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Sample records for lentiviral short hairpin

  1. Lentiviral delivery of short hairpin RNAs

    Science.gov (United States)

    Manjunath, N; Haoquan, Wu; Sandesh, Subramanya; Premlata, Shankar

    2009-01-01

    In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a number of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose. However, induction of long-term shRNA expression can also cause toxicities by inducing off target effects and interference with the endogenous micro RNA (miRNA) pathway that regulates cellular gene expression. Recently, several advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities. In this review we describe some of these advances, focusing on the progress made in the development of lentiviral shRNA delivery strategies to combat viral infections. PMID:19341774

  2. SHARP-2 gene silencing by lentiviral-based short hairpin RNA interference prolonged rat kidney transplant recipients' survival time.

    Science.gov (United States)

    Shou, Z; Xiao, H; Xu, Y; Wang, Y; Yang, Y; Jiang, H; Chen, J; Yamada, K; Miyamoto, K

    2009-01-01

    Split- and hairy-related protein-2 (SHARP-2) controls the expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), which both play a key role in transplant rejection. This study was designed to investigate whether SHARP-2 short hairpin RNA interference (shRNAi) could prolong the survival of rat kidney transplant recipients. A lentiviral-based shRNAi construct, LV-SHARP-2iC, showed a SHARP-2 gene silencing efficiency of 84% in normal rat kidney cells. In activated T-cells, SHARP-2 gene silencing with the LV-SHARP-2iC construct resulted in 61% and 69% down-regulation of IL-2 and IFN-gamma, respectively, compared with a scramble control construct. When donor kidney was perfused with 5 x 10(7) transforming units of the LV-SHARP-2iC construct, the median survival time of the transplant recipients was prolonged by 4 - 5 days compared with control groups. In conclusion, recombinant lentiviral LV-SHARP-2iC construct effectively silenced SHARP-2 gene expression, which reduced IL-2 and IFN-gamma mRNA expression and prolonged rat kidney transplant recipients' survival.

  3. An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.

    Science.gov (United States)

    Adams, Felix F; Heckl, Dirk; Hoffmann, Thomas; Talbot, Steven R; Kloos, Arnold; Thol, Felicitas; Heuser, Michael; Zuber, Johannes; Schambach, Axel; Schwarzer, Adrian

    2017-09-01

    RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

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    Orit Wolstein

    2014-01-01

    Full Text Available Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC. CCR5-targeted shRNA (sh5 and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

  5. Short hairpin RNA expression for enhancing the resistance of ...

    African Journals Online (AJOL)

    Short hairpin RNA expression for enhancing the resistance of Bombyx mori (Bm) to nucleopolyhedrovirus in vitro and in vivo. Roy Bhaskar, Fang Zhou, Shuang Liang, Wan-Fu Yue, Yan-shan Niu, Yun- gen Miao ...

  6. Design of extended short hairpin RNAs for HIV-1 inhibition

    NARCIS (Netherlands)

    Liu, Ying Poi; Haasnoot, Joost; Berkhout, Ben

    2007-01-01

    RNA interference (RNAi) targeted towards viral mRNAs is widely used to block virus replication in mammalian cells. The specific antiviral RNAi response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular expression of short hairpin RNAs (shRNAs). For

  7. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  8. Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication

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    Heinrich Jochen

    2009-04-01

    Full Text Available Abstract The Herpes simplex virus (HSV is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as and small interfering ribonucleic acids (siRNAs for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs, was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.

  9. Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors

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    Kruithof Egbert KO

    2004-08-01

    Full Text Available Abstract Background RNA interference (RNAi can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs, used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2 mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death. Results As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers or more increased expression of the oligoadenylate synthase-1 (OAS1 interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing. Conclusions Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained

  10. Is TNF-a-targeted short hairpin RNA (shRNA) a novel potential therapeutic tool in psoriasis treatment?

    DEFF Research Database (Denmark)

    Stenderup, Karin; Jakobsen, Maria; Rosada, Cecilia

    2008-01-01

      TNF-α is a well known target in psoriasis treatment and biological treatments targeting TNF-a are already clinically used against psoriasis and psoriasis arthritis. Attention is however given to a novel therapeutic tool: RNA interference that controls gene silencing. This study investigates...... the efficiency of targeting TNF-a with specific short hairpin RNA (shRNA) and explores its potential in treating psoriasis. ShRNAs targeting human TNF-α mRNA were generated. Their efficiency in down-regulating TNF-a protein expression was evaluated using a Renilla luciferase screening-assay and a transient co...... TNF-a shRNA was used to transduce HEK293 cells and verify vector-derived TNF-a knockdown in vitro. In vivo, psoriasis skin was exposed to lentiviral TNF-a shRNAs by a single intra-dermal injection. Psoriasis skin for the in vivo study was obtained from psoriatic plaque skin biopsies that were...

  11. Short hairpin-looped oligodeoxynucleotides reduce hepatitis C virus replication

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    Broecker Felix

    2012-07-01

    Full Text Available Abstract Background Persistent infection with hepatitis C virus (HCV is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Standard therapy consists of a combination of interferon-alpha and ribavirin, but many patients respond poorly, especially those infected with HCV genotypes 1 and 4. Furthermore, standard therapy is associated with severe side-effects. Thus, alternative therapeutic approaches against HCV are needed. Findings Here, we studied the effect of a new class of antiviral agents against HCV, short, partially double-stranded oligodeoxynucleotides (ODNs, on viral replication. We targeted the 5’ nontranslated region (5’ NTR of the HCV genome that has previously been shown as effective target for small interfering RNAs (siRNAs in vitro. One of the investigated ODNs, ODN 320, significantly and efficiently reduced replication of HCV replicons in a sequence-, time- and dose-dependent manner. ODN 320 targets a genomic region highly conserved among different HCV genotypes and might thus be able to inhibit a broad range of genotypes and subtypes. Conclusions ODNs provide an additional approach for inhibition of HCV, might be superior to siRNAs in terms of stability and cellular delivery, and suitable against HCV resistant to standard therapy. This study underlines the potential of partially double-stranded ODNs as antiviral agents.

  12. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  13. A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

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    Kivork Christine

    2007-10-01

    Full Text Available Abstract Background Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.

  14. Blocking the Wnt/β-Catenin Pathway by Lentivirus-Mediated Short Hairpin RNA Targeting β-Catenin Gene Suppresses Silica-Induced Lung Fibrosis in Mice

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    Wang, Xin; Dai, Wujing; Wang, Yanrang; Gu, Qing; Yang, Deyi; Zhang, Ming

    2015-01-01

    Silicosis is a form of occupational lung disease caused by inhalation of crystalline silica dust. While the pathogenesis of silicosis is not clearly understood, the Wnt/β-catenin signaling pathway is thought to play a major role in lung fibrosis. To explore the role of Wnt/β-catenin pathway in silicosis, we blocked Wnt/β-catenin pathway both in silica-treated MLE-12 cells (a mouse pulmonary epithelial cell line) and in a mouse silicosis model by using a lentiviral vector expressing a short hairpin RNA silencing β-catenin (Lv-shβ-catenin). In vitro, Lv-shβ-catenin significantly decreased the expression of β-catenin, MMP2 and MMP9, and secretion of TGF-β1. In vivo, intratracheal treatment with Lv-shβ-catenin significantly reduced expression of β-catenin in the lung and levels of TGF-β1 in bronchoalveolar lavage fluid, and notably attenuated pulmonary fibrosis as evidenced by hydroxyproline content and collagen I\\III synthesis in silica-administered mice. These results indicate that blockade of the Wnt/β-catenin pathway can prevent the development of silica-induced lung fibrosis. Thus Wnt/β-catenin pathway may be a target in prevention and treatment of silicosis. PMID:26340635

  15. Novel guanidinylated bioresponsive poly(amidoamines designed for short hairpin RNA delivery

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    Yu J

    2016-12-01

    Full Text Available Jiankun Yu,1 Jinmin Zhang,1 Haonan Xing,1 Yanping Sun,1 Zhen Yang,1 Tianzhi Yang,2 Cuifang Cai,1 Xiaoyun Zhao,3 Li Yang,1 Pingtian Ding1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China; 2Department of Basic Pharmaceutical Sciences, School of Pharmacy, Husson University, Bangor, ME, USA; 3Department of Microbiology and Cell Biology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China Abstract: Two different disulfide (SS-containing poly(amidoamine (PAA polymers were constructed using guanidino (Gua-containing monomers (ie, arginine [Arg] and agmatine [Agm] and N,N'-cystamine bisacrylamide (CBA by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the

  16. Construction of equalized short hairpin RNA library from human brain cDNA.

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    Xu, Lei; Li, Jingqi; Liu, Li; Lu, Lixia; Gao, Jingxia; Li, Xueli

    2007-02-20

    Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.

  17. Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance.

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    Chou, Shuli; Shao, Changxuan; Wang, Jiajun; Shan, Anshan; Xu, Lin; Dong, Na; Li, Zhongyu

    2016-01-01

    The β-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple β-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded β-hairpin peptides with the same β-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded β-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8μM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple β-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded β-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide

  18. Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

    International Nuclear Information System (INIS)

    Flanagan, Sheryl A.; Cooper, Kristin S.; Mannava, Sudha; Nikiforov, Mikhail A.; Shewach, Donna S.

    2012-01-01

    Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2′-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound (≥90%) and prolonged (≥8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA

  19. Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

    Energy Technology Data Exchange (ETDEWEB)

    Flanagan, Sheryl A., E-mail: sflan@umich.edu [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Cooper, Kristin S. [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Mannava, Sudha; Nikiforov, Mikhail A. [Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York (United States); Shewach, Donna S. [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2012-12-01

    Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2 Prime -deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound ({>=}90%) and prolonged ({>=}8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA

  20. Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

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    Gene-Errol E Ringpis

    Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

  1. Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis

    International Nuclear Information System (INIS)

    Di Fulvio, Mauricio; Henkels, Karen M.; Gomez-Cambronero, Julian

    2007-01-01

    Grb2 is an SH2-SH3 protein adaptor responsible for linking growth factor receptors with intracellular signaling cascades. To study the role of Grb2 in cell growth, we have generated a new COS7 cell line (COS7 shGrb2 ), based on RNAi technology, as null mutations in mammalian Grb2 genes are lethal in early development. This novel cell line continuously expresses a short hairpin RNA that targets endogenous Grb2. Stable COS7 shGrb2 cells had the shGrb2 integrated into the genomic DNA and carried on SiL construct (made refractory to the shRNA-mediated interference), but not with an SH2-deficient mutant (R86K). Thus, a viable knock-down and rescue protocol has demonstrated that Grb2 is crucial for cell proliferation

  2. Induction of RNAi Responses by Short Left-Handed Hairpin RNAi Triggers.

    Science.gov (United States)

    Hagopian, Jonathan C; Hamil, Alexander S; van den Berg, Arjen; Meade, Bryan R; Eguchi, Akiko; Palm-Apergi, Caroline; Dowdy, Steven F

    2017-10-01

    Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.

  3. Inhibition of porcine transmissible gastroenteritis virus infection in porcine kidney cells using short hairpin RNAs targeting the membrane gene.

    Science.gov (United States)

    Wang, Li; Dai, Xianjin; Song, Han; Yuan, Peng; Yang, Zhou; Dong, Wei; Song, Zhenhui

    2017-04-01

    The membrane (M) protein is the most abundant component of the porcine transmissible gastroenteritis virus (TGEV) particle. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, three plasmids (pRNAT-1, pRNAT-2, and pRNAT-3) expressing short hairpin RNAs were designed to target three different coding regions of the M gene of TGEV. The plasmids were constructed and transiently transfected into a porcine kidney cells, PK-15, to determine whether these constructs inhibited TGEV production. The analysis of cytopathic effects demonstrated that pRNAT-2 and pRNAT-3 could protect PK-15 cells against pathological changes specifically and efficiently. Additionally, indirect immunofluorescence and 50% tissue culture infectious dose (TCID 50 ) assays showed that pRNAT-2 and pRNAT-3 inhibited the multiplication of the virus at the protein level effectively. Quantitative real-time PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the three plasmids were reduced by 13, 68, and 70%, respectively. This is the first report showing that RNAi targeting of the M gene. Our results could promote studies of the specific function of viral genes associated with TGEV infection and might provide a theoretical basis for potential therapeutic applications.

  4. Comparison of approaches for efficient gene silencing induced by microRNA-based short hairpin RNA and indicator gene expression.

    Science.gov (United States)

    Shan, Z X; Lin, Q X; Deng, C Y; Zhou, Z L; Tan, H H; Fu, Y H; Li, X H; Zhu, J N; Mai, L P; Kuang, S J; Lin, S G; Yu, X Y

    2010-04-01

    MicroRNA-based short hairpin RNAs (shRNAs) are natural inducers of RNA interference and have been increasingly used in shRNA expression strategies. In the present study, we compared the efficiencies of exogenous green fluorescence protein (GFP) and endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown and red fluorescent protein (RFP) indicator expression mediated by three differently designed plasmids. RFP was introduced either at the 5' end, at the 3' end of the human mir155-based target gene (TG) (e.g., GFP or GAPDH) shRNA expression cassette (EC), or at the 3' end of the chimeric intron-containing TG shRNA EC. Comparisons with the control vector showed an obvious reduction of GFP or GAPDH expression with the various shRNA expression plasmids (P < 0.05). When RFP was located at the 5' end or at the 3' end of the TG shRNA EC, RFP expression was low; whereas when RFP was connected with the chimeric intron-containing TG shRNA EC, RFP expression was high. Taken together, this study demonstrated an efficient plasmid design for both TG silencing induced by microRNA-based shRNA and indicator gene expression in vitro.

  5. Significant inhibition of Tembusu virus envelope and NS5 gene using an adenovirus-mediated short hairpin RNA delivery system.

