Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

The advancements,challenges, and future implications of the CRISPR/Cas9 system in swine research

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.     

多轮次胁迫驯化及多因子复合筛选丁醇高产菌

【目的】从陕西省石泉县玉米地土壤中分离获得一株产丁醇菌株并提高其丁醇耐受性和丁醇产量。【方法】采用自行设计的多因子复合筛选方法和丁醇胁迫驯化处理,在获得丁醇高产菌株的同时提高菌株的丁醇耐受性。【结果】野生菌株D64经多轮次丁醇胁迫驯化处理和多因子复合筛选,分离获得突变株T64,其丁醇耐受性明显提高,能在丁醇浓度为20 g/L的复合筛选培养基上正常生长,发酵7%玉米醪丁醇产量由13.35 g/L提高到15.18 g/L,总溶剂(丙酮、丁醇、乙醇)达到21.8 g/L。【结论】采用长时间且丁醇浓度呈梯度渐进增加的胁迫驯化方式,可使菌种在丁醇的环境中不断进化并有效地提高菌株对丁醇的耐受性。多因子复合筛选方法较其他单一因子筛选方法更为有效,能较快获得丁醇高产菌。     

A high-throughput screen to identify novel calcineurin inhibitors

Calcineurin is a eukaryotic protein phosphatase important for many signalling and developmental processes in cells. Inhibitors of this enzyme are used clinically and there is interest in identifying novel inhibitors for therapeutic applications. This report describes a high-throughput assay that can be used to screen natural or chemical libraries of compounds to identify new calcineurin inhibitors. The microtitre plate assay is based on a yeast reporter strain and was validated with known inhibitors and tested in a pilot screen of bacterial extracts.     

阿克苏地区传统酸乳中乳酸菌筛选及安全性初步评价

为挖掘新疆传统发酵乳制品中的优质乳酸菌资源,获得具有良好益生特性及食用安全性的乳酸菌菌株,对新疆阿克苏地区托木尔峰自然保护区周围村镇传统酸乳进行了样品采集、乳酸菌的分离、筛选及菌株生长特性、抗氧化活性和抑菌等功能特性的分析,并进行溶血性和有害代谢物质等食用安全性检测.16S rRNA测序结果显示,8份代表样品共获得12...     

A new helicase assay based on graphene oxide for anti-viral drug development

Recently, graphene oxide (GO), one of the carbon nanomaterials, has received much attention due to its unique physical and chemical properties and high potential in many research areas, including applications as a biosensor and drug delivery vehicle. Various GO-based biosensors have been developed, largely based on its surface adsorption properties for detecting biomolecules, such as nucleotides and peptides, and real-time monitoring of enzymatic reactions. In this review, we discuss recent advances in GO-based biosensors focusing on a novel assay platform for helicase activity, which was also employed in high-throughput screening to discover selective helicase inhibitors.     

3D QSAR pharmacophore-based virtual screening for the identification of potential inhibitors of tyrosinase

Abstract

Tyrosinase plays an important role in melanin biosynthesis and protects skin against ultraviolet radiations. Functional deficiency of tyrosinase results in serious dermatological diseases. Tyrosinase also participates in neuromelanin formation in the human brain, which leads to neurodegeneration resulting in Parkinson’s disease. In fruits and vegetables, tyrosinase plays a critical role in senescence, causing undesired browning that results in faster deterioration and shorter shelf lines. The only commercially available tyrosinase is mushroom tyrosinase and it shows the highest homology to the mammalian tyrosinase. Although kojic acid is currently used as a tyrosinase inhibitor, they have serious side effects such as dermatitis, carcinogenesis and hepatotoxicity. Therefore, in order to develop a more active and safer tyrosinase inhibitor, 3D QSAR pharmacophore models were generated based on experimentally known inhibitors. The pharmacophore model, Hypo1, was developed with a large cost difference, high correlation coefficient and low RMS deviation. Hypo1 showed a good spatial arrangement; consisting of five-point features including two hydrogen bond acceptor, one hydrogen bond donor and two hydrophobic features. Hypo1 was further validated by cost analysis, test set and Fisher’s randomisation method. Hypo1 was used as a 3D query for screening the in-house drug-like databases, and the hits were further selected by applying ADMET, Lipinski’s rule of five and fit value criteria. To identify binding conformations, the obtained hits were subjected to molecular docking. Finally, molecular dynamics simulations revealed the appropriate binding modes of hit compounds. To conclude, we propose the final three hit compounds with new structural scaffolds as a virtual candidate as tyrosinase inhibitors.

Communicated by Ramaswamy H. Sarma     

中央戈壁石下生物土壤结皮中细菌群落结构和多样性

【背景】戈壁石下生物土壤结皮(Biological soil crusts,BSCs)中的微生物在干旱、半干旱地区生态系统物质循环中起重要作用,其细菌群落结构和多样性因气候和地理环境的不同而存在较大差异。中央戈壁面积大,其石下BSCs中细菌群落结构和多样性却知之甚少。【目的】比较中央戈壁石下BSCs和周围裸土中细菌群落结构和多样性的异同,揭示环境因子对它们的影响以及细菌之间的相互作用关系。【方法】利用Illumina MiSeq对16S rRNA基因进行高通量测序,使用生物信息学分析方法分析细菌群落结构和多样性及其影响因素;基于CoNet软件绘制物种共现性网络图,利用Cytoscape 3.5.1软件将网络图可视化。【结果】石下BSCs的速效磷(Available phosphorus,AP)、速效氮(Available nitrogen,AN)和叶绿素a (Chlorophyll a,Chl a)的含量均明显高于周围裸土(最高高出471%),而pH则略低于周围裸土;石下BSCs中细菌的最优势菌门为Cyanobacteria(45.85%-53.77%),优势属(9.25%-18.42%)有Trichocoleus、Chroococcidiopsis和属于Cyanobacteria纲的1个未知属;临近裸土中细菌的最优势菌门为Actinobacteria (38.82%-44.69%),优势属(5%)有Arthrobacter、Rubrobacter和其它3个属于放线菌门或酸杆菌门的未知属;在土壤理化因子中,AP对石下细菌群落组成的影响最大;除Actinophytocola属和Cyanobacteria纲中的未知属外,其他微生物之间均存在较强的互作关系,其中以共存的互作关系为主(约占60%),点度中心性、接近中心性和中介中心性均较高的节点都属于Cyanobacteria门和α-Proteobacteria纲。【结论】中央戈壁石下BSCs中的细菌群落结构和多样性明显不同于临近裸土,Cyanobacteria门和α-Proteobacteria纲的细菌是该地区石下早期BSCs形成和发育最初及最重要的驱动力。     

以酵母嗜杀系统为基础的抗病毒药物筛选模型的建立

根据嗜杀酵母T158c/S14a中L-A病毒-1移码效率改变影响M1病毒的存活,导致K1毒素减少,在低pH的美蓝平板上用杯碟法通过抑菌圈的大小检测酵母K1毒素的嗜杀活性,建立了一个以酵母嗜杀系统为基础的抗病毒药物筛选模型。研究了杯碟法检测酵母毒素嗜杀活性的各种条件。对不同pH和温度下酵母的嗜杀活性进行了研究,确定了模型用于筛选的最适pH范围为4.3~4.7,最适温度范围为20~22℃。运用该模型研究了几种中药对嗜杀活性的抑制作用,发现了金银花和升麻具有一定的抗病毒作用。该模型为抗病毒药物的高通量初筛奠定了基础。