Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

白细胞介素12对小鼠肝癌基因治疗的实验研究

目的：研究白细胞介素１２对小鼠肝癌细胞基因治疗效果。方法：利用脑心肌炎病毒（ＥＭＣＶ）及脊髓灰质炎（Ｐｏ－ｌｉｏ）病毒内核糖体进入位点（ＩＲＥＳ），连接ｍＩＬ－１２ Ｐ４０及ｐ３５ ｃＤＮＡｓ和筛选基因新霉术磷酸转移酶（ＮｅｏＲ），克隆至逆转录病毒载体ｐＧＣＥＮ中，使三个基因同时受逆转录病毒载体５’端ＬＴＲ启动子控制，转录至同一ｍＲＮＡ转录本上，从而构建成多顺反子逆转录病毒载体，即ｐＧＣＥＮ／ｍＩＬ－１２。在ＬｉｐｏｆｅｃｔＡＭＩＮＥ介导下将ｐＧＣＥＮ／ｍＩＬ－１２转染包装细胞ＰＡ３１７，Ｇ４１８筛选，直至出现阳性克隆（命名为：Ｍ４５／ｍＩＬ－１２），挑取抗性克隆，扩大培养，收集上清，用小鼠成纤维细胞ＮＩＨ３Ｔ３测定病毒滴度。然后用重组逆转录病毒感染小鼠肝癌细胞ＭＭ４５Ｔ．Ｌｉ，Ｇ４１８筛选，直至出现阳性克隆，扩大培养，对阳性克隆进行鉴定。将６０Ｃｏ照射（６０ Ｇｙ）Ｍ４５／ｍＩＬ－１２细胞对荷瘤小鼠进行瘤内接种，每周一次，连续治疗三次，观察其治疗效果。结果：病毒上清中重组逆转录病毒滴度为５×１０~５ＣＦＵ／ｍｌ。 ＰＣＲ及ＲＴ－ＰＣＲ证明外源基因已整合至小鼠肝癌细胞基因组中，以及外源基因在ｍＲＮＡ水平上的表达。

人白细胞介素12的cDNA克隆及双顺反子腺病毒载体的构建

目的 :克隆中国人IL 12的 p40和p35亚基cDNA ,构建含hIL 12双顺反子的腺病毒载体。 方法 :采用RT PCR从上海地区人骨髓有核细胞中克隆IL 12的双亚基cDNA ,并插入质粒 pCI中进行全基因序列分析。利用质粒 p△ElsplA和IRES序列构建同时表达p35和 p40亚基的腺病毒载体。酶切鉴定构建物中基因插入的正确性。结果 :上海地区人骨髓有核细胞中IL 12的 p40和p35cDNA基因同已报道的北京地区人DC细胞中IL 12的 p40和国外报道的NKSF ,CLMF的 p40和p35序列各有异同 ,构建的IL 12双亚基同时表达的腺病毒载体酶切鉴定正确。 结论 :人IL 12的 p40和 p35基因可能存在多态性 ,同时表达IL 12双亚基的腺病毒载体构建成功 ,对开展人IL 12基因治疗肿瘤具有重要意义。

人膜补体调节蛋白MCP、CD59在稳定转染的细胞中的共表达及功能研究

采取内核糖体进入位点（IRES）策略构建含人的膜补体调节蛋白基因MCP和CD59的cDNA的双顺反子真核表达载体pcDNA3-MCPIRESCD59，以磷酸钙沉淀法转染NIH3T3细胞，用G418筛选阳性克隆，并研究MCP和CD59双基因在稳定细胞系中的共表达及保护功能。PCR实验结果显示双基因稳定整合在异源细胞NIH3T3的染色体上，RT-PER及Western印迹实验分别从RNA水平和蛋白质水平证实了人补体调节蛋白分子MCP和CD59在细胞系中皆获得同步表达。检测连续传代30次的NIH3T3 pcDNA3-MCPIRESCD59，结果表明人MCP和CD59基因仍稳定整合在细胞基因组中，并未随着传代而丢失，为稳定的转双基因细胞系。补体依赖的细胞毒反应表明，peDNA3-MCPIRESCD59转染细胞由于MCP和CD59的共表达获得了高于MCP或CD59单一表达时所提供的保护功效，能更好地抑制人补体依赖的细胞毒作用的发生，保护宿主细胞免受人补体的攻击。以上结果表明，所构建的双基因重组表达载体实现了不同人补体调节蛋白基因高效转移和高水平共表达，在克服超急性排斥反应的基因治疗中有潜在的应用价值。

RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform

We have previously reported that the human ACAT1 gene produces a chimeric mRNA through the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACAT1 and a novel enzymatically active 56-kDa isoform, respectively, with the latter being authentically present in human cells, including human monocyte- derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops （nt 1255-1268, 1286-1342 and 1355-1384） were tested individually by transfecting expression plasmids into cells that contained the wild-type, deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame （ORF） or to the ORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop135s-1384 from chromosome 1 was dispensable. The results of experi- ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site （IRES）. Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACAT1 transcript.

Emerging role of microRNAs in liver diseases

MicroRNAs are a class of small non-coding RNAs that are found in plants, animals, and some viruses. They modulate the gene function at the post-transcriptional level and act as a fine tuner of various processes, such as development, proliferation, cell signaling, and apopto-sis. They are associated with different types and stages of cancer. Recent studies have shown the involvement of microRNAs in liver diseases caused by various factors, such as Hepatitis C, Hepatitis B, metabolic disorders, and by drug abuse. This review highlights the role of microRNAs in liver diseases and their potential use as therapeutic molecules.

Molecular Characterization of Indian Species of Steinernema （Nematoda： Steinernematidae） Based on Restriction Fragment Length Polymorphism Profile of Internal Transcribed Spacer Region of Ribosomal DNA

Restriction fragment length polymorphism （RFLP） profiles of the amplified products of Internal Transcribed Spacer （ITS） region of rDNA using four restriction enzymes （Alul, Rsal, HinfI and HhaI） revealed distinctness of six Indian isolates of Steinernema one each from Maharashtra （IARI-EPN-mh）, Himachal Pradesh （IARI-EPN-hp）, Dehradun （IARI-EPN-dhdl）, Jharkhand （IARI-EPN-jhl） and two from Madhya Pradesh （IARI-EPN-bpll ＆ IARI-EPN-gwll）, when compared with the only native species Steinernema thermophilum. One of the restriction enzyme, Rsal could differentiate all the six species/strains from one another. The three restriction enzymes yielded patterns which were of diagnostic value but Rsal appeared to be the best diagnostic marker for differentiating these isolates. A tree constructed based upon the band sharing amongst the isolates, produced trichotomy which placed strains from Madhya Pradesh and Jharkhand in one group showing 94% homology, one strain from Bhopal （M.P） formed separate clade along with S. thermophilum with 72% similarity. These isolates, from Maharashtra, Himachal Pradesh and Dehradun, showed only 51% similarity with the S. thermophilum by forming separate clade.

Molecular Characterization of Pythium Spp. Isolated from Tomato Seedlings in the Syrian Coast

Tomato seedlings damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2012. Samples were collected from infected seedlings growing in greenhouses in the Syrian coastal region. Isolation of fungi was done in the laboratories of the Agronomical Reaserch Center, in Lattakia and the molecular analyses were done in the Biotechnology Center at Tishreen University, Lattakia, Syria, during the years 2012, 2013. Eight isolates ofPythium sp. obtained were purified using hyphal tip method （named P1, P2, P3, P4, P5, P6, P7 and P8）. Isolates were morphologically identified by optical microscope, then molecularly Characterized using genus specific ITS primers. The results of morphological characterization of pathogenic species suggested the detection of Pythium aphanidermatum, P. ultimum. The analysis of DNAs from the different isolates with ITS primers, recognizing the inter transcript spacer of nuclear ribosomal DNA proved that the eight, isolates were belonging to the species P. ultimum. The complete sequences of ribosomal DNA internal transcribed spacers regions of selected isolates were determined and submitted to GenBank. The GenBank-BLAST homology search revealed P. ultimum as the most similar sequence （〉 96% identity） with GenBank entry AB355596.