    Science.gov (United States)

    Wang, Hongzhi; Feng, Qiang; Wei, Lei; Zhuo, Liling; Chen, Hao; Diao, Youxiang; Tang, Yi

    2017-10-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus, which was first isolated in the tropics during the 1970s. Recently, a disease characterized by ovarian haemorrhage and neurological symptoms was observed in ducks in China, which threatens poultry production. However, there is no suitable vaccination strategy or effective antiviral drugs to combat TMUV infections. Consequently, there is an urgent need to develop a new anti-TMUV therapy. In this study, we report an efficient short hairpin RNA (shRNA) delivery strategy for the inhibition of TMUV production using an adenovirus vector system. Using specifically designed shRNAs based on the E and NS5 protein genes of TMUV, the vector-expressed viral genes, TMUV RNA replication and infectious virus production were downregulated at different levels in Vero cells, where the shRNA (NS52) was highly effective in inhibiting TMUV. Using the human adenovirus type 5 shRNA delivery system, the recombinant adenovirus (rAd-NS52) inhibited TMUV multiplication with high efficiency. Furthermore, the significant dose-dependent inhibition of viral RNA copies induced by rAd-NS52 was found in TMUV-infected cells, which could last for at least 96h post infection. Our results indicated that the adenovirus-mediated delivery of shRNAs could play an active role in future TMUV antiviral therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

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    Kevin J Paik

    Full Text Available The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

  7. Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

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    Chih-ming Chen

    2009-07-01

    Full Text Available RNA interference (RNAi is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi and pReporter (pRe constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex (RISC, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30 enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

  8. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

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    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  9. Short hairpin RNA targeting insulin-like growth factor binding protein-3 restores the bioavailability of insulin-like growth factor-1 in diabetic rats

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    Zhang-Yan Zhou

    2016-02-01

    Full Text Available ABSTRACT Purpose To investigate whether intracavernosal injection of short hairpin RNA for IGFBP-3 could improve erectile function in streptozotocin-induced diabetic rats. Materials and methods After 12 weeks of IGFBP-3 short hairpin RNA injection treatment, intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated. The expression of IGFBP-3 and IGF-1 at mRNA and protein levels were detected by quantitative real-time PCR analysis and Western blot, respectively. The concentration of cavernous cyclic guanosine monophosphate was detected by enzyme-linked immunosorbent assay. Results At 12 weeks after intracavernous administration of IGFBP-3 shRNA, the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic group (P<0.05. Cavernous IGFBP-3 expression at both mRNA and protein levels was significantly inhibited. At the same time, cavernous IGF-1 expression was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01. Cavernous cyclic guanosine monophosphate concentration was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01. Conclusions Gene transfer of IGFBP-3 shRNA could improve erectile function via the restoration of cavernous IGF-1 bioavailability and an increase of cavernous cGMP concentration in the pathogenesis of erectile dysfunction in streptozotocin-induced diabetic rats.

  10. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

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    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  11. Adeno-Associated Viral Vector-Mediated mTOR Inhibition by Short Hairpin RNA Suppresses Laser-Induced Choroidal Neovascularization

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    Tae Kwann Park

    2017-09-01

    Full Text Available Choroidal neovascularization (CNV is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF, the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA, which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV. Keywords: retinal neovascularization, choroidal neovascularization, adeno-associated virus, mTOR, RNA interference, mTOR shRNA, autophagy

  12. Lentiviral Vector Mediated Claudin1 Silencing Inhibits Epithelial to Mesenchymal Transition in Breast Cancer Cells

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    Xianqi Zhao

    2015-06-01

    Full Text Available Breast cancer has a high incidence and mortality rate worldwide. Several viral vectors including lentiviral, adenoviral and adeno-associated viral vectors have been used in gene therapy for various forms of human cancer, and have shown promising effects in controlling tumor development. Claudin1 (CLDN1 is a member of the tetraspan transmembrane protein family that plays a major role in tight junctions and is associated with tumor metastasis. However, the role of CLDN1 in breast cancer is largely unexplored. In this study, we tested the therapeutic potential of silencing CLDN1 expression in two breast cancer (MDA-MB-231 and MCF7 cell lines using lentiviral vector mediated RNA interference. We found that a CLDN1 short hairpin (shRNA construct efficiently silenced CLDN1 expression in both breast cancer cell lines, and CLDN1 knockdown resulted in reduced cell proliferation, survival, migration and invasion. Furthermore, silencing CLDN1 inhibited epithelial to mesenchymal transition (EMT by upregulating the epithelial cell marker, E-cadherin, and downregulating mesenchymal markers, smooth muscle cell alpha-actin (SMA and Snai2. Our data demonstrated that lentiviral vector mediated CLDN1 RNA interference has great potential in breast cancer gene therapy by inhibiting EMT and controlling tumor cell growth.

  13. Efficacy Analysis of Combinatorial siRNAs against HIV Derived from One Double Hairpin RNA Precursor.

    Science.gov (United States)

    Liu, Chang; Liang, Zhipin; Kong, Xiaohong

    2017-01-01

    Combinatorial small interfering RNA duplexes (siRNAs) have the potential to be a gene therapy against HIV-1, and some studies have reported that transient combinatorial siRNA expression represses HIV replication, but the effects of long-term siRNA expression on HIV replication have not been studied in detail. In this study, HIV-1 replication under the influence of stable combinatorial siRNA expression from a single RNA transcript was analyzed. First, a series of cassettes encoding short hairpin RNA (shRNA)/long hairpin RNA (lhRNA)/double long hairpins (dlhRNA) was constructed and subjected to an analysis of inhibitory efficacy. Next, an optimized dlhRNA encoding cassette was selected and inserted into lentiviral delivery vector FG12. Transient dlhRNA expression reduced replication of HIV-1 in TZM-bl cells and CD4+ T cells successfully. HIV-1 susceptible TZM-bl cells were transducted with the dlhRNA expressing lentiviral vector and sorted by fluorescence-activated cell sorting to obtain stable dlhRNA expressing cells. The generation of four anti-HIV siRNAs in these dlhRNA expressing cells was verified by stem-loop RT-PCR assay. dlhRNA expression did not activate a non-specific interferon response. The dlhRNA expressing cells were also challenged with HIV-1 NL4-3, which revealed that stable expression of combinatorial siRNAs repressed HIV-1 replication for 8 days, after which HIV-1 overcame the inhibitory effect of siRNA expression by expressing mutant versions of RNAi targets. The results of this evaluation of the long-term inhibitory effects of combinatorial siRNAs against HIV-1 provide a reference for researchers who utilize combinatorial RNA interference against HIV-1 or other error-prone viruses.

  14. A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha

    DEFF Research Database (Denmark)

    Aagaard, Lars; Amarzguioui, Mohammed; Sun, Guihua

    2007-01-01

    RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. During the past 3 years a number of systems for conditional RNAi have been...... developed that allow controlled expression of short hairpin RNA (shRNA) triggers of RNAi. The simplest strategy relies on tet-operable polymerase III–promoted shRNAs and co-expression of the tetracycline regulatory protein, TetR. In this study we have combined these features into a single lentiviral vector...

  15. In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene.

    Science.gov (United States)

    He, Lei; Zhang, Yan-ming; Dong, Ling-juan; Cheng, Min; Wang, Jing; Tang, Qing-hai; Wang, Gang

    2012-08-28

    Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined. Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.

  16. In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene

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    He Lei

    2012-08-01

    Full Text Available Abstract Background Transmissible gastroenteritis (TGE is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs targeting the ORF 7 gene of TGEV on virus replication was examined. Results Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml. Stable swine testicular (ST cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. Conclusions Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.

  17. Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo

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    Yao Hailan

    2012-08-01

    Full Text Available Abstract Background Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. Methods To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B expressed by a recombinant vector (pGCL-2B or a recombinant lentivirus (Lenti-2B were tansfected in HeLa cells or transduced in mice infected with CVB3. Results ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. Conclusions Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

  18. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)

    2014-05-09

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  19. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

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    L. Ding

    2014-06-01

    Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  20. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    International Nuclear Information System (INIS)

    Ding, L.; Wu, J.P.; Xu, G.; Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W.

    2014-01-01

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis

  1. A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells

    International Nuclear Information System (INIS)

    Hung, C.-F.; Cheng, T.-L.; Wu, R.-H.; Teng, C.-F.; Chang, W.-T.

    2006-01-01

    RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells

  2. Lentiviral Delivery of Proteins for Genome Engineering.

    Science.gov (United States)

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2016-01-01

    Viruses have evolved to traverse cellular barriers and travel to the nucleus by mechanisms that involve active transport through the cytoplasm and viral quirks to resist cellular restriction factors and innate immune responses. Virus-derived vector systems exploit the capacity of viruses to ferry genetic information into cells, and now - more than three decades after the discovery of HIV - lentiviral vectors based on HIV-1 have become instrumental in biomedical research and gene therapies that require genomic insertion of transgenes. By now, the efficacy of lentiviral gene delivery to stem cells, cells of the immune system including T cells, hepatic cells, and many other therapeutically relevant cell types is well established. Along with nucleic acids, HIV-1 virions carry the enzymatic tools that are essential for early steps of infection. Such capacity to package enzymes, even proteins of nonviral origin, has unveiled new ways of exploiting cellular intrusion of HIV-1. Based on early findings demonstrating the packaging of heterologous proteins into virus particles as part of the Gag and GagPol polypeptides, we have established lentiviral protein transduction for delivery of DNA transposases and designer nucleases. This strategy for delivering genome-engineering proteins facilitates high enzymatic activity within a short time frame and may potentially improve the safety of genome editing. Exploiting the full potential of lentiviral vectors, incorporation of foreign protein can be combined with the delivery of DNA transposons or a donor sequence for homology-directed repair in so-called 'all-in-one' lentiviral vectors. Here, we briefly describe intracellular restrictions that may affect lentiviral gene and protein delivery and review the current status of lentiviral particles as carriers of tool kits for genome engineering.

  3. Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

    Science.gov (United States)

    Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

    2016-08-16

    Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

  4. HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2005-01-01

    Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

  5. Increased Engraftment of Human Short Term Repopulating Hematopoietic Cells in NOD/SCID/IL2rγnull Mice by Lentiviral Expression of NUP98-HOXA10HD.

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    Allistair Abraham

    Full Text Available Techniques to expand human hematopoietic stem cells ex-vivo could be beneficial to the fields of clinical hematopoietic stem cell transplantation and gene therapy targeted at hematopoietic stem cells. NUP98-HOXA10HD is a relatively newly discovered fusion gene that in mouse transplant experiments has been shown to increase numbers of hematopoietic stem cells. We evaluated whether this fusion gene could be used to expand engrafting human primitive CD34+ cells in an immunodeficient mouse model. Gene transfer was achieved using a lentiviral based vector. The engraftment of mobilized peripheral blood human CD34+ cells grown in culture for one week after gene transfer was evaluated 3-4 months after transplant and found to be 2-3 fold higher in the NUP98-HOXA10HD groups as compared to controls. These data suggest an expansive effect at least at the short term human repopulating cell level. Further evaluation in long term repopulating models and investment in a NUP98-HOXA10HD protein seems worthy of consideration. Additionally, the results here provide strong impetus to utilize NUP98-HOXA10HD as a tool to search for underlying genes and pathways involved in hematopoietic stem cell expansion that can be enhanced and have an even more potent expansive effect.

  6. Increased Engraftment of Human Short Term Repopulating Hematopoietic Cells in NOD/SCID/IL2rγnull Mice by Lentiviral Expression of NUP98-HOXA10HD.

    Science.gov (United States)

    Abraham, Allistair; Kim, Yoon-Sang; Zhao, Huifen; Humphries, Keith; Persons, Derek A

    2016-01-01

    Techniques to expand human hematopoietic stem cells ex-vivo could be beneficial to the fields of clinical hematopoietic stem cell transplantation and gene therapy targeted at hematopoietic stem cells. NUP98-HOXA10HD is a relatively newly discovered fusion gene that in mouse transplant experiments has been shown to increase numbers of hematopoietic stem cells. We evaluated whether this fusion gene could be used to expand engrafting human primitive CD34+ cells in an immunodeficient mouse model. Gene transfer was achieved using a lentiviral based vector. The engraftment of mobilized peripheral blood human CD34+ cells grown in culture for one week after gene transfer was evaluated 3-4 months after transplant and found to be 2-3 fold higher in the NUP98-HOXA10HD groups as compared to controls. These data suggest an expansive effect at least at the short term human repopulating cell level. Further evaluation in long term repopulating models and investment in a NUP98-HOXA10HD protein seems worthy of consideration. Additionally, the results here provide strong impetus to utilize NUP98-HOXA10HD as a tool to search for underlying genes and pathways involved in hematopoietic stem cell expansion that can be enhanced and have an even more potent expansive effect.

  7. Alleviation of Trigeminal Nociception Using p75 Neurotrophin Receptor Targeted Lentiviral Interference Therapy.

    Science.gov (United States)

    O'Leary, Valerie B; O'Connell, Marie; Antyborzec, Inga; Ntziachristos, Vasilis; Oliver Dolly, J; Ovsepian, Saak V

    2018-02-09

    Acute and chronic trigeminal (TG) neuropathies are the cause of considerable distress, with limited treatments available at present. Nociceptive neurons enriched with the vanilloid type 1 receptor (VR1) partake in pain sensation and sensitization in the TG system. While VR1 blockers with anti-nociceptive potential are of substantial medical interest, their use remains limited due to poor selectivity and lack of cell-targeting capabilities. This study describes a methodology for the alleviation of nociception via targeted depletion of VR1 in TG sensory neurons in rats. In cultured TG ganglion neurons, VR1 expression was virtually abolished by lentiviral short hairpin RNA (LV-VR1). By decorating GFP encoding LV (LV-GFP) and LV-VR1 with IgG192 for targeting TG sensory neurons enriched with the p75 neurotrophin receptor (p75NTR), transduction of a reporter GFP and VR1 depletion was achieved after injection of targeted vectors into the whisker pad. In IgG192/LV-VR1-injected rats, the behavioral response to capsaicin exposure as well as Erk 1/2 phosphorylation and VR1 current activation by capsaicin were significantly reduced. This pioneering investigation, thus, provides a proof of principle for a means of attenuating TG nociception, revealing therapeutic potential.

  8. Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

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    Yu-Ping Zheng

    2016-01-01

    Full Text Available AIM: To investigate the effects of lentivirus (LV mediated integrin-linked kinase (ILK RNA interference (RNAi on biological behaviors of human lens epithelial cells (LECs. METHODS: Human cataract LECs and immortalized human LEC line, human lens epithelial (HLE B-3 cells were transfected by lentiviral vector expressing ILK-specific short hairpin RNA (shRNA and then stimulated by transforming growth factor- (TGF-, the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction (RT-PCR and Western blot methods; biological behaviors including cell cycle and apoptosis, cell morphology, -smooth muscle actin (SMA stress fiber formation and cell migration were examined. RESULTS: Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-shRNA vector; flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells. Less -SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs. CONCLUSION: The present study demonstrated that ILK was an important regulator for LECs proliferation and migration. LV mediated ILK RNAi is an effective way to decrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis, as well as, to prevent cell migration by inhibiting TGF- induced -SMA stress fiber formation. Thus, LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

  9. Gene Therapy for Neuropathic Pain by Silencing of TNF-α Expression with Lentiviral Vectors Targeting the Dorsal Root Ganglion in Mice

    Science.gov (United States)

    Ogawa, Nobuhiro; Kawai, Hiromichi; Terashima, Tomoya; Kojima, Hideto; Oka, Kazuhiro; Chan, Lawrence; Maegawa, Hiroshi

    2014-01-01

    Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α in vitro, we selected shRNA3 to construct LV-TNF-shRNA3 for in vivo study. We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain. PMID:24642694

  10. Exquisite allele discrimination by toehold hairpin primers

    Science.gov (United States)

    Byrom, Michelle; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.

