Purification and partial characterization of glutathione S-transferases from three field populations of Panonychus citri (Acari: Tetranychidae)

Glutathione S-transferases (GSTs) play central roles in phase II detoxification of both xenobiotics (drugs, insecticides, and herbicides) and endogenous compounds in almost all living organisms. In this study, we successfully purified the GSTs from the citrus red mite, Panonychus citri, by affinity chromatography on Glutathione Sepharose 4B and compared the biochemical characterizations of the purified GSTs from three field populations [beibei (BB), wanzhou (WZ), and zhongxian (ZX)]. SDS–PAGE revealed that the molecular weight of GSTs from three populations consisted of two subunits of 27.3 and 26.1 kDa. The specific activity of the purified GSTs from the WZ and ZX populations was increased 1.5- and 3.8-fold, respectively, compared with the BB population. Accordingly, the pyridaben susceptibility of WZ and ZX populations was less compared with BB population. Kinetic analyses showed that the WZ and ZX populations had higher substrate specificity compared with the BB population based on the values of k _cat and k _cat /K _m to both reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). The in vitro inhibition studies of GSTs indicated that the I ₅₀ values of pyridaben from WZ and ZX populations of P. citri expressed 1.6- and 4.4-fold decreases, respectively, compared to the I ₅₀ value of pyridaben from the BB population. In conclusion, all evidence suggested that the purified GSTs may partially contribute to the susceptibility of acaricide pyridaben in field populations of P. citri.     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

矮化香蕉及其野生型GA3ox基因的结构特点和表达分析

香蕉的矮化突变是香蕉无性繁殖后代最常见的表型变异之一,但其变异的分子调控机理目前尚未研究清楚; 而内源赤霉素是影响植物株高的重要激素之一,GA3-氧化酶是赤霉素生物合成后期的关键酶。为探究GA3-氧化酶编码基因对香蕉矮化的分子调控机理,该研究以威廉斯B6矮化突变体及其野生型亲本为材料,通过RT-PCR技术克隆得到矮化香蕉及其野生型亲本GA3ox基因的全长cDNA序列,并对其推测的氨基酸序列进行比对分析,同时利用qRT-PCR技术对GA3ox基因在不同组织中的表达水平差异进行分析。结果表明:(1)矮化香蕉GA3ox-A和野生型香蕉GA3ox-G的ORF长度均为864 bp,均编码287个氨基酸,经序列比对分析发现两条氨基酸序列之间存在5个位点的差异,从而产生具有不同性质的蛋白质。(2)氨基酸序列同源性分析表明,矮化香蕉GA3ox的氨基酸序列与油棕、海枣、椰子的同源性最高。(3)qRT-PCR显示,GA3ox基因在矮化香蕉叶片和茎秆中的表达水平整体上低于野生型,其中GA3ox在野生型茎秆中的表达水平是矮化植株的2.2～32倍。综上推测,GA3ox基因可能对香蕉茎杆的矮化变异具有重要的调控作用。该研究结果为揭示香蕉矮化突变的分子机制与筛选优良矮化香蕉株系奠定了基础。     

Retraction Note: miR-195-5p/NOTCH2-mediated EMT modulates IL-4 secretion in colorectal cancer to affect M2-like TAM polarization

Background

Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising.

Methods

Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes.

Results

An integrated diagnostic model called “Combined method” was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

     

Meiofauna promotes litter decomposition in stream ecosystems depending on leaf species

Litter decomposition, a fundamental process of nutrient cycling and energy flow in freshwater ecosystems, is driven by a diverse array of decomposers. As an important component of the heterotrophic food web, meiofauna can provide a trophic link between leaf‐associated microbes (i.e., bacteria and fungi)/plant detritus and macroinvertebrates, though their contribution to litter decomposition is not well understood. To investigate the role of different decomposer communities in litter decomposition, especially meiofauna, we compared the litter decomposition of three leaf species with different lignin to nitrogen ratios in litter bags with different mesh sizes (0.05, 0.25, and 2 mm) in a forested stream, in China for 78 days. The meiofauna significantly enhanced the decomposition of leaves of high‐and medium‐ quality, while decreasing (negative effect) or increasing (positive effect) the fungal biomass and diversity. Macrofauna and meiofauna together contributed to the decomposition of low‐quality leaf species. The presence of meiofauna and macrofauna triggered different aspects of the microbial community, with their effects on litter decomposition varying as a function of leaf quality. This study reveals that the meiofauna increased the trophic complexity and modulated their interactions with microbes, highlighting the important yet underestimated role of meiofauna in detritus‐based ecosystems.     