含mIL-12基因多顺反子逆转录病毒载体构建及其在肝癌细胞中的表达

目的 构建含小鼠白细胞介素 12双亚基及新霉素磷酸转移酶基因多顺反子逆转录病毒载体 ,并观察其在小鼠肝癌细胞中的表达。方法 利用脑心肌炎病毒 (EMCV)及脊髓灰质炎 (Polio)病毒内核糖体进入位点 (IRES) ,连接mIL 12p40及p35cDNAs和筛选基因新霉素磷酸转移酶 (NeoR) ,克隆至逆转录病毒载体pGCEN中 ,使三个基因同时受逆转录病毒载体 5′端LTR启动子控制 ,转录至同一mRNA转录本上 ,通过不同机制翻译成蛋白质 ,从而构建成多顺反子逆转录病毒载体 ,即pGCEN/mIL 12。在LipofectAMINE介导下将pGCEN/mIL 12转染包装细胞PA317,G418筛选 ,直至出现阳性克隆 ,挑取抗性克隆 ,扩大培养 ,收集上清 ,用小鼠成纤维细胞NIH3T3测定病毒滴度。然后用重组转录病毒感染小鼠肝癌细胞MM45T .Li,G418筛选 ,直至出现抗性克隆 ,扩大培养 ,对阳性克隆进行鉴定。结果 由PA317包装细胞产生的重组逆转录病毒的滴度为 5× 10 5CFU/ml,将其感染小鼠肝癌细胞MM45T .Li,后经PCR及Southernblot证明 ,外源基因已整合至小鼠肝癌细胞基因组中 ,RT PCR及Northernblot分析外源基因在mRNA水平上的表达 ,并证实mIL 12p40及p35cDNA和NeoR基因转录在同一mRNA上。ELISA显示mIL 12的表达量 48h为 10ng/ 10 6细胞。并且M45 /mIL

含I-A^dα和β链cDNA的三顺反子逆转录病毒载体构建及真核表达

为了协同表达MHCII类分子的两个链α和 β ,利用脑心肌炎病毒和脊髓灰质炎病毒的内核糖体进入位点 ,构建了含MHCII类分子α和 β链及选择标记新霉素磷酸转移酶基因的三顺反子逆转录病毒载体。经包装细胞包装成重组逆转录病毒后 ,感染NIH3T3、MM45T Li、COS7细胞 ,经PCR、RT PCR、Southern印迹、Northern印迹及流式细胞术在多水平上证实了α链和 β链的基因表达和MHCII类分子的正确组装。

Four new species of the genus Hymenoscyphus (fungi) based on morphology and molecular data

Four new species of Hymenoscyphus (H. brevicellulus, H. hyaloexcipulus, H. microcaudatus, and H. subsymmetricus) and a new Chinese record (H. subpallescens) are described. These five species share common characteristics: small apothecia (<3mm in diameter); hymenium whitish, pale yellow, to yellow in color; ectal excipulum of textura prismatica; asci arising from simple septa; ascospores scutuloid and guttulate; saprophytic nutrition; and leaf habitats, except for H. subsymmetricus, which grows on herbaceous stems. Phylogenetic analyses of internal transcribed spacer nuclear ribosomal DNA sequences, the universal DNA barcode for fungi, for 16 species in the genus indicated that these taxa were closely related to H. microserotinus, in accordance with their morphological features, but represented independent species. The distinguishing features of each new species from its relatives are discussed, and their phylogenetic relationships explored.