    2014-01-01

    The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a ‘yes/no’ answer. PMID:24990378

  11. Lentiviral Delivery of RNAi Effectors Against HIV-1

    NARCIS (Netherlands)

    Liu, Ying Poi; Berkhout, Ben

    2009-01-01

    RNA interference (RNAi) holds great promise as gene therapy approach against viral pathogens, including HIV-1. A specific anti-HIV-1 response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular transgene expression of short hairpin RNAs (shRNAs) or

  12. Short-hairpin RNA-induced suppression of adenine nucleotide translocase-2 in breast cancer cells restores their susceptibility to TRAIL-induced apoptosis by activating JNK and modulating TRAIL receptor expression

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    Kim Chul-Woo

    2010-09-01

    Full Text Available Abstract Background Tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL; apo2 ligand induces apoptosis in cancer cells but has little effect on normal cells. However, many cancer cell types are resistant to TRAIL-induced apoptosis, limiting the clinical utility of TRAIL as an anti-cancer agent. We previously reported that the suppression of adenine nucleotide translocase-2 (ANT2 by short-hairpin RNA (shRNA induces apoptosis of breast cancer cells, which frequently express high levels of ANT2. In the present study, we examined the effect of RNA shRNA-induced suppression of ANT2 on the resistance of breast cancer cells to TRAIL-induced apoptosis in vitro and in vivo. Results ANT2 shRNA treatment sensitized MCF7, T47 D, and BT474 cells to TRAIL-induced apoptosis by up-regulating the expression of TRAIL death receptors 4 and 5 (DR4 and DR5 and down-regulating the TRAIL decoy receptor 2 (DcR2. In MCF7 cells, ANT2 knockdown activated the stress kinase c-Jun N-terminal kinase (JNK, subsequently stabilizing and increasing the transcriptional activity of p53 by phosphorylating it at Thr81; it also enhanced the expression and activity of DNA methyltransferase 1 (DNMT1. ANT2 shRNA-induced overexpression of DR4/DR5 and TRAIL sensitization were blocked by a p53 inhibitor, suggesting that p53 activation plays an important role in the transcriptional up-regulation of DR4/DR5. However, ANT2 knockdown also up-regulated DR4/DR5 in the p53-mutant cell lines BT474 and T47 D. In MCF7 cells, ANT2 shRNA treatment led to DcR2 promoter methylation and concomitant down-regulation of DcR2 expression, consistent with the observed activation of DNMT1. Treatment of the cells with a demethylating agent or JNK inhibitor prevented the ANT2 shRNA-induced down-regulation of DcR2 and activation of both p53 and DNMT1. In in vivo experiments using nude mice, ANT2 shRNA caused TRAIL-resistant MCF7 xenografts to undergo TRAIL-induced cell death, up-regulated DR4/DR5

  13. Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

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    Jakobsen Maria

    2011-02-01

    Full Text Available Abstract Background Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα, interleukin-12 (IL-12, and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs. Methods Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels. Results Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by

  14. Lentiviral vectors in cancer immunotherapy.

    Science.gov (United States)

    Oldham, Robyn Aa; Berinstein, Elliot M; Medin, Jeffrey A

    2015-01-01

    Basic science advances in cancer immunotherapy have resulted in various treatments that have recently shown success in the clinic. Many of these therapies require the insertion of genes into cells to directly kill them or to redirect the host's cells to induce potent immune responses. Other analogous therapies work by modifying effector cells for improved targeting and enhanced killing of tumor cells. Initial studies done using γ-retroviruses were promising, but safety concerns centered on the potential for insertional mutagenesis have highlighted the desire to develop other options for gene delivery. Lentiviral vectors (LVs) have been identified as potentially more effective and safer alternative delivery vehicles. LVs are now in use in clinical trials for many different types of inherited and acquired disorders, including cancer. This review will discuss current knowledge of LVs and the applications of this viral vector-based delivery vehicle to cancer immunotherapy.

  15. Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing

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    Karine Cambon

    2017-06-01

    Full Text Available Huntington’s disease (HD is an autosomal dominant neurodegenerative disorder resulting from a polyglutamine expansion in the huntingtin (HTT protein. There is currently no cure for this disease, but recent studies suggest that RNAi to downregulate the expression of both normal and mutant HTT is a promising therapeutic approach. We previously developed a small hairpin RNA (shRNA, vectorized in an HIV-1-derived lentiviral vector (LV, that reduced pathology in an HD rodent model. Here, we modified this vector for preclinical development by using a tat-independent third-generation LV (pCCL backbone and removing the original reporter genes. We demonstrate that this novel vector efficiently downregulated HTT expression in vitro in striatal neurons derived from induced pluripotent stem cells (iPSCs of HD patients. It reduced two major pathological HD hallmarks while triggering a minimal inflammatory response, up to 6 weeks after injection, when administered by stereotaxic surgery in the striatum of an in vivo rodent HD model. Further assessment of this shRNA vector in vitro showed proper processing by the endogenous silencing machinery, and we analyzed gene expression changes to identify potential off-targets. These preclinical data suggest that this new shRNA vector fulfills primary biosafety and efficiency requirements for further development in the clinic as a cure for HD.

  16. Hairpins under tension: RNA versus DNA.

    Science.gov (United States)

    Bercy, Mathilde; Bockelmann, Ulrich

    2015-11-16

    We use optical tweezers to control the folding and unfolding of individual DNA and RNA hairpins by force. Four hairpin molecules are studied in comparison: two DNA and two RNA ones. We observe that the conformational dynamics is slower for the RNA hairpins than for their DNA counterparts. Our results indicate that structures made of RNA are dynamically more stable. This difference might contribute to the fact that DNA and RNA play fundamentally different biological roles in spite of chemical similarity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Ultrafast Unzipping of a Beta-Hairpin Peptide

    NARCIS (Netherlands)

    Zinth, W.; Schrader, T.E.; Schreier, W.J.; Koller, F.O.; Cordes, T.; Babitzki, G.; Denschlag, R.; Tavan, P.; Löweneck, M.; Dong, Shou-Liang; Moroder, L.; Renner, C.; Corkum, Paul; Jonas, David M.; Miller, R.J. Dwayne; Weiner, Andrew M.

    2007-01-01

    Light induced switching of a beta-hairpin structure is investigated by femtosecond IR spectroscopy. While the unzipping process comprises ultrafast kinetics and is finished within 1 ns, the folding into the hairpin structure is a much slower process.

  18. Design and function of triplex hairpin ribozymes.

    Science.gov (United States)

    Aquino-Jarquin, Guillermo; Rojas-Hernández, Ramiro; Alvarez-Salas, Luis Marat

    2010-01-01

    Triplex ribozymes allow for the individual activity of multiple trans-acting ribozymes producing higher target cleavage relative to tandem-expressed RZs. A triplex expression system based on a single hairpin ribozyme for the multiple expression (multiplex) vectors can be engineered to target RNAs with single or multiple antisense-accessible sites. System construction relies on triplex expression modules consisting of hairpin ribozyme cassettes flanked by ribozymes lacking catalytic domains. Multiplex vectors can be generated with single or multiple specificity by tandem cloning of triplex expression modules. Triplex ribozymes are initially tested in vitro using cis- and trans-cleavage assays against radioactive-labeled targets. In addition, triplex ribozymes are tested for cis and trans cleavage in vivo by transfection in cultured cells followed by ribonuclease protection assays (RPAs) and RT-PCR. The use of triplex configurations with multiplex ribozymes will provide the basis for the development of future RZ-based therapies and technologies.

  19. An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.

    Science.gov (United States)

    Lee, Su Jin; Hyun, Soonsil; Kieft, Jeffrey S; Yu, Jaehoon

    2009-02-18

    The hairpin RNA motif is one of the most frequently observed secondary structures and is often targeted by therapeutic agents. An amphiphilic peptide with seven lysine and eight leucine residues and its derivatives were designed for use as ligands against RNA hairpin motifs. We hypothesized that variations in both the hydrophobic leucine-rich and hydrophilic lysine-rich spheres of these amphiphilic peptides would create extra attractive interactions with hairpin RNA targets. A series of alanine-scanned peptides were probed to identify the most influential lysine residues in the hydrophilic sphere. The binding affinities of these modified peptides with several hairpins, such as RRE, TAR from HIV, a short hairpin from IRES of HCV, and a hairpin from the 16S A-site stem from rRNA, were determined. Since the hairpin from IRES of HCV was the most susceptible to the initial series of alanine-scanned peptides, studies investigating how further variations in the peptides effect binding employed the IRES hairpin. Next, the important Lys residues were substituted by shorter chain amines, such as ornithine, to place the peptide deeper into the hairpin groove. In a few cases, a 70-fold improved binding was observed for peptides that contained the specifically located shorter amine side chains. To further explore changes in binding affinities brought about by alterations in the hydrophobic sphere, tryptophan residues were introduced in place of leucine. A few peptides with tryptophan in specific positions also displayed 70-fold improved binding affinities. Finally, double mutant peptides incorporating both specifically located shorter amine side chains in the hydrophilic region and tryptophan residues in the hydrophobic region were synthesized. The binding affinities of peptides containing the simple double modification were observed to be 80 times lower, and their binding specificities were increased 40-fold. The results of this effort provide important information about

  20. Construction of lentiviral shRNA expression vector targeting ...

    African Journals Online (AJOL)

    DNA oligo was cloned into lentiviral expression vector, and then polymerase chain reaction (PCR) and sequencing analyses were conducted to verify the constructs. The verified vectors were co-transfected into 293FT cells that could produce lentiviral. shRNA lentiviruses from the selected constructs were propagated and ...

  1. Lentiviral Vector Gene Transfer to Porcine Airways

    Directory of Open Access Journals (Sweden)

    Patrick L Sinn

    2012-01-01

    Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

  2. How hairpin vortices emerge from exact invariant solutions

    Science.gov (United States)

    Schneider, Tobias M.; Farano, Mirko; de Palma, Pietro; Robinet, Jean-Christoph; Cherubini, Stefania

    2017-11-01

    Hairpin vortices are among the most commonly observed flow structures in wall-bounded shear flows. However, within the dynamical system approach to turbulence, those structures have not yet been described. They are not captured by known exact invariant solutions of the Navier-Stokes equations nor have other state-space structures supporting hairpins been identified. We show that hairpin structures are observed along an optimally growing trajectory leaving a well known exact traveling wave solution of plane Poiseuille flow. The perturbation triggering hairpins does not correspond to an unstable mode of the exact traveling wave but lies in the stable manifold where non-normality causes strong transient amplification.

  3. On hairpin vortices in a transitional boundary layer

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    Uruba Václav

    2012-04-01

    Full Text Available In the presented paper the results of experiments on transitional boundary layer are presented. The boundary layer was generated on smooth flat wall with zero pressure gradient forming one side of the channel of rectangular cross section. The hairpin vortices, packets of hairpin vortices, turbulent spots and calmed regions were experimentally investigated using time-resolved PIV technique.

  4. Envelope determinants of equine lentiviral vaccine protection.

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    Jodi K Craigo

    Full Text Available Lentiviral envelope (Env antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0, and roughly 40% protection against challenge by virus (EV13 with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes

  5. Envelope Determinants of Equine Lentiviral Vaccine Protection

    Science.gov (United States)

    Craigo, Jodi K.; Ezzelarab, Corin; Cook, Sheila J.; Chong, Liu; Horohov, David; Issel, Charles J.; Montelaro, Ronald C.

    2013-01-01

    Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for

  6. Cyclophilin A interacts with diverse lentiviral capsids

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    Emerman Michael

    2006-10-01

    Full Text Available Abstract Background The capsid (CA protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA. This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. Results We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. Conclusion These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

  7. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

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    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  8. "Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt; Ferapontova, Elena E

    2011-03-01

    A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

  9. ncRNAclassifier: a tool for detection and classification of transposable element sequences in RNA hairpins

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    Tempel Sébastien

    2012-09-01

    Full Text Available Abstract Background Inverted repeat genes encode precursor RNAs characterized by hairpin structures. These RNA hairpins are then metabolized by biosynthetic pathways to produce functional small RNAs. In eukaryotic genomes, short non-autonomous transposable elements can have similar size and hairpin structures as non-coding precursor RNAs. This resemblance leads to problems annotating small RNAs. Results We mapped all microRNA precursors from miRBASE to several genomes and studied the repetition and dispersion of the corresponding loci. We then searched for repetitive elements overlapping these loci. We developed an automatic method called ncRNAclassifier to classify pre-ncRNAs according to their relationship with transposable elements (TEs. We showed that there is a correlation between the number of scattered occurrences of ncRNA precursor candidates and the presence of TEs. We applied ncRNAclassifier on six chordate genomes and report our findings. Among the 1,426 human and 721 mouse pre-miRNAs of miRBase, we identified 235 and 68 mis-annotated pre-miRNAs respectively corresponding completely to TEs. Conclusions We provide a tool enabling the identification of repetitive elements in precursor ncRNA sequences. ncRNAclassifier is available at http://EvryRNA.ibisc.univ-evry.fr.

  10. Transient β-hairpin formation in α-synuclein monomer revealed by coarse-grained molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hang; Ma, Wen [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Han, Wei [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Schulten, Klaus, E-mail: kschulte@ks.uiuc.edu [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)

    2015-12-28

    Parkinson’s disease, originating from the intrinsically disordered peptide α-synuclein, is a common neurodegenerative disorder that affects more than 5% of the population above age 85. It remains unclear how α-synuclein monomers undergo conformational changes leading to aggregation and formation of fibrils characteristic for the disease. In the present study, we perform molecular dynamics simulations (over 180 μs in aggregated time) using a hybrid-resolution model, Proteins with Atomic details in Coarse-grained Environment (PACE), to characterize in atomic detail structural ensembles of wild type and mutant monomeric α-synuclein in aqueous solution. The simulations reproduce structural properties of α-synuclein characterized in experiments, such as secondary structure content, long-range contacts, chemical shifts, and {sup 3}J(H{sub N}H{sub C{sub α}})-coupling constants. Most notably, the simulations reveal that a short fragment encompassing region 38-53, adjacent to the non-amyloid-β component region, exhibits a high probability of forming a β-hairpin; this fragment, when isolated from the remainder of α-synuclein, fluctuates frequently into its β-hairpin conformation. Two disease-prone mutations, namely, A30P and A53T, significantly accelerate the formation of a β-hairpin in the stated fragment. We conclude that the formation of a β-hairpin in region 38-53 is a key event during α-synuclein aggregation. We predict further that the G47V mutation impedes the formation of a turn in the β-hairpin and slows down β-hairpin formation, thereby retarding α-synuclein aggregation.

