Electrotransformation of Thermoanaerobacter ethanolicus JW200

Electrotransformation of Thermoanaerobacter ethanolicus JW200 was achieved using the plasmid, pTE16, and a pUC-based suicide vector, pTEA2. The construct pTE16 is based on the Escherichia coli-Clostridium perfringens shuttle vector pJIR715 and contains a thermostable chloramphenicol (Cm) resistance cassette. Evidence supporting transformation was provided by extracting plasmid pTE16 from presumptive transformants of T. ethanolicus and by PCR specific to the chloramphenicol acetyltransferase (cat) gene on the vector pTEA2. Transformation frequencies of plasmid pTE16 and pTEA2 were 50 ± 7.4 and 30 ± 4.2 transformants per μg plasmid DNA. The results provide the first unequivocal gene transfer method functional in T. ethanolicus.     

内蒙古不同类型草地叶面积指数遥感估算

叶面积指数(Leaf Area Index,LAI)是重要的植被结构参数,反演LAI是植被遥感的重要研究内容之一。根据在内蒙古呼伦贝尔和锡林浩特草原利用LAI 2000观测的草地LAI,比较了不同植被指数(SR、RSR、EVI、NDVI、SAVI和ARVI)估算不同类型草地LAI的能力,建立了基于Landsat-5 TM遥感数据的LAI估算模型,并利用LAI观测数据对模型进行了检验,生成了研究区内草地LAI分布图,据此对MODIS LAI产品一致性进行了评价。结果表明,在呼伦贝尔和锡林浩特两个研究区,RSR与LAI的相关性最高(R²分别为0.628、0.728,RMSE分别为0.512、0.490),在低密度草地,RSR的优势更为明显;验证表明,根据RSR建立的LAI估算模型的精度可达70%;利用TM数据生成的两个地区的LAI(TM LAI)空间变化明显,锡林浩特草地的LAI值整体上低于呼伦贝尔草地;在呼伦贝尔和锡林浩特,MODIS LAI产品与TM LAI一致性分别为0.566,0.323,MODIS LAI产品高估了呼伦贝尔草地LAI值,而在锡林浩特研究区则存在低估现象。     

LINC00839 Promotes Neuroblastoma Progression by Sponging miR-454-3p to Up-Regulate NEUROD1

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Increasing long non-coding RNAs (lncRNAs) are reported to be associated with NB tumorigenesis and aggressiveness. Here, we attempted to investigate the biological functions of LINC00839 in NB progression as well as its possible pathogenic mechanisms. Public microarray datasets were applied to unearth the abnormally expressed lncRNAs in NB. RT-qPCR analysis was used to measure the expression of LINC00839, miR-454-3p, and neuronal differentiation 1 (NEUROD1) mRNA. The protein level was determined by a western blot assay. CCK-8, plate clone formation, EdU, wound-healing scratch, and transwell assays were employed to evaluate cell proliferation, migration, and invasion. Xenografts were developed in nude mice to determine the effects of LINC00839 on NB tumor growth. Dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments were performed to identify the interaction between miR-454-3p and LINC00839 or NEUROD1. According to GSE datasets (GSE16237 and GSE16476), LINC00839 was found as a potential driver of NB progression. LINC00839 expression was higher in NB tumor tissues and cells. Also, LINC00839 expression was positively correlated with MYCN amplification, advanced INSS stages, and worse prognosis. Silencing of LINC00839 suppressed cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, LINC00839 could act as a sponge of miR-454-3p to facilitate the expression of its target NEUROD1. Moreover, miR-454-3p was demonstrated to exert an anti-cancer activity in NB. More importantly, the tumor-suppressive properties mediated by LINC00839 knockdown were significantly counteracted by the inhibition of miR-454-3p or overexpression of NEUROD1. Our study demonstrates that LINC00839 exerts an oncogenic role in NB through sponging miR-454-3p to up-regulate NEUROD1 expression, deepening our comprehension of lncRNA involved in NB and providing access to the possibility of LINC00839 as a therapeutic target for NB.

