Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

用Hela229细胞培养法研究呼吸道肺炎衣原体感染

本文用Ｈｅｌａ２２９细胞培养与单克隆抗体免疫荧光法对９４０例咽拭子及２２４例纤支镜取材标本进行肺炎衣原体分离鉴定。结果正常组、上呼吸道感染组、下呼吸道感染组、肺部肿瘤组的咽拭子标本分离率分别为０％（０／２４８）,２．１３％（１０／４６８）,２．１％（３／１４６）,１．２８％（１／７８）。上呼吸道感染组和下呼吸道感染组的分离率均高于正常组和肺部肿瘤组。前二组与正常组的差异有显著性意义（Ｐ＜０．０５）,与肿瘤组的差异无显著性意义（Ｐ＞０．０５）。下呼吸道感染组、肺部肿瘤组的纤支镜取材分离率分别为１０．９６％（     

shRNA‑mediated knockdown of KNTC1 inhibits non-small-cell lung cancer through regulating PSMB8

In view of the important roles played by Kinetochore proteins in mitosis, we believed that they may contribute to the development and progression of human cancers, which has been reported recently elsewhere. Kinetochore-associated 1 (KNTC1) participates in the segregation of sister chromatids during mitosis, the effects of which on non-small-cell lung cancer (NSCLC) remain unclear. Here, we sought to identify the biological significance of KNTC1 in NSCLC. KNTC1 protein expression in NSCLC tissues was investigated by immunohistochemistry. Lentivirus delivered short hairpin RNA (shRNA) was utilized to establish KNTC1 silence NSCLC cell lines. The effects of KNTC1 depletion on NSCLC cell proliferation, migration, apoptosis, and tumor formation were analyzed by MTT assay, wound-healing assay, transwell assay, flow cytometry assay, and in nude mouse models in vivo. After KNTC1 reduction, NSCLC cell viability, proliferation, migration, and invasion were restrained. A xenograft tumor model was also provided to demonstrate the inhibited tumorigenesis in NSCLC. In addition, the downstream mechanism analysis indicated that KNTC1 depletion was positively associated with PSMB8. The findings of the present study suggested that KNTC1 may have a pivotal role in mediating NSCLC progression and may act as a novel therapeutic target for NSCLC.Subject terms: Non-small-cell lung cancer, Cell migration     

土地利用变化对三峡库区重庆段植被净初级生产力的影响

研究土地利用变化对区域植被净初级生产力(Net Primary Productivity,NPP)的影响对于明确区域植被固碳能力与土地利用变化的关系,以及维持生态系统结构稳定具有重要意义。以三峡库区重庆段为例,基于2000—2015年MOD17A3数据和土地利用数据,分析研究区NPP时空分布特征并从景观生态学的角度探讨土地利用变化对区域植被NPP的影响。研究表明:(1)NPP年均值16年间波动不大,空间分布上从东到西逐渐减少;(2)研究期内林地面积增加,耕地和草地面积减小,而NPP总量从25.6 TgC增加到了28.5 TgC,其中耕地NPP约占总量的44%,林地次之(40%),草地最少(14%),2000—2005年、2005—2010年、2010—2015年土地利用变化对NPP变化的贡献率分别为26.49%、59.76%、17.27%;(3)区域生态景观指数中的香农多样性指数SHDI、斑块密度PD与NPP呈正相关,而聚合度AI与NPP呈负相关,景观格局类型和景观格局变化均影响区域植被NPP的增长。要提高区域植被NPP,需优化土地利用格局,增加景观异质性和斑块密度,重视培育幼龄林,并控制成熟林的数量。     

基于mtDNA D-loop分析高原鳅群体遗传多样性和系统发育

本研究以高原鳅属6个群体共82尾个体的线粒体DNA D-loop高变区为标记,采用分子生物信息学方法分析其遗传多样性及系统发育关系.结果表明,在570 bp的mtDNA D-loop中共检测到149个多态位点,由此界定了 39个单倍型.在6个群体中,单倍型多样度(Hd)由高到低依次为细尾高原鳅(T.stenura)(0...     

Quantitative regulation of the thermal stability of enveloped virus vaccines by surface charge engineering to prevent the self-aggregation of attachment glycoproteins

The development of thermostable vaccines can relieve the bottleneck of existing vaccines caused by thermal instability and subsequent poor efficacy, which is one of the predominant reasons for the millions of deaths caused by vaccine-preventable diseases. Research into the mechanism of viral thermostability may provide strategies for developing thermostable vaccines. Using Newcastle disease virus (NDV) as model, we identified the negative surface charge of attachment glycoprotein as a novel determinant of viral thermostability. It prevented the temperature-induced aggregation of glycoprotein and subsequent detachment from virion surface. Then structural stability of virion surface was improved and virus could bind to and infect cells efficiently after heat-treatment. Employing the approach of surface charge engineering, thermal stability of NDV and influenza A virus (IAV) vaccines was successfully improved. The increase in the level of vaccine thermal stability was determined by the value-added in the negative surface charge of the attachment glycoprotein. The engineered live and inactivated vaccines could be used efficiently after storage at 37°C for at least 10 and 60 days, respectively. Thus, our results revealed a novel surface-charge-mediated link between HN protein and NDV thermostability, which could be used to design thermal stable NDV and IAV vaccines rationally.     

A loosened gating mechanism of RIG-I leads to autoimmune disorders

DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-I_E510V and RIG-I_Q517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N_1-2-2′-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.