  11. Short (

    NARCIS (Netherlands)

    Telleman, Gerdien; den Hartog, Laurens

    2013-01-01

    Aim: This systematic review assessed the implant survival rate of short (<10 mm) dental implants installed in partially edentulous patients. A case report of a short implant in the posterior region have been added. Materials and methods: A search was conducted in the electronic databases of MEDLINE

  12. Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

    Directory of Open Access Journals (Sweden)

    Shen-shen ZHI

    2011-02-01

    Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

  13. Hybridization-based biosensor containing hairpin probes and use thereof

    Science.gov (United States)

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2010-10-12

    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  14. scid cells efficiently integrate hairpin and linear DNA substrates.

    Science.gov (United States)

    Staunton, J E; Weaver, D T

    1994-01-01

    The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination. Images PMID:8196630

  15. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    Science.gov (United States)

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Inhibition of HIV-1 lentiviral particles infectivity by Gynostemma ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... simian immunodeficiency virus: implications for the safety of lentiviral vectors. Hum. Genet. Ther. 11: 2403-2413. Williams JE (2001). Review of antiviral and immuno modulating properties of plants of the Peruvian rainforest with a particular emphasis on Una de Gato and Sangre de Grado. Altern. Med. Rev.

  17. Lentiviral vectors for treating and modeling human CNS disorders.

    Science.gov (United States)

    Azzouz, Mimoun; Kingsman, Susan M; Mazarakis, Nicholas D

    2004-09-01

    Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral-mediated overexpression of mutated human alpha-synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non-primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. Copyright 2004 John Wiley & Sons, Ltd.

  18. The feasibility of incorporating Vpx into lentiviral gene therapy vectors

    Directory of Open Access Journals (Sweden)

    Samantha A McAllery

    2016-01-01

    Full Text Available While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT occurs in HIV-1 reservoirs during lentiviral gene delivery. The viral protein Vpx is capable of increasing lentiviral RT by antagonizing the restriction factor SAMHD1. Incorporating Vpx into lentiviral vectors could substantially increase gene delivery into the HIV-1 reservoir. The feasibility of this Vpx approach was tested in resting cell models utilizing macrophages and dendritic cells. Our results showed Vpx exposure led to increased permissiveness of cells over a period that exceeded 2 weeks. Consequently, significant lower potency of HIV-1 antiretrovirals inhibiting RT and integration was observed. When Vpx was incorporated with anti-HIV-1 genes inhibiting either pre-RT or post-RT stages of the viral life-cycle, transduction levels significantly increased. However, a stronger antiviral effect was only observed with constructs that inhibit pre-RT stages of the viral life cycle. In conclusion this study demonstrates a way to overcome the major delivery obstacle of gene delivery into HIV-1 reservoir cell types. Importantly, incorporating Vpx with pre-RT anti-HIV-1 genes, demonstrated the greatest protection against HIV-1 infection.

  19. Inhibition of HIV-1 lentiviral particles infectivity by Gynostemma ...

    African Journals Online (AJOL)

    These claims motivated the study in which the inhibition of viral vector infectivity of HeLa cells was assessed flow cytometrically by measuring the expression of green fluorescent protein (GFP) transgene incorporated in the lentiviral vector construct. An infectious VSV-G-pseudotyped, human immunodeficiency virus type ...

  20. Design and Potential of Non-Integrating Lentiviral Vectors

    Directory of Open Access Journals (Sweden)

    Aaron Shaw

    2014-01-01

    Full Text Available Lentiviral vectors have demonstrated promising results in clinical trials that target cells of the hematopoietic system. For these applications, they are the vectors of choice since they provide stable integration into cells that will undergo extensive expansion in vivo. Unfortunately, integration can have unintended consequences including dysregulated cell growth. Therefore, lentiviral vectors that do not integrate are predicted to have a safer profile compared to integrating vectors and should be considered for applications where transient expression is required or for sustained episomal expression such as in quiescent cells. In this review, the system for generating lentiviral vectors will be described and used to illustrate how alterations in the viral integrase or vector Long Terminal Repeats have been used to generate vectors that lack the ability to integrate. In addition to their safety advantages, these non-integrating lentiviral vectors can be used when persistent expression would have adverse consequences. Vectors are currently in development for use in vaccinations, cancer therapy, site-directed gene insertions, gene disruption strategies, and cell reprogramming. Preclinical work will be described that illustrates the potential of this unique vector system in human gene therapy.

  1. Whirlwinds and hairpins in the atmospheric surface layer

    NARCIS (Netherlands)

    Oncley, Steven P.; Hartogensis, O.K.; Tong, Chenning

    2016-01-01

    Vortices in the atmospheric surface layer are characterized using observations at unprecedented resolution from a fixed array of 31 turbulence sensors. During the day, these vortices likely are dust devils, though no visual observations are available for confirmation. At night, hairpin vortices

  2. Modeling the mechanism of CLN025 beta-hairpin formation

    Science.gov (United States)

    McKiernan, Keri A.; Husic, Brooke E.; Pande, Vijay S.

    2017-09-01

    Beta-hairpins are substructures found in proteins that can lend insight into more complex systems. Furthermore, the folding of beta-hairpins is a valuable test case for benchmarking experimental and theoretical methods. Here, we simulate the folding of CLN025, a miniprotein with a beta-hairpin structure, at its experimental melting temperature using a range of state-of-the-art protein force fields. We construct Markov state models in order to examine the thermodynamics, kinetics, mechanism, and rate-determining step of folding. Mechanistically, we find the folding process is rate-limited by the formation of the turn region hydrogen bonds, which occurs following the downhill hydrophobic collapse of the extended denatured protein. These results are presented in the context of established and contradictory theories of the beta-hairpin folding process. Furthermore, our analysis suggests that the AMBER-FB15 force field, at this temperature, best describes the characteristics of the full experimental CLN025 conformational ensemble, while the AMBER ff99SB-ILDN and CHARMM22* force fields display a tendency to overstabilize the native state.

  3. Method of identifying hairpin DNA probes by partial fold analysis

    Science.gov (United States)

    Miller, Benjamin L [Penfield, NY; Strohsahl, Christopher M [Saugerties, NY

    2009-10-06

    Method of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

  4. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes.

    Science.gov (United States)

    Qian, Wei; Wang, Yong; Li, Rui-Fu; Zhou, Xin; Liu, Jing; Peng, Dai-Zhi

    2017-03-03

    BACKGROUND Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. MATERIAL AND METHODS In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. RESULTS The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. CONCLUSIONS This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.

  5. Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

    DEFF Research Database (Denmark)

    Kushon, S A; Jordan, J P; Seifert, J L

    2001-01-01

    structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic......-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary...

  6. Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

    Science.gov (United States)

    Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M; Yue, Junming

    2017-01-01

    CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

  7. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

    Directory of Open Access Journals (Sweden)

    Tatjana P Kristensen

    2014-03-01

    Full Text Available The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural

  8. Neuron-specific RNA interference using lentiviral vectors

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup; Marion, Ingrid van; Hasholt, Lis

    2009-01-01

    demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show...... that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages. CONCLUSIONS: In the present study, we developed vectors for cell-specific RNAi for use...

  9. Low titer lentiviral transgenesis in rodents with simian immundeficiency virus vector.

    Science.gov (United States)

    Bender, Balázs; Hoffmann, Orsolya Ivett; Negre, Didier; Kvell, Krisztián; Bősze, Zsuzsanna; Hiripi, László

    2013-09-01

    Efficient production of transgenic animals using low-titer lentiviral constructs remains challenging. Here we demonstrate that microinjection of simian immundeficiency virus-derived lentiviral constructs can produce transgenic mice and rats with high efficiency even when using low-titer virus preparations.

  10. Construction of a novel lentiviral vector carrying human B-domain ...

    African Journals Online (AJOL)

    ... integration were detected in all cell lines after transfection. A novel lentiviral vector carrying human FVIII³BD was constructed, which was able to transfect different mammalian cell types accompanied by high-level activity. This lentiviral vector may provide a theoretical basis for the gene therapy of patients with hemophilia ...

  11. Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors

    DEFF Research Database (Denmark)

    Jakobsson, J; Nielsen, T Tolstrup; Staflin, K

    2006-01-01

    Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped le......Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV...... and human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells....

  12. Efficient transgenesis in farm animals by lentiviral vectors

    Science.gov (United States)

    Hofmann, Andreas; Kessler, Barbara; Ewerling, Sonja; Weppert, Myriam; Vogg, Barbara; Ludwig, Harald; Stojkovic, Miodrag; Boelhauve, Marc; Brem, Gottfried; Wolf, Eckhard; Pfeifer, Alexander

    2003-01-01

    Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively. PMID:14566324

  13. Lentiviral Vectors for Cancer Immunotherapy and Clinical Applications

    Directory of Open Access Journals (Sweden)

    David Escors

    2013-07-01

    Full Text Available The success of immunotherapy against infectious diseases has shown us the powerful potential that such a treatment offers, and substantial work has been done to apply this strategy in the fight against cancer. Cancer is however a fiercer opponent than pathogen-caused diseases due to natural tolerance towards tumour associated antigens and tumour-induced immunosuppression. Recent gene therapy clinical trials with viral vectors have shown clinical efficacy in the correction of genetic diseases, HIV and cancer. The first successful gene therapy clinical trials were carried out with onco(g-retroviral vectors but oncogenesis by insertional mutagenesis appeared as a serious complication. Lentiviral vectors have emerged as a potentially safer strategy, and recently the first clinical trial of patients with advanced leukemia using lentiviral vectors has proven successful. Additionally, therapeutic lentivectors have shown clinical efficacy for the treatment of HIV, X-linked adrenoleukodystrophy, and b-thalassaemia. This review aims at describing lentivectors and how they can be utilized to boost anti-tumour immune responses by manipulating the effector immune cells.

  14. Lentiviral Vectors for Cancer Immunotherapy and Clinical Applications

    Energy Technology Data Exchange (ETDEWEB)

    Liechtenstein, Therese, E-mail: t.liechtenstein.12@ucl.ac.uk [University College London, 5 University Street, London, WC1E 6JF (United Kingdom); Perez-Janices, Noemi; Escors, David [University College London, 5 University Street, London, WC1E 6JF (United Kingdom); Navarrabiomed Fundacion Miguel Servet, 3 Irunlarrea St., Hospital Complex of Navarra, 31008 Pamplona, Navarra (Spain)

    2013-07-02

    The success of immunotherapy against infectious diseases has shown us the powerful potential that such a treatment offers, and substantial work has been done to apply this strategy in the fight against cancer. Cancer is however a fiercer opponent than pathogen-caused diseases due to natural tolerance towards tumour associated antigens and tumour-induced immunosuppression. Recent gene therapy clinical trials with viral vectors have shown clinical efficacy in the correction of genetic diseases, HIV and cancer. The first successful gene therapy clinical trials were carried out with onco(γ-)retroviral vectors but oncogenesis by insertional mutagenesis appeared as a serious complication. Lentiviral vectors have emerged as a potentially safer strategy, and recently the first clinical trial of patients with advanced leukemia using lentiviral vectors has proven successful. Additionally, therapeutic lentivectors have shown clinical efficacy for the treatment of HIV, X-linked adrenoleukodystrophy, and β-thalassaemia. This review aims at describing lentivectors and how they can be utilized to boost anti-tumour immune responses by manipulating the effector immune cells.

  15. Lentiviral Vectors for Cancer Immunotherapy and Clinical Applications

    International Nuclear Information System (INIS)

    Liechtenstein, Therese; Perez-Janices, Noemi; Escors, David

    2013-01-01

    The success of immunotherapy against infectious diseases has shown us the powerful potential that such a treatment offers, and substantial work has been done to apply this strategy in the fight against cancer. Cancer is however a fiercer opponent than pathogen-caused diseases due to natural tolerance towards tumour associated antigens and tumour-induced immunosuppression. Recent gene therapy clinical trials with viral vectors have shown clinical efficacy in the correction of genetic diseases, HIV and cancer. The first successful gene therapy clinical trials were carried out with onco(γ-)retroviral vectors but oncogenesis by insertional mutagenesis appeared as a serious complication. Lentiviral vectors have emerged as a potentially safer strategy, and recently the first clinical trial of patients with advanced leukemia using lentiviral vectors has proven successful. Additionally, therapeutic lentivectors have shown clinical efficacy for the treatment of HIV, X-linked adrenoleukodystrophy, and β-thalassaemia. This review aims at describing lentivectors and how they can be utilized to boost anti-tumour immune responses by manipulating the effector immune cells

  16. Hairpin DNA probe with 5'-TCC/CCC-3' overhangs for the creation of silver nanoclusters and miRNA assay.

    Science.gov (United States)

    Xia, Xiaodong; Hao, Yuanqiang; Hu, Shengqiang; Wang, Jianxiu

    2014-01-15

    A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine residues and Ag(+), a short cytosine-rich oligonucleotide (ODN) with only six bases 5'-TCCCCC-3' served as an efficient scaffold for the creation of the AgNCs. The AgNCs displayed a bright red emission when excited at 545nm. Such ODN base-stabilized AgNCs have been exploited for miRNA sensing. Overhangs of TCC at the 5' end (5'-TCC) and CCC at the 3' end (CCC-3') (denoted as 5'-TCC/CCC-3') appended to the hairpin ODN probe which also contains recognition sequences for target miRNA were included. Interestingly, the AgNCs/hairpin ODN probe showed similar spectral properties as that templated by 5'-TCCCCC-3'. The formation of the hairpin ODN probe/miRNA duplex separated the 5'-TCC/CCC-3' overhangs, thus disturbing the optical property or structure of the AgNCs. As a result, fluorescence quenching of the AgNCs/hairpin ODN probe was obtained, which allows for facile determination of target miRNA. The proposed method is simple and cost-effective, holding great promise for clinical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Structural modeling of DNA mini-hairpin molecules with various loop sequences

    Science.gov (United States)

    Nakamura, Shugo; Hirose, Hitoshi; Ikeguchi, Mitsunori; Shimizu, Kentaro

    1999-07-01

    We modeled the structures of DNA fragments dGC(GNA)GC (N = A, G, T, and C), which are known to form mini-hairpins, and dGC(GAC)GC and dGC(GAG)GC, which are known not to form mini-hairpins, by using global optimization of conformational energy. For all of the fragments, we obtained mini-hairpins which had similar backbone structures and positions of bases. The conformational energy differences between the energy-optimized structures were small. Our results suggest that the optimized energy values are not sufficient to discriminate which fragments form thermo-stable mini-hairpins and which do not.

