Tumour‐associated macrophages as a novel target of VEGI‐251 in cancer therapy

Tumour‐associated macrophages (TAMs), which possess M2‐like characters and are derived from immature monocytes in the circulatory system, represent a predominant population of inflammatory cells in solid tumours. TAM infiltration in tumour microenvironment can be used as an important prognostic marker in many cancer types and is a potential target for cancer prevention or treatment. VEGI‐251 not only is involved in the inhibition of tumour angiogenesis, but also participates in the regulation of host immunity. This work aimed to investigate the involvement of VEGI‐251 in the regulation of specific antitumour immunity. We found that recombinant human VEGI‐251(rhVEGI‐251) efficiently mediated the elimination of TAMs in tumour tissue in mice, and induced apoptosis of purified TAMs in vitro. During this process, caspase‐8 and caspase‐3 were activated, leading to PARP cleavage and apoptosis. Most importantly, we further elucidated the mechanism underlying VEGI‐251‐triggered TAM apoptosis, which suggests that ASK1, an intermediate component of the VEGI‐251, activates the JNK pathway via TRAF2 in a potentially DR3‐dependent manner in the process of TAM apoptosis. Collectively, our findings provide new insights into the basic mechanisms underlying the actions of VEGI‐251 that might lead to future development of antitumour therapeutic strategies using VEGI‐251 to target TAMs.     

Determination of the genetic architecture of seed size and shape via linkage and association analysis in soybean (Glycine max L. Merr.)

Seed-size traits, which are controlled by multiple genes in soybean, play an important role in determining seed yield, quality and appearance. However, the molecular mechanisms controlling the size of soybean seeds remain unclear, and little research has been done to investigate these mechanisms. In this study, we performed a genetic analysis to determine the genetic architecture of soybean seed size and shape via linkage and association analyses. We used 184 recombinant inbred lines (RILs) and 219 cultivated soybean accessions to evaluate seed length, seed width and seed height as seed-size traits, and their ratios of these values as seed-shape traits. Our results showed that all six traits had high heritability ranging from 92.46 to 98.47 %. Linkage analysis in the RILs identified 12 quantitative traits loci (QTLs), with five of these QTLs being associated with seed size, five with seed shape and two with the two first principal components of our principal component analysis (PCA). Association analysis in the 219 accessions detected 41 single nucleotide polymorphism (SNP)-trait associations, with 20 of these SNPs being associated with seed-size traits, seven with seed-shape traits and 14 with the two first principal components of our PCA. This analysis reveals that seed-size and seed-shape may be controlled by different genetic factors. Our results provide a greater understanding of phenotypic structure and genetic architecture of soybean seed, and the QTLs detected in this study form a basis for future fine mapping, quantitative trait gene cloning and molecular breeding in soybean.     

杀粉蝶菌素A1生物合成基因簇中甲基转移酶PieB2的功能

【目的】研究杀粉蝶菌素A1产生菌中甲基转移酶基因pieB2的功能。【方法】利用接合转移和同源重组双交换的方法,构建pieB2基因缺失突变株,以及利用接合转移的方法,构建回补菌株。通过高保真PCR克隆pieB2基因到表达载体pET28a上,构建质粒pJTU5997,转化入大肠杆菌E.coliBL21(DE3)/pLysE中诱导表达。利用高效液相色谱检测PieB2的体外酶活。【结果】获得了pieB2基因缺失的双交换突变株。发酵结果显示,该突变株不再产生杀粉蝶菌素A1,而是积累了一种脱甲基产物。N-末端融合组氨酸标签的PieB2在大肠杆菌中获得可溶性表达,通过体外催化证明了PieB2甲基转移酶的功能。【结论】体内遗传实验和体外生化实验证明了PieB2作为甲基转移酶在杀粉蝶菌素A1合成中的作用。     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。     

假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰

[目的]DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。[方法]首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spfBCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了SpfB蛋白与DNA结合序列。[结果]spfB基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spfB基因的回补能够恢复该表型,证明spfB基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在△spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spfBCDE的启动子区域5''-TGTTTGT-3''相结合。[结论]SpfB作为转录调控因子负调控磷硫酰化修饰基因spfBCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。     

