Successive chromosome walking by compatible ends ligation inverse PCR

Here we describe an advanced polymerase chain reaction (PCR) technique, the compatible ends ligation inverse PCR (CELI-PCR) for chromosome walking. In CELI-PCR, several restriction enzymes, which produce compatible cohesive ends, were used to digest target DNA simultaneously or sequentially to produce DNA fragments of suitable size. DNA fragments were then easily circularized and PCR amplification could be carried out efficiently. The previous limitations of inverse PCR were overcome, such as unavailable restriction sites, poor template DNA circularization, and low amplification efficiency. Therefore, successive chromosome walking was performed successfully. Our work, isolating a 11,395-bp fragment from Gossypium hirsutum, was presented as an example to describe how CELI-PCR was carried out.     

不同供氮水平的水稻叶尖的傅里叶转换红外光谱

四个氮素水平处理的盆栽水稻(Oryza sativa L.)的叶尖在不同生育期均表现出明显的傅里叶转换红外光谱差异.新定义的光谱指数((A3400-A1653)/(A3400+A1653),A为某频率处的吸收值)随着施氮水平的提高而降低.结果表明,傅里叶转换红外光谱可用于诊断植物的氮素状况.     

Phage displayed peptides to avian H5N1 virus distinguished the virus from other viruses

The purpose of the current study was to identify potential ligands and develop a novel diagnostic test to highly pathogenic avian influenza A virus (HPAI), subtype H5N1 viruses using phage display technology. The H5N1 viruses were used as an immobilized target in a biopanning process using a 12-mer phage display random peptide library. After five rounds of panning, three phages expressing peptides HAWDPIPARDPF, AAWHLIVALAPN or ATSHLHVRLPSK had a specific binding activity to H5N1 viruses were isolated. Putative binding motifs to H5N1 viruses were identified by DNA sequencing. In terms of the minimum quantity of viruses, the phage-based ELISA was better than antiserum-based ELISA and a manual, semi-quantitative endpoint RT-PCR for detecting H5N1 viruses. More importantly, the selected phages bearing the specific peptides to H5N1 viruses were capable of differentiating this virus from other avian viruses in enzyme-linked immunosorbent assays.     

FBXW7 suppresses epithelial‐mesenchymal transition and chemo‐resistance of non‐small‐cell lung cancer cells by targeting snai1 for ubiquitin‐dependent degradation

Objectives

FBXW7 acts as a tumour suppressor by targeting at various oncoproteins for ubiquitin‐mediated degradation. However, the clinical significance and the involving regulatory mechanisms of FBXW7 manipulation of NSCLC regeneration and therapy response are not clear.

Materials and Methods

Immunohistochemical staining and qRT‐PCR were applied to detect FBXW7 and Snai1 expression in 100 samples of NSCLC and matched tumour‐adjacent tissues. FBXW7 manipulation of cancer biological functions were studied by using MTT assay, immunoblotting, flow cytometry, transwells, wound healing assay, and sphere‐formation assays. Immunofluorescence and co‐immunoprecipitation were used to analyse the possible interaction between Snai1 and FBXW7.

Results

We detected the decreased FBXW7 expression in majority of the NSCLC tissues, and lower FBXW7 level was correlated with advanced TNM stage. Furthermore, those patients with decreased FBXW7 expression tend to have both poorer 5‐year survival outcomes, and shorter disease‐free survival, comparing to those with higher FBXW7 levels. Functionally, we found that FBXW7 enforcement suppressed NSCLC progression by inducing cell growth arrest, increasing chemo‐sensitivity and inhibiting Epithelial‐mesenchymal Transition (EMT) progress. Results further showed that FBXW7 could interact with Snai1 directly to degrade its expression through ubiquitylating alternation in NSCLC, which could be partially abrogated by restoring Snai1 expression.

Conclusions

FBXW7 conduction of tumour suppression was partly through degrading Snai1 directly for ubiquitylating regulation in NSCLC

     

小鼠pαMHC-EGFP胚胎干细胞株的构建

应用电穿孔方法将含有α肌球蛋白重链启动子的pαMHC-EGFP载体转染到D3系小鼠胚胎千细胞,应用200μg／ml新霉素进行药物选择。采用悬浮培养法,体外诱导分化心肌细胞。荧光显微镜下,观察到第7天和第8天拟胚体中出现“跳动”的心肌细胞并同时有绿色荧光蛋白的表达。同时与D3系小鼠胚胎干细胞比较心肌细胞分化率的变化无显著差异（P〉0．05）。该细胞株在分化心肌细胞的同时,具有绿色荧光蛋白的标记,因而利于对心肌细胞的识别和纯化。     

Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice

It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda) is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid^−/−) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid^−/− mice. Their induction efficiency was similar to that of wild-type (Aid^+/+) iPS cells. The Aid^−/− iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid^+/+ and Aid^−/− iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation.     

Fabrication of Bioinspired Structured Superhydrophobic and Superoleophilic Copper Mesh for Efficient Oil-water Separation

Oily water treatment has attracted the attention of many researchers.The development of effective and cheap oil/water separation materials is urgent for treating this problem.Herein,inspired by superhydrophobic typical plant leaves such as lotus,red rose and marigold,superhydrophobic and superoleophilic copper mesh was fabricated by etching and then surface modification with 1-dodecanethiol (HS(CH2)11CH3).A rough silver layer is formed on the mesh surface after immersion.The obtained mesh surface exhibits superhydrophobicity and superoleophilicity and the static water contact angle was 153° ± 3°.In addition,the as-prepared copper mesh shows self-cleaning character with water and chemical stability.The as-prepared copper foam can easily remove the organic solvents either on water or underwater.We demonstrate that by using the as-prepared mesh,oils can be absorbed and separated,and that high separation efficiencies of larger than 92％ are retained for various oils.Thus,such superhydrophobic and superoleophilic copper mesh is a very promising material for the application of oil spill cleanup and industrial oily wastewater treatment.     

Quantitative evaluation of siRNA delivery in vivo

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.