  18. Design of a new hairpin DNAzyme: The activity controlled by TMPyP ...

    African Journals Online (AJOL)

    The catalytic activity of the 10-23 hairpin DNAzyme decreased and even inactivated due to the enhanced stability of G-quadruplex structure by TMPyP4 molecules. This DNAzyme is controllable to cleavage substrate and has some potential significance in gene therapy. Key words: 10-23 DNAzyme, hairpin DNAzyme, ...

  19. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

    Directory of Open Access Journals (Sweden)

    Christelle Folio

    2017-11-01

    Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

  20. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline.

    Science.gov (United States)

    Folio, Christelle; Sierra, Natalia; Dujardin, Marie; Alvarez, Guzman; Guillon, Christophe

    2017-11-09

    Feline immunodeficiency virus (FIV) is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS) in cats and wild felines. Its capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg-Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

  1. Hairpin packet structure of a turbulent boundary layer in inclined wall-normal/spanwise planes

    Science.gov (United States)

    Lee, Jae Hwa; Sung, Hyung Jin

    2009-11-01

    Turbulent coherent structures associated with hairpin packet motions have been scrutinized using the instantaneous flow fields obtained from the direct numerical simulation (DNS) of a turbulent boundary layer (TBL). The Reynolds number based on the momentum thickness was varied in the range Reθ=890˜2560. This study focused on the hairpin packet motions in inclined wall-normal/spanwise planes. The hairpin vortex signature associated with the hairpin leg components in the vertical inclined plane consists of a counter-rotating vortex pair, upward and downward motions and a stagnation point induced by the Q2 and Q4 events. These hairpin signatures were observed in the instantaneous flow field, in the two-point correlations and in the conditionally averaged flow fields, respectively. We considered three inclined planes (45^o, 90^o, and 135^o) to investigate the spatial characteristics of the hairpin packet motions in the log and wake regions. The statistical flow fields showed that significantly different flow patterns are induced by the intersections of the three inclined planes with the hairpin packet motions.

  2. HBS-Tools for Hairpin Bisulfite Sequencing Data Processing and Analysis

    Directory of Open Access Journals (Sweden)

    Ming-an Sun

    2015-01-01

    Full Text Available The emerging genome-wide hairpin bisulfite sequencing (hairpin-BS-Seq technique enables the determination of the methylation pattern for DNA double strands simultaneously. Compared with traditional bisulfite sequencing (BS-Seq techniques, hairpin-BS-Seq can determine methylation fidelity and increase mapping efficiency. However, no computational tool has been designed for the analysis of hairpin-BS-Seq data yet. Here we present HBS-tools, a set of command line based tools for the preprocessing, mapping, methylation calling, and summarizing of genome-wide hairpin-BS-Seq data. It accepts paired-end hairpin-BS-Seq reads to recover the original (pre-bisulfite-converted sequences using global alignment and then calls the methylation statuses for cytosines on both DNA strands after mapping the original sequences to the reference genome. After applying to hairpin-BS-Seq datasets, we found that HBS-tools have a reduced mapping time and improved mapping efficiency compared with state-of-the-art mapping tools. The HBS-tools source scripts, along with user guide and testing data, are freely available for download.

  3. Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Masoud, Rula; Drory, Hagai; Plavner, Noa; Liber, Miran; Nir, Eyal

    2013-10-10

    The dynamics of two DNA hairpins (5'-TCGCCT-A31-AGGCGA-3' and 5'-TCGCCG-A31-CGGCGA-3') were studied using immobilization-based and diffusion-based single-molecule fluorescence techniques. The techniques enabled separated and detailed investigation of the states and of the transition reactions. Only two states, open and closed, were identified from analysis of the FRET histograms; metastable states with lifetimes longer than the technique resolution (0.3 ms) were not observed. The opening and closing reaction rates were determined directly from the FRET time trajectories, and the Gibbs free energies of these states and of the transition state were calculated using the Kramer theory. The rates, which are undoubtedly of transitions between the fully closed and the fully open states and ranged from 2 to 90 s(-1), were lower (∼10-fold) than the rates previously determined from fluorescence correlation spectroscopy. The heights of the barriers for closing were almost identical for the two hairpins. The barrier for opening the hairpin with the stronger stem was higher (4.3 kJ/mol) than that for the hairpin with the weaker stem, in very good agreement with the difference in stability calculated by the nearest-neighbor method. The barrier for closing the hairpin decreased (∼8 kJ/mol) and the barrier for opening increased (∼4 kJ/mol) with increasing NaCl concentration (10-100 mM), indicating that higher ionic strength stabilizes the folded state with respect to the transition state and stabilizes the transition state relative to the unfolded state. The very good agreements in the dynamics measured for free hairpins, for hairpins anchored to origami, and for hairpins anchored to the coverslip and the very good agreement between the two single-molecule techniques demonstrate that neither the origami nor the coverslip influence the hairpin dynamics, supporting a previous demonstration that origami can serve as a platform for biophysical investigations.

  4. Structural basis for functional tetramerization of lentiviral integrase.

    Directory of Open Access Journals (Sweden)

    Stephen Hare

    2009-07-01

    Full Text Available Experimental evidence suggests that a tetramer of integrase (IN is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.

  5. Desmin-regulated lentiviral vectors for skeletal muscle gene transfer.

    Science.gov (United States)

    Talbot, Gillian E; Waddington, Simon N; Bales, Olivia; Tchen, Rose C; Antoniou, Michael N

    2010-03-01

    Lentiviral vectors (LVs) are highly attractive as a gene therapy agent as they are able to stably integrate their genomes in both dividing and nondividing cells and, in principle, provide long-term therapeutic benefit. However, their performance in skeletal muscle in adult animals has, to date, been disappointing. In order to gain clearer insight into their utility in this tissue type, we have conducted an extensive quantitative comparison of constitutive and muscle-specific promoter activities in skeletal muscle and nonmuscle systems following LV delivery in cell lines and neonatal mice. Our data show that LV delivery to hind leg skeletal muscle of neonatal mouse results in long-term transgene expression in adulthood. We find that the human desmin (DES) promoter/enhancer is the first muscle-specific control region to match the activity of the highly active constitutive human cytomegalovirus (hCMV) promoter/enhancer in skeletal muscle within a LV context both in vitro and in vivo. Furthermore, the DES promoter/enhancer provides six- to eightfold greater expression per viral copy than the muscle-specific human muscle creatine kinase (CKM) promoter/enhancer. DES also confers a more reproducible and tissue-specific transgene expression profile compared to CKM and is therefore a highly attractive regulatory element for use in muscle gene therapy vectors.

  6. Targeting lentiviral vectors to antigen-specific immunoglobulins.

    Science.gov (United States)

    Ziegler, Leslie; Yang, Lili; Joo, Kye il; Yang, Haiguang; Baltimore, David; Wang, Pin

    2008-09-01

    Gene transfer into B cells by lentivectors can provide an alternative approach to managing B lymphocyte malignancies and autoreactive B cell-mediated autoimmune diseases. These pathogenic B cell populations can be distinguished by their surface expression of monospecific immunoglobulin. Development of a novel vector system to deliver genes to these specific B cells could improve the safety and efficacy of gene therapy. We have developed an efficient method to target lentivectors to monospecific immunoglobulin-expressing cells in vitro and in vivo. We were able to incorporate a model antigen CD20 and a fusogenic protein derived from the Sindbis virus as two distinct molecules into the lentiviral surface. This engineered vector could specifically bind to cells expressing surface immunoglobulin recognizing CD20 (alphaCD20), resulting in efficient transduction of target cells in a cognate antigen-dependent manner in vitro, and in vivo in a xenografted tumor model. Tumor suppression was observed in vivo, using the engineered lentivector to deliver a suicide gene to a xenografted tumor expressing alphaCD20. These results show the feasibility of engineering lentivectors to target immunoglobulin- specific cells to deliver a therapeutic effect. Such targeting lentivectors also could potentially be used to genetically mark antigen-specific B cells in vivo to study their B cell biology.

  7. Lentiviral vectors for the treatment of primary immunodeficiencies.

    Science.gov (United States)

    Farinelli, Giada; Capo, Valentina; Scaramuzza, Samantha; Aiuti, Alessandro

    2014-07-01

    In the last years important progress has been made in the treatment of several primary immunodeficiency disorders (PIDs) with gene therapy. Hematopoietic stem cell (HSC) gene therapy indeed represents a valid alternative to conventional transplantation when a compatible donor is not available and recent success confirmed the great potential of this approach. First clinical trials performed with gamma retroviral vectors were promising and guaranteed clinical benefits to the patients. On the other hand, the outcome of severe adverse events as the development of hematological abnormalities highlighted the necessity to develop a safer platform to deliver the therapeutic gene. Self-inactivating (SIN) lentiviral vectors (LVVs) were studied to overcome this hurdle through their preferable integration pattern into the host genome. In this review, we describe the recent advancements achieved both in vitro and at preclinical level with LVVs for the treatment of Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), ADA deficiency (ADA-SCID), Artemis deficiency, RAG1/2 deficiency, X-linked severe combined immunodeficiency (γchain deficiency, SCIDX1), X-linked lymphoproliferative disease (XLP) and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.

  8. Spatial confinement induces hairpins in nicked circular DNA

    Science.gov (United States)

    Japaridze, Aleksandre; Orlandini, Enzo; Smith, Kathleen Beth; Gmür, Lucas; Valle, Francesco; Micheletti, Cristian

    2017-01-01

    Abstract In living cells, DNA is highly confined in space with the help of condensing agents, DNA binding proteins and high levels of supercoiling. Due to challenges associated with experimentally studying DNA under confinement, little is known about the impact of spatial confinement on the local structure of the DNA. Here, we have used well characterized slits of different sizes to collect high resolution atomic force microscopy images of confined circular DNA with the aim of assessing the impact of the spatial confinement on global and local conformational properties of DNA. Our findings, supported by numerical simulations, indicate that confinement imposes a large mechanical stress on the DNA as evidenced by a pronounced anisotropy and tangent–tangent correlation function with respect to non-constrained DNA. For the strongest confinement we observed nanometer sized hairpins and interwound structures associated with the nicked sites in the DNA sequence. Based on these findings, we propose that spatial DNA confinement in vivo can promote the formation of localized defects at mechanically weak sites that could be co-opted for biological regulatory functions. PMID:28201616

  9. Complexity Results and the Growths of Hairpin Completions of Regular Languages (Extended Abstract)

    Science.gov (United States)

    Diekert, Volker; Kopecki, Steffen

    The hairpin completion is a natural operation on formal languages which has been inspired by molecular phenomena in biology and by DNA-computing. In 2009 we presented in [6] a (polynomial time) decision algorithm to decide regularity of the hairpin completion. In this paper we provide four new results: 1.) We show that the decision problem is NL-complete. 2.) There is a polynomial time decision algorithm which runs in time O(n8), this improves [6], which provided O(n^{20}). 3.) For the one-sided case (which is closer to DNA computing) the time is O(n2), only. 4.) The hairpin completion is unambiguous linear context-free. This result allows to compute the growth (generating function) of the hairpin completion and to compare it with the growth of the underlying regular language.

  10. Different Fluorophore Labeling Strategies and Designs Affect Millisecond Kinetics of DNA Hairpins

    Directory of Open Access Journals (Sweden)

    Andreas Hartmann

    2014-09-01

    Full Text Available Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have revealed unprecedented information at various time scales varying from milliseconds to seconds. The choice and the attachment of fluorophores is a pivotal requirement for single-molecule FRET experiments. One particularly well-studied millisecond conformational change is the opening and closing of DNA hairpin structures. In this study, we addressed the influence of base- and terminal-labeled fluorophores as well as the fluorophore DNA interactions on the extracted kinetic information of the DNA hairpin. Gibbs free energies varied from ∆G0 = −3.6 kJ/mol to ∆G0 = −0.2 kJ/mol for the identical DNA hairpin modifying only the labeling scheme and design of the DNA sample. In general, the base-labeled DNA hairpin is significantly destabilized compared to the terminal-labeled DNA hairpin and fluorophore DNA interactions additionally stabilize the closed state of the DNA hairpin. Careful controls and variations of fluorophore attachment chemistry are essential for a mostly undisturbed measurement of the underlying energy landscape of biomolecules.

  11. Lentiviral vectors can be used for full-length dystrophin gene therapy.

    Science.gov (United States)

    Counsell, John R; Asgarian, Zeinab; Meng, Jinhong; Ferrer, Veronica; Vink, Conrad A; Howe, Steven J; Waddington, Simon N; Thrasher, Adrian J; Muntoni, Francesco; Morgan, Jennifer E; Danos, Olivier

    2017-03-06

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

  12. Thermosensitivity of the reverse transcription process as an inactivation mechanism of lentiviral vectors.

    Science.gov (United States)

    Carmo, M; Dias, J D; Panet, A; Coroadinha, A S; Carrondo, M J T; Alves, P M; Cruz, P E

    2009-10-01

    Lentiviral vectors are an important tool for gene transfer research and gene therapy purposes. However, the low stability of these vectors affects their production, storage, and efficacy in preclinical and clinical settings. In the present work the mechanism underlying the thermosensitivity of lentiviral vectors was evaluated. For lentiviral vectors pseudotyped with amphotropic and RDpro envelopes, the capacity to perform reverse transcription was lost rapidly at 37 degrees C, in high correlation with the loss of infectivity. The vector with RDpro envelope presented a higher level of stability than that with amphotropic envelope for both the reverse transcription process and viral infectivity. Reverse transcriptase enzyme inactivation and viral template RNA degradation were not implicated in the loss of the viral capacity to perform reverse transcription. Furthermore, early entry steps in the infection process do not determine the rate of viral inactivation, as the amount of viral RNA and p24 protein entering the cells decreased slowly for both vectors. Taken together, it can be concluded that the reverse transcription process is thermolabile and thus determines the rate of lentiviral inactivation. Strategies to stabilize the reverse transcription process should be pursued to improve the applicability of lentiviral vectors in gene therapy.

