Abstract:Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently,triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their af?nity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modi?ed oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modi?ed oligonucleotides passed ef?ciently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more ef?ciently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modi?ed oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-mod-i?ed oligonucleotides has strong potential as a new strategy for HBV inhibition.

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	1. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription
	Sun Zhen,Chen Hongyan,Lu Daru
	Biology 04 January 2013
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	Abstract:Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently,triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their af?nity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modi?ed oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modi?ed oligonucleotides passed ef?ciently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more ef?ciently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modi?ed oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-mod-i?ed oligonucleotides has strong potential as a new strategy for HBV inhibition.
	TO cite this article:Sun Zhen,Chen Hongyan,Lu Daru. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription[OL].[ 4 January 2013] http://en.paper.edu.cn/en_releasepaper/content/4511934


	2. Cloning and Functional characterization of three Superoxide Dismutases genes from halophyte Salicornia europaea and Thellungiella halophila
	Wu Fan ,Mei Yu,Lu Maolong,Juan Li,Wang Rongfu,Wang Xianlei,Sun Qizhen,Jie Zang,Kai Xu,Hu Ji
	Biology 17 April 2012
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	Abstract:In order to study the halophyte salt-tolerance mechanism, we cloned the manganese (Mn) and Cu/Zn superoxide dismutase (SOD) full-length cDNAs from Salicornia europaea by RACE method for the first time, sequence analysis indicated that the MnSOD gene (GenBank accession number: JQ061158) comprises an open reading frame of 699 bp, encoding 233-amino acid polypeptide with a predicted molecular mass of 25.7 kDa. Correspondingly, the Cu/ZnSOD gene (GenBank accession number: JQ061160) consists of an open reading frame of 684 bp which encodes a protein of 228 amino acids with a predicted molecular mass of 23.3 kDa. The prokaryotic expression vectors pET30-SeMSD, pET30-SeCSD and pET30-ThMSD were constructed, and the target proteins were expressed successfully in BL21 Escherichia coli. Through optimization of the inducing concentration of Isopropyl β-D-Thiogalactopyranoside (IPTG), we tested the salt tolerance of these three superoxide dismutases under 6.5% and 7.5% NaCl, and the results demonstrate that the recombinants BL21 (pET30-SeMSD) and BL21 (pET30-ThMSD) show better tolerance to salinity stress in comparison with the control stain BL21 (pET30), but the recombinant BL21 (pET30-SeCSD) has not displayed increased salt tolerance.
	TO cite this article:Wu Fan ,Mei Yu,Lu Maolong, et al. Cloning and Functional characterization of three Superoxide Dismutases genes from halophyte Salicornia europaea and Thellungiella halophila[OL].[17 April 2012] http://en.paper.edu.cn/en_releasepaper/content/4474413

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	3. The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum
	Liu Shuyang ,Guoxiong Peng,Yuxian Xia
	Biology 22 March 2012
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	Abstract:Background: The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was cloned from the locust-specific entomopathogenic fungus Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse factors from environment and host insect. Results: Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium), Czapek-dox and PDA plates, respectively, the cAMP levels was also reduced in PD liquid culture. Knockdown of MaAC by RNAi led to a great reduction in fungal growth in the hemolymph of locusts after injection and topical inoculation, thus demonstrating that MaAC encodes an adenylate cyclase and is required for virulence of M. acridum. Virulence assay indicated that the effect of MaAC on the virulence was mainly inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. Conclusion: MaAC affects virulence, primarily by fungal growth inside the insect, and is required for tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects the fungal virulence via vegetative growth and tolerance against oxidative stress, osmotic stress and locust fever.
	TO cite this article:Liu Shuyang ,Guoxiong Peng,Yuxian Xia. The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum[OL].[22 March 2012] http://en.paper.edu.cn/en_releasepaper/content/4471763


	4. The binding and unwinding properties of the Bloom helicase catalytic core to the G4DNA structure
	LUO Heng,XU Houqiang,CAI Mingjuan,CHEN Xiang,DING Mei,LI Kun,MENG Huihui
	Biology 13 March 2012
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	Abstract:G4DNA, which widely exists in the structure of the telomeres in normal cells, plays a pivotal part in the process of prolonging the telomere DNA by catalyzing the enzyme telomerase. Bloom (BLM) helicase, an important member of the RecQ DNA helicase family, plays an important role in DNA metabolism, including DNA replication, repair, transcription, and recombination. The unwinding of G4DNA requires DNA helicase participation, which is crucial for maintaining chromosomal stability. This study was conducted to determine the DNA-binding and unwinding properties of the BLM helicase catalytic core to G4DNA using fluorescence polarization and the electrophoretic mobility shift assay (EMSA). The results revealed that the BLM helicase catalytic core could bind and unwind G4DNA. The molecular affinity of G4DNA binding by the helicase was dependent on the single-stranded DNA (ssDNA) terminals in the G4DNA; the helicase binds to the G4DNA where one helicase monomer covers approximately 10 nucleotides at the 3' or 5' ssDNA tail. The unwinding of G4DNA was dependent on the presence of a 3' ssDNA tail and ATP; the G4DNA with only a 3' ssDNA tail was the most favorable substrate to be unwound by the BLM helicase catalytic core, and required 3' ssDNA tails of at least 10 nt in length for efficient unwinding. The blunt-ended G4DNA was loosely bound and partly unwound by the helicase catalytic core. The experimental results presented are beneficial to further the understanding the functionality of BLM helicase in cells.
	TO cite this article:LUO Heng,XU Houqiang,CAI Mingjuan, et al. The binding and unwinding properties of the Bloom helicase catalytic core to the G4DNA structure[OL].[13 March 2012] http://en.paper.edu.cn/en_releasepaper/content/4471423


