Effects of an electric field and zero valent iron on anaerobic treatment of azo dye wastewater and microbial community structures

A zero valent iron (ZVI) bed with a pair of electrodes was packed in an anaerobic reactor aiming at enhancing treatment of azo dye wastewater. The experiments were carried out in three reactors operated in parallel: an electric field enhanced ZVI-anaerobic reactor (R1), a ZVI-anaerobic reactor (R2) and a common anaerobic reactor (R3). R1 presented the highest performance in removal of COD and color. Raising voltage in R1 further improved its performance. Scanning electron microscopy images displayed that the structure of granular sludge from R1 was intact after being fed with the high dye concentration, while that of R3 was broken. Fluorescence in situ hybridization analysis indicated that the abundance of methanogens in R1 was significantly greater than that in the other two reactors. Denaturing gradient gel electrophoresis showed that the coupling of electric field and ZVI increased the diversity of microbial community and especially enhanced bacterial strains responsible for decolorization.     

Smyd1干扰真核表达质粒及其稳定转染H9c2细胞系的构建

运用RNA干扰技术(RNA interference RNAi)构建pSUPER.retro-Smyd1真核表达质粒,经鉴定后用脂质体法转染H9c2细胞,通过G418筛选出稳定表达pSUPER.retro-Smyd1的细胞系,最后经Western blot及RT-PCR实验鉴定其干扰效果。经鉴定,通过H9c2细胞系构建的pSUPER.retro-Smyd1干扰细胞系的干扰效果显著。因此,本实验成功构建了Smyd1干扰真核表达质粒及其稳定转染的H9c2细胞系,为进一步研究Smyd1基因在心脏发育中的作用奠定了良好的实验基础。     

猪GM-CSF基因的克隆及其真核表达载体的构建

采用PCR方法从猪外周血液淋巴细胞cDNA中扩增出与预期设计大小相符的GM-CSF基因特异性条带,PCR产物经EcoR Ⅰ和Xho Ⅰ双酶切后,插入到载体pIRES2-EGFP构建成真核表达载体pIRES2-EGFP-GM-CSF.经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较,证实了重组质粒的正确性.将构建好的真核表达质粒转染到山羊胎儿成纤维细胞中进行瞬时表达,荧光检测证实细胞转染成功.pIRES2-EGFP-pGM-CSF真核表达载体的成功构建为下一步在细胞水平研究GM-CSF蛋白功能以及进一步将其开发为高档疫苗佐方剂奠定了基础.     

Design of Experiment in CHO and HEK transient transfection condition optimization

Transient gene expression (TGE) is a well-established enabling technology for rapid generation of recombinant proteins, with Human Embryonic Kidney (HEK) and Chinese Hamster Ovary (CHO) cell lines and polyethyleneimine (PEI) as the transfection reagent being its most popular components. However, despite considerable progress made in the field, volumetric titers can still be a limiting factor causing the manipulation of increasing quantities of culture media and DNA. Here, we report a systematic analysis of TGE conditions and their influence on yields and protein quality. Guided by Design of Experiments (DoE), we conclude that TGE yields with one test antibody can be maximized by a parallel increase of cell density - 2.4 to 3.0 × 10(6)cells/mL - and PEI concentration - 24 to 30 mg/L - while maintaining a 1:1 ratio of heavy chain and light chain encoding plasmids. Interestingly, we also show that in these conditions, DNA concentration can be maintained in the 1mg/L range, thereby limiting the need for large DNA preparations. Our optimized settings for PEI-mediated TGE in HEK and CHO cells evaluated on several proteins are generally applicable to recombinant antibodies and proteins.     

尘螨变应原Derf1真核表达载体的构建及转染CHO细胞

目的：构建尘螨变应原Der f1真核表达载体,转染真核细胞并进行蛋白表达。方法：根据Genebank中Der f1基因的核酸序列（AB034946）,设计引物,采用PCR法,从保存的JM109工程菌中扩增Der f1编码基因,克隆到真核表达质粒pcDNA3.1/myc-his A上,以脂质体法转染CHO细胞,经G418筛选,进行稳定表达细胞株的筛选和鉴定。结果：将目的基因Der f1成功连接到pcDNA3.1/myc-hisA-Derf1并转染CHO细胞,获得稳定表达的CHO细胞株。结论：成功构建了尘螨变应原Der f1真核表达载体,并转染CHO细胞表达蛋白质。     

Recruitment of the intracellular Ca by ultrashort electric stimuli: The impact of pulse duration

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca²⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca²⁺ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca²⁺ can be controlled by the duration of the stimulus.     

Derivation of the clonal‐cell lines from Siberian sturgeon (Acipenser baerii) head‐kidney cell lines and its applicability to foreign gene expression and virus culture

This study was conducted to establish and characterize the clonal‐cell lines from Siberian sturgeon Acipenser baerii head‐kidney tissues and to evaluate its applicability as a research tool. From the culture of A. baerii head‐kidney derived cells, 10 cell lines were established first and then eight clonal‐cell lines were derived from clonal growth and colony expansion of two cell lines that showed significant high colony‐forming ability. All eight clonal‐cell lines were morphologically similar and grew stably under monolayer culture but their growth rates were significantly different. They possessed diploid DNA contents, expressed epithelial cell‐related genes and showed strong anchorage dependency to substrates. When a clonal‐cell line was transfected separately with three plasmid vectors including fluorescent reporter genes driven by cytomegalovirus, marine medaka Oryzias dancena β‐actin or A. baerii β‐actin promoter, the cell lines expressed fluorescent signals regardless of promoter types. The cells harbouring foreign genes could be expanded to stable cell lines under drug selection and then they additionally could form the extensively proliferating colonies at low‐density culture. Finally, the clonal‐cell lines showed the susceptibility to viral haemorrhagic septicaemia virus (VHSV). Collectively, the clonal‐cell lines from A. baerii head kidney were established and these cell lines will be able to provide an excellent in vitro system for various biological studies in this fish species.     

Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum

CRISPR-Cas9 mediated genome editing is addressing key limitations in the transfection of malaria parasites. While this method has already simplified the needed molecular cloning and reduced the time required to generate mutants in the human pathogen Plasmodium falciparum, optimal selection of required guide RNAs and guidelines for successful transfections have not been well characterised, leading workers to use time-consuming trial and error approaches. We used a genome-wide computational approach to create a comprehensive and publicly accessible database of possible guide RNA sequences in the P. falciparum genome. For each guide, we report on-target efficiency and specificity scores as well as information about the genomic site relevant to optimal design of CRISPR-Cas9 transfections to modify, disrupt, or conditionally knockdown any gene. As many antimalarial drug and vaccine targets are encoded by multigene families, we also developed a new paralog specificity score that should facilitate modification of either a single family member of interest or multiple paralogs that serve overlapping roles. Finally, we tabulated features of successful transfections in our laboratory, providing broadly useful guidelines for parasite transfections. Molecular studies aimed at understanding parasite biology or characterising drug and vaccine targets in P. falciparum should be facilitated by this comprehensive database.