  13. Packaging of HCV-RNA into lentiviral vector

    Energy Technology Data Exchange (ETDEWEB)

    Caval, Vincent [INSERM U966, Universite Francois Rabelais de Tours, Faculte de Medecine, 10 Bd. Tonnelle, 37000 Tours (France); Piver, Eric [INSERM U966, Universite Francois Rabelais de Tours, Faculte de Medecine, 10 Bd. Tonnelle, 37000 Tours (France); Service de Biochimie et Biologie Moleculaire, CHRU de Tours (France); Ivanyi-Nagy, Roland; Darlix, Jean-Luc [LaboRetro, ENS-Lyon INSERM, U758, 46 Allee d' Italie, 69364 Lyon (France); Pages, Jean-Christophe, E-mail: jean-christophe.pages@univ-tours.fr [INSERM U966, Universite Francois Rabelais de Tours, Faculte de Medecine, 10 Bd. Tonnelle, 37000 Tours (France); Service de Biochimie et Biologie Moleculaire, CHRU de Tours (France)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Description of HCV-RNA Core-D1 interactions. Black-Right-Pointing-Pointer In vivo evaluation of the packaging of HCV genome. Black-Right-Pointing-Pointer Determination of the role of the three basic sub-domains of D1. Black-Right-Pointing-Pointer Heterologous system involving HIV-1 vector particles to mobilise HCV genome. Black-Right-Pointing-Pointer Full length mobilisation of HCV genome and HCV-receptor-independent entry. -- Abstract: The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.

  14. Highly efficient retrograde gene transfer into motor neurons by a lentiviral vector pseudotyped with fusion glycoprotein.

    Directory of Open Access Journals (Sweden)

    Miyabi Hirano

    Full Text Available The development of gene therapy techniques to introduce transgenes that promote neuronal survival and protection provides effective therapeutic approaches for neurological and neurodegenerative diseases. Intramuscular injection of adenoviral and adeno-associated viral vectors, as well as lentiviral vectors pseudotyped with rabies virus glycoprotein (RV-G, permits gene delivery into motor neurons in animal models for motor neuron diseases. Recently, we developed a vector with highly efficient retrograde gene transfer (HiRet by pseudotyping a human immunodeficiency virus type 1 (HIV-1-based vector with fusion glycoprotein B type (FuG-B or a variant of FuG-B (FuG-B2, in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G. We have also developed another vector showing neuron-specific retrograde gene transfer (NeuRet with fusion glycoprotein C type, in which the short C-terminal segment of the extracellular domain and transmembrane/cytoplasmic domains of RV-G was substituted with the corresponding regions of VSV-G. These two vectors afford the high efficiency of retrograde gene transfer into different neuronal populations in the brain. Here we investigated the efficiency of the HiRet (with FuG-B2 and NeuRet vectors for retrograde gene transfer into motor neurons in the spinal cord and hindbrain in mice after intramuscular injection and compared it with the efficiency of the RV-G pseudotype of the HIV-1-based vector. The main highlight of our results is that the HiRet vector shows the most efficient retrograde gene transfer into both spinal cord and hindbrain motor neurons, offering its promising use as a gene therapeutic approach for the treatment of motor neuron diseases.

  15. Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

    Czech Academy of Sciences Publication Activity Database

    Sarnová, Lenka; Malík, Radek; Sedláček, Radislav; Svoboda, Petr

    2010-01-01

    Roč. 9, č. 8 (2010), s. 1-10 ISSN 1477-5751 R&D Projects: GA MŠk ME09039 Grant - others:EMBO SDIG(DE) project 1483 Institutional research plan: CEZ:AV0Z50520514 Keywords : transgenic RNAi * shRNA * oocyte Subject RIV: EB - Genetics ; Molecular Biology http://www.jnrbm.com/content/9/1/8

  16. Application of short hairpin RNAs (shRNAs) to study gene function in ...

    African Journals Online (AJOL)

    Yomi

    2010-12-29

    Dec 29, 2010 ... Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W (2002). Human. Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. The EMBO J. 21(21): 5875-5885. Zhang H, Kolb FA, Jaskiewicz L, Westhof E, Filipowicz W (2004). Single processing center models for human Dicer and ...

  17. Size estimation of complexes consisting of hairpin DNAs bound to an assembler-strand. [Abstract only

    Science.gov (United States)

    Baumann, Ulrich

    1994-01-01

    In connection with a model of a primitive translation based entirely on nucleic acids the structural requirements and interactions of small nucleic acids were studied. As shown previously hairpins bind to a complementary single-strand by base pairs of their loop nucleotides if the loop contains at least five nucleotides (Baumann et al 1987). Here, the question is approached to which degree a single-strand, the assembler-strand, is occupied with hairpin molecules. A gapless occupation would allow the close proximity necessary for the assumed peptide bond formation by hairpins bearing an amino acid at their 3'-end. Oligo(dC)(sub n), n = 12, 15, 18, and the investigated hairpins form complexes which are detectable under nondenaturing electrophoretic conditions. The size range is estimated to be 3-4 hairpin molecules to one assembler molecule when oligo(dC)(sub n), n = 15, 18, is used. Due to the limitation of the method, dependence of the migration velocity on the shape which is unique and thus limits the use of marker nucleic acids, and due to the uncertainty if the ends of the assembler form base pairs, a gapless occupation of the assembler is conceivable but not proven.

  18. An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation.

    Directory of Open Access Journals (Sweden)

    Ke Shi

    Full Text Available Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting-rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.

  19. Constructing multi-resolution Markov State Models (MSMs) to elucidate RNA hairpin folding mechanisms.

    Science.gov (United States)

    Huang, Xuhui; Yao, Yuan; Bowman, Gregory R; Sun, Jian; Guibas, Leonidas J; Carlsson, Gunnar; Pande, Vijay S

    2010-01-01

    Simulating biologically relevant timescales at atomic resolution is a challenging task since typical atomistic simulations are at least two orders of magnitude shorter. Markov State Models (MSMs) provide one means of overcoming this gap without sacrificing atomic resolution by extracting long time dynamics from short simulations. MSMs coarse grain space by dividing conformational space into long-lived, or metastable, states. This is equivalent to coarse graining time by integrating out fast motions within metastable states. By varying the degree of coarse graining one can vary the resolution of an MSM; therefore, MSMs are inherently multi-resolution. Here we introduce a new algorithm Super-level-set Hierarchical Clustering (SHC), to our knowledge, the first algorithm focused on constructing MSMs at multiple resolutions. The key insight of this algorithm is to generate a set of super levels covering different density regions of phase space, then cluster each super level separately, and finally recombine this information into a single MSM. SHC is able to produce MSMs at different resolutions using different super density level sets. To demonstrate the power of this algorithm we apply it to a small RNA hairpin, generating MSMs at four different resolutions. We validate these MSMs by showing that they are able to reproduce the original simulation data. Furthermore, long time folding dynamics are extracted from these models. The results show that there are no metastable on-pathway intermediate states. Instead, the folded state serves as a hub directly connected to multiple unfolded/misfolded states which are separated from each other by large free energy barriers.

  20. Effect of DNA hairpin loops on the twist of planar DNA origami tiles.

    Science.gov (United States)

    Li, Zhe; Wang, Lei; Yan, Hao; Liu, Yan

    2012-01-31

    The development of scaffolded DNA origami, a technique in which a long single-stranded viral genome is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides, represents an important milestone in DNA nanotechnology. Recent findings have revealed that two-dimensional (2D) DNA origami structures based on the original design parameters adopt a global twist with respect to the tile plane, which may be because the conformation of the constituent DNA (10.67 bp/turn) deviates from the natural B-type helical twist (10.4 bp/turn). Here we aim to characterize the effects of DNA hairpin loops on the overall curvature of the tile and explore their ability to control, and ultimately eliminate any unwanted curvature. A series of dumbbell-shaped DNA loops were selectively displayed on the surface of DNA origami tiles with the expectation that repulsive interactions among the neighboring dumbbell loops and between the loops and the DNA origami tile would influence the structural features of the underlying tiles. A systematic, atomic force microscopy (AFM) study of how the number and position of the DNA loops influenced the global twist of the structure was performed, and several structural models to explain the results were proposed. The observations unambiguously revealed that the first generation of rectangular shaped origami tiles adopt a conformation in which the upper right (corner 2) and bottom left (corner 4) corners bend upward out of the plane, causing linear superstructures attached by these corners to form twisted ribbons. Our experimental observations are consistent with the twist model predicted by the DNA mechanical property simulation software CanDo. Through the systematic design and organization of various numbers of dumbbell loops on both surfaces of the tile, a nearly planar rectangular origami tile was achieved. © 2011 American Chemical Society

  1. Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

    DEFF Research Database (Denmark)

    Nielsen, Troels T; Jakobsson, Johan; Rosenqvist, Nina

    2009-01-01

    BACKGROUND: Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are bounda...

  2. Mouse transplant models for evaluating the oncogenic risk of a self-inactivating XSCID lentiviral vector.

    Directory of Open Access Journals (Sweden)

    Sheng Zhou

    Full Text Available Hematopoietic stem cell gene therapy requires the use of integrating retroviral vectors in order to stably transmit a therapeutic gene to mature blood cells. Human clinical trials have shown that some vector integration events lead to disrupted regulation of proto-oncogenes resulting in disordered hematopoiesis including T-cell leukemia. Newer vectors have been designed to decrease the incidence of these adverse events but require appropriate pre-clinical assays to demonstrate safety. We have used two distinct mouse serial transplant assays to evaluate the safety of a self-inactivating lentiviral vector intended for use in X-linked severe combined immunodeficiency (XSCID gene therapy trials. These experiments entailed 28 months of total follow-up and included 386 mice. There were no cases in which the XSCID lentiviral vector clearly caused hematopoietic malignancies, although a single case of B cell malignancy was observed that contained the lentiviral vector as a likely passenger event. In contrast, a SFFV-DsRed γ-retroviral vector resulted in clonal transformation events in multiple secondary recipients. Non-specific pathology not related to vector insertions was noted including T cell leukemias arising from irradiated recipient cells. Overall, this comprehensive study of mouse transplant safety assays demonstrate the relative safety of the XSCID lentiviral vector but also highlight the limitations of these assays.

  3. Direct gene transfer in the Gottingen minipig CNS using stereotaxic lentiviral microinjections

    DEFF Research Database (Denmark)

    GLUD, AN; Hedegaard, Claus; nielsen, MS

    2010-01-01

    We aim to induce direct viral mediated gene transfer in the substantia nigra (SN) of the Gottingen minipig using MRI guided stereotaxic injections of lentiviral vectors encoding enhanced green fluorescent protein (EGFP). Nine female Gottingen minipigs were injected unilaterally into the SN with 6...

  4. Hairpin formation within the enhancer region of the human enkephalin gene

    Energy Technology Data Exchange (ETDEWEB)

    McMurray, C.T.; Douglass, J.O. (Oregon Health Sciences Univ., Portland (United States)); Wilson, W.D. (Georgia State Univ., Atlanta (United States))

    1991-01-15

    The 3{prime},5{prime}-cyclic adenosine monophosphate (cAMP)-inducible enhancer of the human enkephaline gene is located within an imperfect palindrom of 23 base pairs. The authors have found that a 23-base-pair oligonucleotide duplex containing the enhancer undergoes a reversible conformational transition from the duplex to two individual hairpin structures each formed from one strand of the duplex. Each individual hairpin forms with mismatched base pairs, one containing two GT pairs and the other containing two AC pairs. The conformational transition is stabilized by proton transfer to the hairpin containing AC mismatched pairs. The unique physical and thermodynamic properties of the enkephalin enhancer DNA suggest a model in which DNA secondary structure within the enhancer region plays and active role incAMP-inducible activation of the human enkephalin gene via formation of cruciform structures.

  5. Dynamic force spectroscopy of DNA hairpins: I. Force kinetics and free energy landscapes

    International Nuclear Information System (INIS)

    Mossa, A; Manosas, M; Forns, N; Huguet, J M; Ritort, F

    2009-01-01

    We investigate the thermodynamics and kinetics of DNA hairpins that fold/unfold under the action of applied mechanical force. We introduce the concept of the molecular free energy landscape and derive simplified expressions for the force dependent Kramers–Bell rates. To test the theory we have designed a specific DNA hairpin sequence that shows two-state cooperative folding under mechanical tension and carried out pulling experiments using optical tweezers. We show how we can determine the parameters that characterize the molecular free energy landscape of such sequences from rupture force kinetic studies. Finally we combine such kinetic studies with experimental investigations of the Crooks fluctuation relation to derive the free energy of formation of the hairpin at zero force

  6. Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)

    2012-09-05

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

  7. DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

    DEFF Research Database (Denmark)

    Franch, Oskar; Iacovelli, Federico; Falconi, Mattia

    2016-01-01

    In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our...... previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation...

  8. New World feline APOBEC3 potently controls inter-genus lentiviral transmission.

    Science.gov (United States)

    Konno, Yoriyuki; Nagaoka, Shumpei; Kimura, Izumi; Yamamoto, Keisuke; Kagawa, Yumiko; Kumata, Ryuichi; Aso, Hirofumi; Ueda, Mahoko Takahashi; Nakagawa, So; Kobayashi, Tomoko; Koyanagi, Yoshio; Sato, Kei

    2018-04-10

    The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) gene family appears only in mammalian genomes. Some A3 proteins can be incorporated into progeny virions and inhibit lentiviral replication. In turn, the lentiviral viral infectivity factor (Vif) counteracts the A3-mediated antiviral effect by degrading A3 proteins. Recent investigations have suggested that lentiviral vif genes evolved to combat mammalian APOBEC3 proteins, and have further proposed that the Vif-A3 interaction may help determine the co-evolutionary history of cross-species lentiviral transmission in mammals. Here we address the co-evolutionary relationship between two New World felids, the puma (Puma concolor) and the bobcat (Lynx rufus), and their lentiviruses, which are designated puma lentiviruses (PLVs). We demonstrate that PLV-A Vif counteracts the antiviral action of APOBEC3Z3 (A3Z3) of both puma and bobcat, whereas PLV-B Vif counteracts only puma A3Z3. The species specificity of PLV-B Vif is irrespective of the phylogenic relationships of feline species in the genera Puma, Lynx and Acinonyx. We reveal that the amino acid at position 178 in the puma and bobcat A3Z3 is exposed on the protein surface and determines the sensitivity to PLV-B Vif-mediated degradation. Moreover, although both the puma and bobcat A3Z3 genes are polymorphic, their sensitivity/resistance to PLV Vif-mediated degradation is conserved. To the best of our knowledge, this is the first study suggesting that the host A3 protein potently controls inter-genus lentiviral transmission. Our findings provide the first evidence suggesting that the co-evolutionary arms race between lentiviruses and mammals has occurred in the New World.

  9. Vibrational spectral simulation for peptides of mixed secondary structure: Method comparisons with the Trpzip model hairpin

    Czech Academy of Sciences Publication Activity Database

    Bouř, Petr; Keiderling, T. A.