	5. Comparative transcriptome analysis between insect-resistant cotton and non-transgenic cotton (Gossypium hirsutum L.) in response to salt stress
	Fangjiao Xu,Mingliang Jia,Junli Feng,Ying Liang,Yanjun Liang,Jishuang Chen
	Biology 12 March 2012
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	Abstract:Insect-resistant cotton is one of the main transgenic crops in the world. High salinity immensely limits insect-resistant cotton growth and productivity. To determined genes in response to salt stress and clarify reasons of transcription level changes caused by insect-resistant factor, gene expression microarray was used to monitor the differentially expressed genes in insect-resistant cotton CCRI 41 and non-transgenic cotton CCRI 23. Microarray analysis showed that 2341 genes were up-regulated and 3073genes were repressed in CCRI 41 exposed for 24 h to 200 mM NaCl. A total of 7674 salt-resistant genes of CCRI 23 were identified, the expression levels of 2341 genes were enhanced, and the transcript levels of 4718 genes were decreased by 200 mM NaCl. Gene ontology of annotated genes revealed that some of the salt responsive genes involved to important processes as the response to osmotic stress, abscisic acid stimulus and reactive oxygen species were induced by the NaCl treatment. Comparative transcriptome analysis between CCRI 41and CCRI 23 in response to salt stress exhibited that toxin catabolic process and regulation strategy of DNA level played important role in response to salt stress. Quantitative RT-PCR was used to validate 4 selected salt responsive genes. This work represents the first study in disclosing the possible molecular evidence that insect-resistant cotton have weak tolerance against NaCl stress. Comparative transcriptome analysis will assist in laying a foundation for cultivating of insect-resistant and salt-resistant cotton.
	TO cite this article:Fangjiao Xu,Mingliang Jia,Junli Feng, et al. Comparative transcriptome analysis between insect-resistant cotton and non-transgenic cotton (Gossypium hirsutum L.) in response to salt stress[OL].[12 March 2012] http://en.paper.edu.cn/en_releasepaper/content/4471217


	6. Bioinformatics and expression analysis of the amino acid transporter gene family (OsAAT) in rice
	CAI Hongmei
	Biology 09 March 2012
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	Abstract:Nitrogen (N) is one of the most important limiting factors for plant growth and development. Amino acids are the major source of organic N, which is converted from inorganic N absorbed by plant roots from the soil. Amino acid transporters are the principal mediators of organic N distribution and important regulators of resource allocation in plants. Although the complete genomic sequence of rice has already been released, there is still little known about amino acid transporter genes in rice. In this study, 79 OsAAT genes were identified by a database search of the rice genome based upon HMM profiles. A bioinformatics analysis of the complete set of OsAAT genes is presented, including chromosomal location, phylogenetic analysis, gene structure, protein analysis, conserved motifs, protein structures and cis-element analysis of the promoters. In addition, the comprehensive expression profile of OsAAT genes in rice tissues/organs under N starvation conditions was investigated by real-time PCR analysis. Diverse expression patterns of OsAAT genes indicated diverse biological functions of the amino acid transporters and the important roles of OsAAT genes in N uptake, metabolism and distribution during N starvation.
	TO cite this article:CAI Hongmei. Bioinformatics and expression analysis of the amino acid transporter gene family (OsAAT) in rice[OL].[ 9 March 2012] http://en.paper.edu.cn/en_releasepaper/content/4470810


	7. Transcription analysis of rice under nitrogen starvation
	CAI Hongmei,LU Yongen,LIAN Xingming
	Biology 08 March 2012
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	Abstract:Nitrogen is an essential mineral nutrient required for plant growth and development. Insufficient nitrogen (N) supply triggers extensive physiological and biochemical changes in plants. In this study, we used Affymetrix GeneChip rice genome arrays to analyze the dynamics of rice transcriptome under N starvation. N starvation induced or suppressed transcription of 3,518 genes, representing 10.88% of the genome. These changes, mostly transient, affected various cellular metabolic pathways, including stress response, primary and secondary metabolism, molecular transport, regulatory process and organismal development. 462 or 13.1% transcripts for N starvation expressed similarly in root and shoot. Comparative analysis between rice and Arabidopsis identified 73 orthologous groups that responded to N starvation, demonstrated the existence of conserved N stress coupling mechanism among plants.
	TO cite this article:CAI Hongmei,LU Yongen,LIAN Xingming. Transcription analysis of rice under nitrogen starvation[OL].[ 8 March 2012] http://en.paper.edu.cn/en_releasepaper/content/4470665


