Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis.     

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH₂-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10⁻³ mol/L and 4.09 × 10⁻³ μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.     

Quality by design approach for viral clearance by protein a chromatography

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus‐like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X‐MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design‐of‐experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95–103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

玉蝉花繁殖生态学研究

通过野外调查、人工授粉和室内种子萌发实验等,对天目山玉蝉花（Iris ensata）种群繁殖生态特征进行了研究。结果显示：玉蝉花自然种群呈聚集型分布,种群更新主要依赖贡献率为0.756的无性繁殖。人工同瓣和异瓣自花授粉座果率分别为10%和20%,结实数分别为0和4±1（n=2）粒/果;同株和异株异花授粉座果率分别为80%和100%,结实数分别为59±7（n=8）和64±9（n=10）粒/果,自交主要来自同株异花授粉,而同株花异熟、雌雄异位和异向等可有效降低自交。自然套袋不结实,不存在无融合生殖。花粉/胚珠比1277±270（n=10）,辅证玉蝉花为兼性异花授粉、异花授粉植物。有效授粉昆虫为蜜蜂（Apismellifica）,访花频率为0.019次.花-1.h-1,单只蜜蜂回访同朵花的比率为3.5%。自然种群立地种子数估算值为368粒/m2,种子宿存,具非深度生理休眠,萌发需光,低温层积60d萌发率达94.9±0.7%。玉蝉花种群繁殖适应对策为k对策,具备自我维持机制,但现正受人为干扰。     

ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选

Neuregulin-1(NRG1,纽兰格林)通过活化ErbB2/ErbB4二聚体具有治疗心衰的作用,目前已完成临床二期。为避免作为心衰治疗药物时同时激活ErbB3并产生副作用,因此用NRG1变异体的方法寻找能对ErbB4专一性激活的配体。文章构建了带有不同筛选标记的ErbB2、ErbB3、ErbB4细胞表达质粒,将ErbB2/ErbB3、ErbB2/ErbB4质粒共转染至CHO细胞,建立了ErbB2/ErbB3特异性表达和ErbB2/ErbB4特异性表达的细胞株。通过与新生大鼠原代心肌细胞比较,证明ErbB2/ErbB4细胞株信号传导功能与心肌细胞相似,NRG1可以激活下游的AKT信号途径、PI3K信号途径,并表现出良好的剂量效应。因此可以通过检测与心肌功能密切相关的下游信号AKT磷酸化水平快速筛选抗心衰药物,并通过与ErbB2/ErbB3信号激活水平比较鉴定其对心肌细胞的特异性。文章还构建了31个不同的NRG1突变体并在大肠杆菌中成功的表达和纯化。将这些突变体用于刺激两个细胞株,通过检测AKT磷酸化水平,发现这些突变体对ErbB2/ErbB3与ErbB2/ErbB4受体的激活能力不同。进一步检测其中5个ErbB2/ErbB4激活特异性发生改变的突变体与两对受体的亲和力,发现这些突变体和ErbB2/ErbB4与ErbB2/ErbB3受体亲和力的变化有一致性。最终筛选到了4个可以更特异性激活ErbB2/ErbB4受体的突变体作为更有效治疗心衰的候选药物。     

基于COI序列的DNA条形码在中国沿海缀锦蛤亚科贝类中的应用分析

该研究探讨了将COI序列应用于中国沿海缀锦蛤亚科贝类物种鉴定的可行性,获得了该亚科5属11种贝类51个个体的43个单倍型序列.碱幕替换饱和性分析表明,颠换未出现饱和现象,而转换在序列分化达到10%至15%时即到达饱和.单倍犁Hap33可能是由杂交引起的,排除此瞥倍型,种内个体间遗传距离在0%到2.02%之间,平均为0.46%,属内不同种个体间遗传距离在17.21%～32.24%之间,平均为24.96%,存在条形码问隙:11种缀锦蛤哑科贝类在邻接树和贝叶斯树上都独立的单系群.该研究表明,基于COI的DNA条形码技术能够将研究所涉及的约98%的缀锦蛤亚科贝类鉴定剑种的水平,因此,利用DNA条形码技术可以对缀锦蛤亚科贝类进行有效地分类鉴定.     

Toll-like receptor 9 agonists up-regulates the expression of cyclooxygenase-2 via activation of NF-κB in prostate cancer cells

CpG-oligonucleotides (CpG-ODNs), mimicking bacterial DNA, have recently been shown to stimulate prostate cancer invasion in vitro via Toll-like receptor 9 (TLR9). Since cyclooxygenase 2 (COX-2), frequently overexpressed in multiple tumor types including prostate cancer, is a causal factor for tumor development, invasion and metastasis, an interesting question is raised whether TLR9 regulates COX-2 expression in prostate cancer cells. To address this question, herein we examined COX-2 expression in PC-3 cells stimulated with different doses and time courses of CpG-ODNs. The regulatory role of NF-κB in TLR9-mediated COX-2 expression was also investigated. CpG-ODN was found to up-regulate the expression of COX-2 in PC-3 cells in a dose- and time-dependent manner, but have little impact on COX-1 expression. Moreover, CpG-ODN also promoted nuclear translocation and activation of NF-κB, which appeared to be required for COX-2 induction by CpG-ODN. Overall, TLR9 up-regulates COX-2 expression in prostate cancer cells, at least partially through the activation of NF-κB, which may be implicated in tumor invasion and metastasis.