    2005-01-01

    Roč. 109, - (2005), 23687-23697 ISSN 1089-5647 R&D Projects: GA AV ČR(CZ) IAA4055104 Grant - others:NSF(US) CHE03-16014 Institutional research plan: CEZ:AV0Z40550506 Keywords : VCD * trpzin model hairpin * peptides Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.679, year: 2003

  10. Folding of single-stranded DNA quadruplexes containing an autonomously stable mini-hairpin loop.

    Science.gov (United States)

    Balkwill, Graham D; Garner, Thomas P; Searle, Mark S

    2009-05-01

    The single-stranded DNA quadruplex motif TG(3)-L(1)-G(3)-L(2)-G(3)-L(3)-G(3)T (where L(1), L(2) and L(3) are the three loop sequences) was used as a template for probing the effects of the loop sequences on stability and folding topology. An autonomously stable mini-hairpin sequence (ACGTAGT) was inserted into the central loop (L(2)) of different sequences with intrinsic propensities to form either parallel or anti-parallel structures. Single nucleotides (T) at positions L(1) and L(3) strongly favour the formation of a parallel structure with the L(2) hairpin insert affecting stability in the same way as a T(7) loop. However, in the context of an anti-parallel quadruplex with T(3) loops in positions L(1) and L(3), the mini-hairpin in the central loop forms a stable structure which enhances the T(m) of the quadruplex by approximately 10 degrees C when compared with the T(7) insert. The CD and UV melting data show that base pairing interactions within the ACGTAGT hairpin loop sequence, when accommodated as a diagonal loop in an anti-parallel structure, can enhance stability and lead to novel quadruplex structures, adding complexity to the folding landscape and expanding the potential repertoire of sequences that are able to regulate gene expression in vivo.

  11. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

    Directory of Open Access Journals (Sweden)

    Gregory Upert

    2012-01-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

  12. Hairpin-like fluorescent probe for imaging of NF-κB transcription factor activity.

    Science.gov (United States)

    Metelev, Valeri; Zhang, Surong; Tabatadze, David; Bogdanov, Alexei

    2011-04-20

    Three oligodeoxyribonucleotides (ODN) covalently labeled with near-infrared (NIR) fluorochromes were synthesized and characterized with a goal of comparing in vitro a hairpin-based and a duplex-based FRET probe designed for the detection of human recombinant NF-κB p50/p65 heterodimer binding to DNA. Using deoxyguanosine phosphoramidite with a phosphorus-linked aminoethylene (diethylene glycol) hydrophilic linker, we synthesized ODNs with internucleoside reactive sites. The hairpin loop amino linker was modified with IRDye 800CW (FRET acceptor), and the 3'-end was modified with Cy5.5 (FRET donor) using a dithio-linker. To obtain a duplex probe, we conjugated Cy5.5 and 800CW to complementary strands at the distance of ten base pairs in the resultant duplex. No quenching of dyes was observed in either probe. The FRET efficiency was higher in the duplex (71%) than in the hairpin (56%) due to a more favorable distance between the donor and the acceptor. However, the hairpin design allowed more precise ratiometric measurement of fluorescence intensity changes as a result of NF-κB p50/p65 binding to the probe. We determined that as a result of binding there was a statistically significant increase of fluorescence intensity of Cy5.5 (donor) due to a decrease of FRET if normalized by 800CW intensity measured independently of FRET. We conclude that the hairpin based probe design allows for the synthesis of a dual fluorescence imaging probe that renders signal changes that are simple to interpret and stoichiometrically correct for detecting transcription factor-DNA interactions.

  13. Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors.

    Science.gov (United States)

    Sachdeva, Rohit; Jönsson, Marie E; Nelander, Jenny; Kirkeby, Agnete; Guibentif, Carolina; Gentner, Bernhard; Naldini, Luigi; Björklund, Anders; Parmar, Malin; Jakobsson, Johan

    2010-06-22

    In this study, we have used a microRNA-regulated lentiviral reporter system to visualize and segregate differentiating neuronal cells in pluripotent cultures. Efficient suppression of transgene expression, specifically in undifferentiated pluripotent cells, was achieved by using a lentiviral vector expressing a fluorescent reporter gene regulated by microRNA-292. Using this strategy, it was possible to track progeny from murine ES, human ES cells, and induced pluripotent stem cells as they differentiated toward the neural lineage. In addition, this strategy was successfully used to FACS purify neuronal progenitors for molecular analysis and transplantation. FACS enrichment reduced tumor formation and increased survival of ES cell-derived neuronal progenitors after transplantation. The properties and versatility of the microRNA-regulated vectors allows broad use of these vectors in stem cell applications.

  14. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    Directory of Open Access Journals (Sweden)

    Dismuke Adria D

    2009-07-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  15. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    Directory of Open Access Journals (Sweden)

    Kohn Aimee

    2009-01-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  16. Lentiviral vectors in neurodegenrative disorders - Aspects in gene therapy and disease models

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup

    2009-01-01

    Neurodegenerative disorders remain a complex group of diseases (i.e. Huntington's disease, HD) that are characterized by progressive loss of neurons resulting in movement disorders, cognitive decline, dementia and death. There is no cure for these diseases and treatment relies on symptomatic relief......, which is most often only satisfactory in the initial phase of the disease. Gene therapy is a novel treatment strategy intended to treat or alleviate disease by genetically modifying cells by introducing nucleic acids into the cells. Lentiviral vectors hold great promise as gene transfer vectors...... and in vivo. Robust gene knock-down was shown using a ubiquitous promoter (CMV) and for the first time neuron specific RNAi was obtained using a neuron specific promoter (NSE). Furthermore, optimization of lentiviral vectors was conducted using an insulator element (cHS4) in order to enhance transgene...

  17. Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting.

    Directory of Open Access Journals (Sweden)

    Heather M Marshall

    2007-12-01

    Full Text Available To replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. Lentiviral integration is favored in active transcription units, which allows efficient viral gene expression after integration, but the mechanisms directing integration targeting are incompletely understood. A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN, and has been reported to be important for HIV infectivity and integration targeting.Here we report studies of lentiviral integration targeting in 1 human cells with intensified RNAi knockdowns of PSIP1/LEDGF/p75, and 2 murine cells with homozygous gene trap mutations in the PSIP1/LEDGF/p75 locus. Infections with vectors derived from equine infections anemia virus (EIAV and HIV were compared. Integration acceptor sites were analyzed by DNA bar coding and pyrosequencing.In both PSIP1/LEDGF/p75-depleted cell lines, reductions were seen in lentiviral infectivity compared to controls. For the human cells, integration was reduced in transcription units in the knockdowns, and this reduction was greater than in our previous studies of human cells less completely depleted for PSIP1/LEDGF/p75. For the homozygous mutant mouse cells, similar reductions in integration in transcription units were seen, paralleling a previous study of a different mutant mouse line. Integration did not become random, however-integration in transcription units in both cell types was still favored, though to a reduced degree. New trends also appeared, including favored integration near CpG islands. In addition, we carried out a bioinformatic study of 15 HIV integration site data sets in different cell types, which showed that the frequency of integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression.

  18. Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

    OpenAIRE

    Warlich, Eva; Kuehle, Johannes; Cantz, Tobias; Brugman, Martijn H; Maetzig, Tobias; Galla, Melanie; Filipczyk, Adam A; Halle, Stephan; Klump, Hannes; Schöler, Hans R; Baum, Christopher; Schroeder, Timm; Schambach, Axel

    2011-01-01

    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iP...

  19. Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and M?ller cells

    OpenAIRE

    Semple-Rowland, Susan L.; Coggin, William E.; Geesey, Mero; Eccles, Kristofer S.; Abraham, Leah; Pachigar, Krunal; Ludlow, Rachel; Khani, Shahrokh C.; Smith, W. Clay

    2010-01-01

    Purpose Growing evidence suggests that successful treatment of many inherited photoreceptor diseases will require multi-protein therapies that not only correct the genetic defects linked to these diseases but also slow or halt the related degenerative phenotypes. To be effective, it is likely that therapeutic protein expression will need to be targeted to specific cell types. The purpose of this study was to develop dual-promoter lentiviral vectors that target expression of two proteins to re...

  20. Elements of lentiviral vector design toward gene therapy for treating mucopolysaccharidosis I

    Directory of Open Access Journals (Sweden)

    Li Ou

    2016-09-01

    Full Text Available Mucopolysaccharidosis type I (MPS I is a lysosomal disease caused by α-l-iduronidase (IDUA deficiency and accumulation of glycosaminoglycans (GAG. Lentiviral vector encoding correct IDUA cDNA could be used for treating MPS I. To optimize the lentiviral vector design, 9 constructs were designed by combinations of various promoters, enhancers, and codon optimization. After in vitro transfection into 293FT cells, 5 constructs achieved the highest IDUA activities (5613 to 7358 nmol/h/mg protein. These 5 candidate vectors were then tested by injection (1 × 107 TU/g into neonatal MPS I mice. After 30 days, one vector, CCEoIDW, achieved the highest IDUA levels: 2.6% of wildtype levels in the brain, 9.9% in the heart, 200% in the liver and 257% in the spleen. CCEoIDW achieved the most significant GAG reduction: down 49% in the brain, 98% in the heart, 100% in the liver and 95% in the spleen. Further, CCEoIDW had the lowest transgene frequency, especially in the gonads (0.03 ± 0.01 copies/100 cells, reducing the risk of insertional mutagenesis and germ-line transmission. Therefore, CCEoIDW is selected as the optimal lentiviral vector for treating MPS I disease and will be applied in large animal preclinical studies. Further, taken both in vitro and in vivo comparisons together, codon optimization, use of EF-1α promoter and woodchuck hepatitis virus posttranscriptional response element (WPRE could enhance transgene expression. These results provided a better understanding of factors contributing efficient transgene expression in lentiviral gene therapies.

  1. [Construction and identification of Nogo extra cellular peptide residues 1-40 gene lentiviral vector].

    Science.gov (United States)

    Yuan, Haifeng; Song, Yueming; Liu, Hao; Zhou, Chunguang; Kong, Qingquan; Liu, Liming; Gong, Quan

    2012-02-01

    To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40 (NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. The DNA fragment of NEP1-40 coding sequence was amplified by PCR with designed primer from the cDNA library including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40, and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene delivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation, and NEP1-40 protein expression was detected in 293T cells by Western blot. The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP 1-40.

  2. Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency.

    Science.gov (United States)

    De Ravin, Suk See; Wu, Xiaolin; Moir, Susan; Anaya-O'Brien, Sandra; Kwatemaa, Nana; Littel, Patricia; Theobald, Narda; Choi, Uimook; Su, Ling; Marquesen, Martha; Hilligoss, Dianne; Lee, Janet; Buckner, Clarissa M; Zarember, Kol A; O'Connor, Geraldine; McVicar, Daniel; Kuhns, Douglas; Throm, Robert E; Zhou, Sheng; Notarangelo, Luigi D; Hanson, I Celine; Cowan, Mort J; Kang, Elizabeth; Hadigan, Coleen; Meagher, Michael; Gray, John T; Sorrentino, Brian P; Malech, Harry L; Kardava, Lela

    2016-04-20

    X-linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations inIL2RGencoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1. Copyright © 2016, American Association for the Advancement of Science.

  3. Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer.

    Science.gov (United States)

    Chandrashekran, Anil; Casimir, Colin; Dibb, Nick; Readhead, Carol; Winston, Robert

    2016-01-01

    Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice.

  4. A lentiviral gene therapy strategy for the in vitro production of feline erythropoietin.

    Directory of Open Access Journals (Sweden)

    Natalia Vapniarsky

    Full Text Available Nonregenerative anemia due to chronic renal failure is a common problem in domestic cats. Unfortunately, administration of recombinant human erythropoietin often only improves anemia temporarily due to antibody development. In this in vitro study, feline erythropoietin cDNA was cloned from feline renal tissue and utilized in the construction of a replication-defective lentiviral vector. The native recombinant feline erythropoietin (rfEPO sequence was confirmed by sequencing. Upon viral vector infection of human 293H cells, Crandall Renal Feline Kidney cell line and primary feline peripheral blood mononuclear cells, bioactive rfEPO protein was produced. The presence of cellular rfEPO cDNA was confirmed by standard PCR, production of abundant rfEPO mRNA was confirmed by real-time PCR, and secretion of rfEPO protein was demonstrated by Western blot analyses, while rfEPO protein bioactivity was confirmed via an MTT proliferation bioassay. This in vitro study demonstrates the feasibility of a replication-defective lentiviral vector delivery system for the in vitro production of biologically active feline erythropoietin. Anemic cats with chronic renal failure represent a potential in vivo application of a lentiviral gene therapy system.

  5. Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Daniel C Farley

    Full Text Available It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02. VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

  6. A guide to approaching regulatory considerations for lentiviral-mediated gene therapies.

    Science.gov (United States)

    White, Michael; Whittaker, Roger; Stoll, Elizabeth Ann

    2017-06-12

    Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well-characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is non-pathogenic and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations, and how they are administered in the United Kingdom, although many of the principles will be similar for other regions including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarises the extant regulatory guidance for gene therapies, categorised as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

  7. β-hairpin-mediated formation of structurally distinct multimers of neurotoxic prion peptides.

    Directory of Open Access Journals (Sweden)

    Andrew C Gill

    Full Text Available Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109-122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109-122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109-122 peptide (A117V, associated with familial prion disease increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106-126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an

  8. β-Hairpin-Mediated Formation of Structurally Distinct Multimers of Neurotoxic Prion Peptides

    Science.gov (United States)

    Gill, Andrew C.

    2014-01-01

    Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A117V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new

  9. [Construction of recombinant lentiviral vector of Tie2-RNAi and its influence on malignant melanoma cells in vitro].

    Science.gov (United States)

    Shan, Xiu-ying; Liu, Zhao-liang; Wang, Biao; Guo, Guo-xiang; Wang, Mei-shui; Zhuang, Fu-lian; Cai, Chuan-shu; Zhang, Ming-feng; Zhang, Yan-ding

    2011-07-01

    To construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells. Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P0.05) between the two lentiviral vectors of Tie2-RNAi. Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.