	8. Two novel antimicrobial peptides from skin secretions of the frog, Rana nigrovittata
	Liu Xiuhong,Liu Rui,Wei Lin,Yang Hailong,Zhang Keyun,Liu Jingze,Lai Ren
	Biology 21 February 2012
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	Abstract:Two novel antimicrobial peptides with similarity to brevinin-2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin-2-RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin-2-RN2), respectively, by Edman degradation. Different from brevinin-2,which is composed of 33 amino acid residues (aa), both brevinin-2-RN1 and -RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465-9) encoding precursors of brevinin-2-RN1 and -RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin-2-RN. Both brevinin-2-RN1 and -RN2 showed strong antimicrobial activities against gram-positive and gram-negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure.
	TO cite this article:Liu Xiuhong,Liu Rui,Wei Lin, et al. Two novel antimicrobial peptides from skin secretions of the frog, Rana nigrovittata[OL].[21 February 2012] http://en.paper.edu.cn/en_releasepaper/content/4465356


	9. A tripartite dsRNA virus infecting Raphanus sativas which represents a new taxon of Chrysoviridae
	LIU Jianning,LI Liqiang,XU Aixia,WANG Ting,ZHU Xiwu,CHEN Jishuang
	Biology 16 February 2012
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	Abstract:The family Chrysoviridae consists of a single genus chrysovirus infecting fungi. Its genome comprises four monocistronic dsRNAs with the largest dsRNA encoding RNA-dependent RNA polymerase, and the other three encoding coat protein, putative protease and a protein with unknown function respectively. In recent years, more and more genome segments-various tentative chrysoviruses have been reported in fungi. In this study, we report a plant tripartite chrysovirus infecting Raphanus sativus. Its entire genome was determined used the modified single primer amplification technique. Sequence analysis revealed that the virus genome is comprised of three dsRNA segments with the sizes of 3638, 3517 and 3299 bp respectively. Each dsRNA segments has a unique sequence and contains a single major open reading frame. In silico analysis revealed that the largest dsRNA encodes RNA-dependent RNA polymerase, and the other two encode coat protein and putative protease, respectively. The phylogenetic analysis of all available chrysoviruses revealed that RasCV1 together with AmaCV formed a new taxon infecting plants, which is closely related to representative chrysoviruses infecting fungi. In addition, the variety of the chrysovirus genome organization is discussed.
	TO cite this article:LIU Jianning,LI Liqiang,XU Aixia, et al. A tripartite dsRNA virus infecting Raphanus sativas which represents a new taxon of Chrysoviridae[OL].[16 February 2012] http://en.paper.edu.cn/en_releasepaper/content/4466779


	10. A Signal Peptide Library for Bacillus subtilis and its Application for Secretion of a Thermostable β-Galactosidase
	GONG Zifeng,XIA Yu
	Biology 05 December 2011
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	Abstract:β-Galactosidase catalyzes the hydrolysis of lactose and is widely used in dairy processing. The thermostable β-galactosidase obtained from Geobacillus stearothermophilus has several advantages and it has potential applications in the dairy industry. In this work, this enzyme was secreted by Bacillus subtilis using the general secretion (Sec) pathway. A signal peptide library was constructed with 20 selected signal peptide coding sequences, and it was used for probing the secretory capacity of the thermostable β-galactosidase in Bacillus subtilis 168. In the 20 recombinants constructed, expression of the target enzyme was observed in 4 plasmids carrying the signal peptide coding sequences from Bacillus subtilis AmyX, NprE, OppA and YweA, respectively. The target enzyme fused with N-terminal signal peptide from NprE achieved extracellular secretion at a level of 64.0 IU/mL, which accounts for 29.6% of the total target enyzme synthesized. This is the first report of the thermostable β-galactosidase from Geobacillus stearothermophilus by a Sec signal peptide in Bacillus subtilis. The results indicated the applicability of the signal peptide library constructed. Yet the low secretion capacity of the target enzyme also suggested that, in dealing with such kind of substrate, the Sec pathway might have no advantage over the twin-arginine translocation pathway.
	TO cite this article:GONG Zifeng,XIA Yu. A Signal Peptide Library for Bacillus subtilis and its Application for Secretion of a Thermostable β-Galactosidase[OL].[ 5 December 2011] http://en.paper.edu.cn/en_releasepaper/content/4453161

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