     

LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma

Long non-coding RNAs (lncRNAs) are a novel class of regulators in multiple cancer biological processes. However, the functions of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) remain largely unknown. In this study, we identified PWAR6 as a frequently down-regulated lncRNA in PDAC samples as well as a panel of pancreatic cancer cell lines. Down-regulated PWAR6 was associated with multiple clinical outcomes, including advanced tumour stage, distant metastasis, and overall survival of PDAC patients. In our cell-based assays, ectopic expression of PWAR6 dramatically repressed PDAC cells proliferation, invasion and migration, accelerated apoptosis, and induced cell cycle arrest at G0/G1 phase. In contrast, depletion of PWAR6 mediated by siRNA exhibited opposite effects on PDAC cell behaviours. In vivo study further validated the anti-tumour role of PWAR6 in PDAC. By taking advantage of available online sources, we also identified YAP1 as a potential PWAR6 target gene. Negative correlation between YAP1 and PWAR6 expressions were observed in both online database and our PDAC samples. Notably, rescue experiments further indicated that YAP1 is an important downstream effector involved in PWAR6-mediated functions. Mechanistically, PWAR6 could bind to methyltransferase EZH2, a core component of Polycomb Repressive Complex 2 (PRC2) in regulating gene expression, and scaffold EZH2 to the promoter region of YAP1, resulting in epigenetic repression of YAP1. In conclusion, our data manifest the vital roles of PWAR6 in PDAC tumorigenesis and underscore the potential of PWAR6 as a promising target for PDAC diagnosis and therapy.     

Red cell genotyping and the future of pretransfusion testing

The ABO blood group, based on molecular biological detection technology, has the advantages of simple operation, high sensitivity, and standardized result interpretation, and is not affected by sample immunological characteristics. However, clinically, performance verification, clinical application scope, quality management, abnormal result processing, and other issues associated with the ABO blood group molecular detection technology are relatively complex, and there is a lack of unified norms and standards. Therefore, from the perspective of the whole process of ABO molecular biology detection, this study aims to provide standardized opinions on important links affecting the detection results, common problems encountered in the detection process, and the assessment and treatment of abnormal results. Finally, a Chinese expert consensus on molecular biological technology based on genotyping and sequencing detection was put forward, which standardizes the detection process, improves the accuracy of results, and promotes the development of technology and broader clinical application.     

长江口外海域微型和小型底栖生物群落结构和时空变化

利用DAPI荧光计数法和Ludox-QPS方法并结合环境因子的分析,研究了2010年和2011年夏季(7月)及秋季(11月)长江口至济州岛沿线的CJ断面沉积物中的微型及小型底栖生物的组成、丰度和生物量及分布特点。结果表明,表层5 cm沉积物中,细菌(108个/cm3)、蓝细菌(106个/cm3)、自养(106个/cm3)和异养微型鞭毛虫(105—106个/cm3)的丰度较其他类群高3个数量级以上;细菌(19—24μg C/cm3)、自养(13—31μg C/cm3)和异养(5—44μg C/cm3)微型鞭毛虫的生物量明显较蓝细菌(1—2μg C/cm3)、小型底栖生物(0.9—1μg C/cm3)、纤毛虫(0.04—0.2μg C/cm3)、异养小鞭毛虫(0.02—0.08μg C/cm3)及硅藻(0.001—0.008μg C/cm3)高。除细菌和蓝细菌外,2011年微型底栖生物和小型底栖生物各类群的丰度和生物量大多高于2010年,且大多为秋季高于夏季。发现的166种纤毛虫中,肉食性纤毛虫所占现存量的比例最大,菌食性次之,藻食性和杂食性最小;肉食性纤毛虫在秋季所占比例高于夏季,而菌食性则相反。统计分析表明,微型底栖生物丰度和生物量年度间具有极显著差异,基于物种-丰度的纤毛虫群落结构年度间呈极显著差异,而小型底栖生物则未见显著差异。微型和小型底栖生物各类群夏季和秋季的分布受多因子而非单一环境因子的影响,长江冲淡水对该断面的微型和小型底栖生物的影响不显著。对微型底栖生物各类群及水体中相应类群的比较分析表明,该海域的微型底栖生物具有明显的数量和功能重要性。     

miRNA-223 Suppresses Mouse Gallstone Formation by Targeting Key Transporters in Hepatobiliary Cholesterol Secretion Pathway

miRNA-223 has been previously reported to play an essential role in hepatic cholesterol homeostasis. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3'' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.