Multiple end joining mechanisms repair a chromosomal DNA break in fission yeast

Non-homologous end joining (NHEJ) is an important mechanism for repairing DNA double-strand breaks (DSBs). The fission yeast Schizosaccharomyces pombe has a conserved set of NHEJ factors including Ku, DNA ligase IV, Xlf1, and Pol4. Their roles in chromosomal DSB repair have not been directly characterized before. Here we used HO endonuclease to create a specific chromosomal DSB in fission yeast and examined the imprecise end joining events allowing cells to survive the continuous expression of HO. Our analysis showed that cell survival was significantly reduced in mutants defective for Ku, ligase IV, or Xlf1. Using Sanger sequencing and Illumina sequencing, we have characterized in depth the repair junction sequences in HO survivors. In wild type cells the majority of repair events were one-nucleotide insertions dependent on Ku, ligase IV, and Pol4. Our data suggest that fission yeast Pol4 is important for gap filling during NHEJ repair and can extend primers in the absence of terminal base pairing with the templates. In Ku and ligase IV mutants, the survivors mainly resulted from two types of alternative end joining events: one used microhomology flanking the HO site to delete sequences of hundreds to thousands of base pairs, the other rejoined the break using the HO-generated overhangs but also introduced one- or two-nucleotide base substitutions. The chromosomal repair assay we describe here should provide a useful tool for further exploration of the end joining repair mechanisms in fission yeast.     

Long Non-coding RNA URHC Regulates Cell Proliferation and Apoptosis via ZAK through the ERK/MAPK Signaling Pathway in Hepatocellular Carcinoma

Long non-coding RNAs (lncRNAs) have previously been implicated in human disease states, especially cancer. Although the aberrant expression of lncRNAs has been observed in cancer, the biological functions and molecular mechanisms underlying aberrantly expressed lncRNAs in hepatocellular carcinoma (HCC) have not been widely established. In the present study, we investigated a novel lncRNA, termed URHC (up-regulated in hepatocellular carcinoma), and evaluated its role in the progression of HCC. Expression profiling using a lncRNA microarray revealed that URHC was highly expressed in 3 HCC cell lines compared to normal hepatocytes. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses confirmed that URHC expression was increased in hepatoma cells and HCC tissues. Moreover, using qRT-PCR, we confirmed that URHC expression was up-regulated in 30 HCC cases (57.7%) and that its higher expression was correlated with poor overall survival. We further demonstrated that URHC inhibition reduced cell proliferation and promoted apoptosis. We hypothesize that URHC may function by regulating the sterile alpha motif and leucine zipper containing kinase AZK (ZAK) gene, which is located near URHC on the same chromosome. We found that ZAK mRNA levels were down-regulated in HCC tissues and the expression levels of ZAK were negatively correlated with those of URHC in the above HCC tissues. Next, we confirmed that URHC down-regulated ZAK, which is involved in URHC-mediated cell proliferation and apoptosis. Furthermore, ERK/MAPK pathway inactivation partially accounted for URHC-ZAK-induced cell growth and apoptosis. Thus, we concluded that high URHC expression can promote cell proliferation and inhibit apoptosis by repressing ZAK expression through inactivation of the ERK/MAPK pathway. These findings may provide a novel mechanism and therapeutic targets for the treatment of HCC.     

人胰高糖素样肽-1及其突变体在大肠杆菌BL21中的表达与生物活性研究

以人胰高糖素样肽-1（human glucagons-like peptide-1,GLP-1））为研究对象,以克隆载体pPblu2SKP/（GLP-1A2G）2基因为模板,利用PCR扩增获得GLP-1与其突变体GLP-1A2G的编码基因,并将其插入原核表达质粒pGEX-4T-1中,利用氯化钙法将其转入表达宿主大肠杆菌（Escherichia coli）BL21（DE3）中诱导表达,经IPTG诱导后,收集菌体。菌体经超声破碎后,裂解液用GST亲和层析纯化得到融合蛋白,经凝血酶酶解,用Superdex G-75凝胶层析,得到GLP-1及其突变体GLP-1A2G的样品,经SDS-PAGE电泳测定,样品纯度〉90%。小鼠糖耐量试验表明,相对于空白对照组而言,GLP-1及其突变体GLP-1A2G可以明显地控制血糖的水平,两者的生物活性并无明显差异。由于突变体GLP-1A2G较GLP-1可以更加有效的抵制DPPⅣ的降解,提示突变体GLP-1A2G较GLP-1在白蛋白融合或PEG修饰等方面具有更大的优势。