泸型酒酒醅细菌群落的发酵演替规律

【目的】考察泸型酒发酵过程中酒醅细菌群落的演替规律,探讨菌群演替与环境因素变化的相关性。【方法】采用高通量测序技术分析泸型酒酒醅细菌群落的演替规律,并运用Mantel test分析不同发酵阶段的细菌群落演替与环境因素变化的相关性。【结果】酒醅发酵过程中有397个属的微生物,其中Lactobacillus、Bacillus、Weissella、Dysgonomonas、Comamonas以及Ruminococcaceae为优势属(相对丰度1.0%)。通过聚类分析可将酒醅发酵过程划分为3个阶段:阶段I (0–5 d),阶段II (6–17 d)和阶段III(18–40d),且3个阶段的酒醅菌群结构差异显著(P0.05)。Metastats分析结果表明,与阶段I相比,阶段II酒醅细菌群落中Lactobacillus和unclassifiedLactobacillaceae相对丰度显著升高(P0.05),而unclassifiedBacillaceae、 Staphylococcus、 Bacillus、 unclassified Enterobacteriaceae、 Lactococcus、Pseudomonas、Thermoactinomyces、Leuconostoc、Staphylococcus相对丰度显著降低(P0.05)。与阶段II相比,阶段III酒醅细菌群落中Lactobacillus相对丰度显著增长(P0.05),Comamonas、Acetobacter、unclassified Bacilli、Clostridium、Bacillus、Ruminococcus、unclassified Porphyromonadaceae和unclassified Streptophyta相对丰度显著下降(P0.05)。结果表明,阶段I的细菌菌群演替与酒醅温度、水分和乙醇浓度变化线性相关(P0.05);阶段II和阶段III的细菌菌群演替与酒醅温度、水分、酸度、乙醇浓度均没有相关性(P0.05)。【结论】泸型酒酒醅中细菌群落在不同发酵阶段结构差异显著,且温度、水分以及乙醇浓度对酒醅发酵前期(0–5 d)细菌群落演替具有重要作用。     

Cloning, purification, and characterization of thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) from a newly characterized thermophile Thermoanaerobacter tengcongensis was expressed in Escherichia coli and purified. Analytical gel filtration suggested that the enzyme exist as a homotetramer in solution. The optimal pH for the forward reaction was found to be 8.0 and the optimal temperature 70 degrees C. The steady-state kinetic characteristics suggest that hypoxanthine is the most effective substrate. This enzyme showed a half-life of 75min at 50 degrees C and no apparent loss of activity after 3 months at 4 degrees C.     

Time-Course Association Mapping of the Grain-Filling Rate in Rice (Oryza sativa L.)

Detecting quantity trait locus (QTLs) and elite alleles that are associated with grain-filling rate (GFR) in rice is essential for promoting the utilization of hybrid japonica rice and improving rice yield. Ninety-five varieties including 58 landraces and 37 elite varieties from the core germplasm collection were genotyped with 263 simple sequence repeat (SSR) markers. The GFR of the 95 varieties was evaluated at five stages, 7, 14, 21, 28 and 35 days after flowering (DAF) both in 2011 and 2012. We found abundant phenotypic and genetic diversity in the studied population. A population structure analysis identified seven subpopulations. A linkage disequilibrium (LD) analysis indicated that the levels of LD ranged from 60.3 cM to 84.8 cM and artificial selection had enhanced the LD. A time-course association analysis detected 31 marker-GFR associations involving 24 SSR markers located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 11 and 12 of rice at five stages. The elite alleles for high GFR at each stage were detected. Fifteen excellent parental combinations were predicted, and the best parental combination ‘Nannongjing62401×Laolaihong’ could theoretically increase 4.086 mg grain^-1 d^-1 at the five stages. Our results demonstrate that the time-course association mapping for GFR in rice could detect elite alleles at different filling stages and that these elite alleles could be used to improve the GFR via pyramiding breeding.     

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3β of Wnt/β-catenin signaling pathway

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR-344 directly targets the 3′ UTR of GSK3β (Glycogen synthase kinase 3 beta). Knockdown of GSK3β with siRNA results in inhibiting 3T3-L1 differentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevates the level of active β-catenin, which is the downstream effector of GSK3β in the Wnt/β-catenin signaling pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3β and subsequently activating the Wnt/β-catenin signaling pathway.