEPO), which is a widely used therapeutic agent for the treatment of anemia and which represents one of the largest biopharmaceuticals markets, is produced from recombinant Chinese hamster ovary cells. rhEPO is a glycoprotein with complex glycan structure, which is responsible for its therapeutic efficacy, including the in vivo activity and half-life. In order to obtain an optimal and consistent glycoform profile of rhEPO and concurrently maintain a high production yield, various approaches in drug development and cell culture technology have been attempted. Recent advances in rhEPO production are classified into three types: the development of improved rhEPO molecules by protein engineering; improvement of production host cells by genetic engineering; and culture condition optimization by fine control of the production mode/system, process parameters, and culture media. In this review, we focus on rhEPO production strategies as they have progressed thus far. Furthermore, the current status of the market and outlook on rhEPO and its derivatives are discussed.     

不同细胞周期大鼠肝实质细胞癌细胞粘弹特性研究

以胸腺嘧啶核苷和秋水仙碱顺序阻断法及胸腺嘧啶核苷双阻断法分别获得同步化Ｇ１期和Ｓ期细胞,从细胞周期角度出发,采用微管吸吮技术对大鼠肝实质细胞癌细胞的粘弹特性进行了测定并以标准线性固体模型对实验数据进行了拟合,结果表明：该细胞具有高弹性和低粘性的总体特征;Ｇ１期细胞与Ｓ期细胞相比具有高Ｋ１值和低μ值的特点,从而显示Ｇ１期细胞比Ｓ期细胞具有更大的强度和更快的被动变形能力。这些结果不仅反映了同步化细胞存在的细胞骨架状态的周期性差异,也提示Ｇ１期细胞可能比Ｓ期细胞更适于在血流中存活和转移。     

Clinical manifestation, imaging, and genotype analysis of two pedigrees with spinocerebellar ataxia

The objective of this study was to analyze the clinical manifestation, imaging characteristics, genotype, and the relationship between the three aforementioned parameters in two pedigrees suffering from spinocerebellar ataxia. To evaluate the clinical manifestation of the two pedigrees and to compare the characteristics, we performed the MRI analysis of some patients from both pedigrees, while 2 ml of the peripheral blood sample was collected for gene analysis. The gene analysis data showed that pedigree 1 was certified spinocerebellar ataxia type-2 (SCA2); the CAG repeats in the proband, proband’s mother, and proband’s brother were 44, 36, and 38, respectively. The MRI revealed brainstem cerebellar atrophy and “cross sign” and “ordinate sign” of pons. Pedigree 2 was certified SCA1; the CAG repeats of the proband, proband’s aunt, and proband’s asymptomatic cousin were 60, 51, and 52, respectively. The MRI revealed cerebellar atrophy in these individuals. We, therefore, concluded that it was difficult to diagnose the SCA subset solely through the clinical manifestation. The imaging characteristics analysis and final diagnosis depended basically on gene analysis data.     

Homology modeling threedimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis

AnxB1,a novel annexin previously isolated from Cysticercus cellulose,shows high thrombi affinity and anticoagulant activity in vivo.In order to investigate the relationship between structure and biological function,a predicted three-dimensional(3D)model of AnxB1 was generated by homology modeling.This model contains four homologous internal-domains and the Cα trace of domain Ⅰ,Ⅱ and IV shows high similarity.Based on the structure characterization,four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E.coli.Two of the mutants,GST-M3 and GST-M4 reserved high anticoagulant activity(p＜0.01 vs.GST).Furthermore,compared with the wild type GST-AnxB1,the immunogenicity of GST-M3 and GST-M4 was reduced significantly(p＜0.01)and the molecular weight was lowered to 27 kD and 34 kD,respectively.These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.     

雷氏大疣蛛毒素—Ⅱ的纯化与初步毒性研究

雷氏大疣蛛是我国最近鉴定的蜘蛛新种。雷氏大疣蛛毒素－Ⅱ就是以其粗毒为材料,利用阴、阳离子交换层析和反相HPLC分离得到并命名的一种新型神经毒素肽,根据质谱分析得知它的相对分子质量为3021．56;通过初步毒性研究,证明它是一个神经毒素。     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

Production and selected fuel properties of biodiesel from promising non-edible oils: Euphorbia lathyris L., Sapium sebiferum L. and Jatropha curcas L

A comparative study on the composition, biodiesel production and fuel properties of non-edible oils from Euphorbia lathyris L. (EL), Sapium sebiferum L. (SS), and Jatropha curcas L. (JC) was conducted. Under optimal conditions, the FAME content and yield of the three oils were greater than 97.5 wt.% and 84.0%, respectively. The best biodiesel was produced from EL due to its high monounsaturation (82.66 wt.%, Cn: 1), low polyunsaturation (6.49 wt.%, Cn: 2, 3) and appropriate proportion of saturated components (8.78 wt.%, Cn: 0). Namely, EL biodiesel possessed a cetane number of 59.6, an oxidation stability of 10.4 h and a cold filter plug point of -11 °C. However, the cetane number (40.2) and oxidative stability (0.8 h) of dewaxed SS kernel oil (DSSK) biodiesel were low due to the high polyunsaturation (72.79 wt.%). In general, the results suggest that E. lathyris L. is a promising species for biodiesel feedstock.