  10. Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation

    Science.gov (United States)

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates. PMID:18375754

  11. Combined High-Speed 3D Scalar and Velocity Reconstruction of Hairpin Vortex

    Science.gov (United States)

    Sabatino, Daniel; Rossmann, Tobias; Zhu, Xuanyu; Thorsen, Mary

    2017-11-01

    The combination of 3D scanning stereoscopic particle image velocimetry (PIV) and 3D Planar Laser Induced Fluorescence (PLIF) is used to create high-speed three-dimensional reconstructions of the scalar and velocity fields of a developing hairpin vortex. The complete description of the regenerating hairpin vortex is needed as transitional boundary layers and turbulent spots are both comprised of and influenced by these vortices. A new high-speed, high power, laser-based imaging system is used which enables both high-speed 3D scanning stereo PIV and PLIF measurements. The experimental system uses a 250 Hz scanning mirror, two high-speed cameras with a 10 kHz frame rate, and a 40 kHz pulsed laser. Individual stereoscopic PIV images and scalar PLIF images are then reconstructed into time-resolved volumetric velocity and scalar data. The results from the volumetric velocity and scalar fields are compared to previous low-speed tomographic PIV data and scalar visualizations to determine the accuracy and fidelity of the high-speed diagnostics. Comparisons between the velocity and scalar field during hairpin development and regeneration are also discussed. Supported by the National Science Foundation under Grant CBET-1531475, Lafayette College,and the McCutcheon Foundation.

  12. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

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    Das Atze T

    2012-07-01

    Full Text Available Abstract Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

  13. 2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

    International Nuclear Information System (INIS)

    El-Yazbi, Amira F.; Loppnow, Glen R.

    2012-01-01

    Highlights: ► Molecular beacon with 2AP bases detects DNA damage in a simple mix-and-read assay. ► Molecular beacons with 2AP bases detect damage at a 17.2 nM limit of detection. ► The 2AP molecular beacon is linear over a 0–3.5 μM concentration range for damage. - Abstract: Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R 2 = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.

  14. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate

    Science.gov (United States)

    Tan, Elliot; Wilson, Timothy J.; Nahas, Michelle K.; Clegg, Robert M.; Lilley, David M. J.; Ha, Taekjip

    2003-01-01

    The natural form of the hairpin ribozyme comprises two major structural elements: a four-way RNA junction and two internal loops carried by adjacent arms of the junction. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. We have used single-molecule spectroscopy to show that the natural form fluctuates among three distinct states: the folded state and two additional, rapidly interconverting states (proximal and distal) that are inherited from the junction. The proximal state juxtaposes the two loop elements, thereby increasing the probability of their interaction and thus accelerating folding by nearly three orders of magnitude and allowing the ribozyme to fold rapidly in physiological conditions. Therefore, the hairpin ribozyme exploits the dynamics of the junction to facilitate the formation of the active site from its other elements. Dynamic interplay between structural elements, as we demonstrate for the hairpin ribozyme, may be a general theme for other functional RNA molecules. PMID:12883002

  15. Conformational dynamics of DNA hairpins at millisecond resolution obtained from analysis of single-molecule FRET histograms.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Berger, Yaron; Liber, Miran; Nir, Eyal

    2013-12-19

    Here we provide high resolution study of DNA hairpin dynamics achieved by probability distribution analysis (PDA) of diffusion-based single-molecule Förster resonance energy transfer (sm-FRET) histograms. The opening and closing rates of three hairpins both free and attached to DNA origami were determined. The agreement with rates previously obtained using the total internal reflection (TIRF) technique and between free hairpins and hairpins attached to origami validated the PDA and demonstrated that the origami had no influence on the hairpin dynamics. From comparison of rates of four DNA hairpins, differing only in stem sequence, and from comparison with rates calculated using nearest-neighbor method and standard transition state theory, we conclude that the unfolding reaction resembles that of melting of DNA duplex with a corresponding sequence and that the folding reaction depends on counterion concentration and not on stem sequence. Our validation and demonstration of the PDA method will encourage its implementation in future high-resolution dynamic studies of freely diffusing biomolecules.

  16. Ex-Vivo Gene Therapy Using Lentiviral Mediated Gene Transfer Into Umbilical Cord Blood Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Hanieh Jalali

    2016-02-01

    Full Text Available Background Introduction of therapeutic genes into the injured site of nervous system can be achieved using transplantation of cellular vehicles containing desired gene. To transfer exogenous genes into the cellular vehicles, lentiviral vectors are one of interested vectors because of advantages such high transduction efficiency of dividing and non-dividing cells. Unrestricted somatic stem cells are subclasses of umbilical cord blood derived stem cells which are appreciate candidates to use as cellular vehicles for ex vivo gene therapy of nervous system. Objectives In current study we investigated the effect of lentiviral vector transduction on the neuronal related features of unrestricted somatic stem cells to indicate the probable and unwanted changes related to transduction procedure. Materials and Methods In this experimental study, lentiviral vector containing green fluorescent protein (GFP were transduced into unrestricted somatic stem cells and its effect was investigated with using MTT assay, qPCR and immunohistochemistry techniques. For statistical comparison of real time PCR results, REST software (2009, Qiagen was used. Results Obtained results showed lentiviral vector transduction did not have cytotoxic effects on unrestricted somatic stem cells and did not change neuronal differentiation capacity of them as well the expression of some neuronal related genes and preserved them in multilineage situation. Conclusions In conclusion, we suggested that lentiviral vectors could be proper vectors to transfer therapeutic gene into unrestricted somatic stem cells to provide a cellular vehicle for ex vivo gene therapy of nervous system disorders.

  17. Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

    Directory of Open Access Journals (Sweden)

    Michele E Murphy

    2016-01-01

    Full Text Available Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform. Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method's specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector.

  18. Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels.

    Directory of Open Access Journals (Sweden)

    Zifu Zhang

    Full Text Available Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13-15 (HH13-15. A total of 80 embryos were injected and 48 G0 chimeras (60% were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR, indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%. We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1% were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7% were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13-15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection.

  19. [RelB silencing in mouse bone-marrow derived dendritic cells mediated by lentiviral vector].

    Science.gov (United States)

    Bao, Jie; Wang, Qian; Zheng, Lei; Qiu, Yu-rong; Zeng, Fang-yin; Yang, Chun-li; Huang, Xian-zhang

    2008-09-01

    To silence RelB gene in mouse bone-marrow derived dendritic cells (DC) utilizing lentiviral vector, a novel tolerogenic dendritic cell with a relatively low expression level RelB was constructed and a new way to treat and prevent autoimmune diseases was explored. Interferential targeting sequence R5 of RelB in mice was designed, synthesized and cloned into lentiviral vectors. Together with viral packaging materials were co-cultured in 293FT cell line to package lentiviral vector. Supernatant fluids were harvested, then virus titer detected. Mouse bone marrow derived DCs were infected by lentivirus particle. RelB gene expression level was detected by RT-PCR and immunofluorescence staining and analyzed by software of geo pro. There are three experiment control groups including immature DC, mature DC and DC infected by a negative independent control of T6. A similar RelB expression was detected by RT-PCR and immunofluorescence staining assay between DC infected virus R5 and immature DC, but was lower than that of mature DC. Significant difference in statistics P < 0.05. A similar RelB expression was detected by RT-PCR and immunofluorescence staining approaches between DC infected virus T6 and mature DC, but was higher than that of immature DC. Significant difference in statistics P < 0.05. RelB gene expressed by mouse bone marrow derived DC was silenced by Lentivirus vector effectively. The lentivirus vector with a low immunogenicity can be used to immunotherapy in vivo and overcome difficult transfection problem of primary DC. A new viral vector of DC immunotherapy can be obtained.

  20. Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung [School of Medicine, Chonnam National University, Gwangju (Korea, Republic of); Kim, Yeon Soo [Schoole of Medicine, Inje University, Seoul (Korea, Republic of)

    2004-07-01

    For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells.

  1. Feline immunodeficiency virus cross-species transmission: Implications for emergence of new lentiviral infections

    Science.gov (United States)

    Lee, Justin; Malmberg, Jennifer L.; Wood, Britta A.; Hladky, Sahaja; Troyer, Ryan; Roelke, Melody; Cunningham, Mark W.; McBride, Roy; Vickers, Winston; Boyce, Walter; Boydston, Erin E.; Serieys, Laurel E.K.; Riley, Seth P D; Crooks, Kevin R.; VandeWoude, Sue

    2016-01-01

    Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of HIV emergence following human exposures to SIV, understanding processes that promote successful cross-species lentiviral transmissions is highly relevant. We have previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) in a small number of animals in California and Florida. In this study we investigate host-specific selection pressures, within-host viral fitness, and inter- vs. intra-species transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analysis of proviral and viral RNA levels demonstrates that PLVA fitness is severely restricted in mountain lions compared to bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but did not detect selection among twenty PLVA isolates from bobcats. These findings support that PLVA is a bobcat-adapted virus, which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to emergence of HIV from nonhuman primates.IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which outcome will occur are complex, and the risk of new virus emergence is therefore difficult to predict. Here

  2. Feline Immunodeficiency Virus Cross-Species Transmission: Implications for Emergence of New Lentiviral Infections.

    Science.gov (United States)

    Lee, Justin; Malmberg, Jennifer L; Wood, Britta A; Hladky, Sahaja; Troyer, Ryan; Roelke, Melody; Cunningham, Mark; McBride, Roy; Vickers, Winston; Boyce, Walter; Boydston, Erin; Serieys, Laurel; Riley, Seth; Crooks, Kevin; VandeWoude, Sue

    2017-03-01

    Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of human immunodeficiency virus (HIV) emergence following human exposures to simian immunodeficiency virus (SIV), an understanding of processes that promote successful cross-species lentiviral transmissions is highly relevant. We previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats ( Lynx rufus ) and mountain lions ( Puma concolor ) for a small number of animals in California and Florida. In this study, we investigate host-specific selection pressures, within-host viral fitness, and inter- versus intraspecies transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analyses of proviral and viral RNA levels demonstrate that PLVA fitness is severely restricted in mountain lions compared to that in bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but we did not detect selection among 20 PLVA isolates from bobcats. These findings support the hypothesis that PLVA is a bobcat-adapted virus which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals that intraspecific PLVA transmission has occurred among panthers ( Puma concolor coryi ) in Florida following the initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to the emergence of HIV from nonhuman primates. IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which

  3. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    International Nuclear Information System (INIS)

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-01-01

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  4. A comparison of foamy and lentiviral vector genotoxicity in SCID-repopulating cells shows foamy vectors are less prone to clonal dominance

    Directory of Open Access Journals (Sweden)

    Elizabeth M Everson

    2016-01-01

    Full Text Available Hematopoietic stem cell (HSC gene therapy using retroviral vectors has immense potential, but vector-mediated genotoxicity limits use in the clinic. Lentiviral vectors are less genotoxic than gammaretroviral vectors and have become the vector of choice in clinical trials. Foamy retroviral vectors have a promising integration profile and are less prone to read-through transcription than gammaretroviral or lentiviral vectors. Here, we directly compared the safety and efficacy of foamy vectors to lentiviral vectors in human CD34+ repopulating cells in immunodeficient mice. To increase their genotoxic potential, foamy and lentiviral vectors with identical transgene cassettes with a known genotoxic spleen focus forming virus promoter were used. Both vectors resulted in efficient marking in vivo and a total of 825 foamy and 460 lentiviral vector unique integration sites were recovered in repopulating cells 19 weeks after transplantation. Foamy vector proviruses were observed less often near RefSeq gene and proto-oncogene transcription start sites than lentiviral vectors. The foamy vector group were also more polyclonal with fewer dominant clones (two out of six mice than the lentiviral vector group (eight out of eight mice, and only lentiviral vectors had integrants near known proto-oncogenes in dominant clones. Our data further support the relative safety of foamy vectors for HSC gene therapy.

  5. Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

    Science.gov (United States)

    Anisenko, Andrey N; Knyazhanskaya, Ekaterina S; Zatsepin, Timofey S; Gottikh, Marina B

    2017-01-01

    Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  6. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

    Science.gov (United States)

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L

    2014-01-01

    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

  7. Lentiviral transgenesis in mice via a simple method of viral concentration.

    Science.gov (United States)

    Cheng, Pei-Hsun; Chang, Yu-Fan; Mao, Su-Han; Lin, Hsiu-Lien; Chen, Chuan-Mu; Yang, Shang-Hsun

    2016-10-01

    Transgenic animals are important in vivo models for biological research. However, low transgenic rates are commonly reported in the literature. Lentiviral transgenesis is a promising method that has greater efficiency with regard to generating transgenic animals, although the transgenic rate of this approach is highly dependent on different transgenes and concentrated lentiviruses. In this study, we modified a method to concentrate lentiviruses using a table centrifuge, commonly available in most laboratories, and carried out analysis of the transgenic efficiency in mice. Based on 26 individual constructs and 627 live pups, we found that the overall transgenic rate was more than 30%, which is higher than obtained with pronuclear microinjection. In addition, we did not find any significant differences in transgenic efficiency when the size of inserts was less than 5000 bp. These results not only show that our modified method can successfully generate transgenic mice but also suggest that this approach could be generally applied to different constructs when the size of inserts is less than 5000 bp. It is anticipated that the results of this study can help encourage the wider laboratory use of lentiviral transgenesis in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

    Science.gov (United States)

    Espana-Agusti, Judit; Tuveson, David A; Adams, David J; Matakidou, Athena

    2015-06-05

    The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.

  9. Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector

    Directory of Open Access Journals (Sweden)

    Bryan P Burke

    2015-01-01

    Full Text Available We described earlier a dual-combination anti-HIV type 1 (HIV-1 lentiviral vector (LVsh5/C46 that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1 vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

  10. Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.

    Science.gov (United States)

    Varga, E; Nemes, C; Davis, R P; Ujhelly, O; Klincumhom, N; Polgar, Z; Muenthaisong, S; Pirity, M K; Dinnyes, A

    2014-04-01

    One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Expression profiles of Vpx/Vpr proteins are co-related with the primate lentiviral lineage

    Directory of Open Access Journals (Sweden)

    Yosuke Sakai

    2016-08-01

    Full Text Available Viruses of human immunodeficiency virus type 2 (HIV-2 and some simian immunodeficiency virus (SIV lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution.

  12. Lentiviral vector induced insertional haploinsufficiency of Ebf1 causes murine leukemia.

    Science.gov (United States)

    Heckl, Dirk; Schwarzer, Adrian; Haemmerle, Reinhard; Steinemann, Doris; Rudolph, Cornelia; Skawran, Britta; Knoess, Sabine; Krause, Johanna; Li, Zhixiong; Schlegelberger, Brigitte; Baum, Christopher; Modlich, Ute

    2012-06-01

    Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with γ-retroviral vectors, due to their integration bias to transcription units in comparison to the γ-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design.

  13. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin.

    Science.gov (United States)

    Choi, Kwang-Hwan; Park, Jin-Kyu; Kim, Hye-Sun; Uh, Kyung-Jun; Son, Dong-Chan; Lee, Chang-Kyu

    2013-01-01

    Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by in vitro differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes.

  14. Use of lentiviral vectors to deliver and express bicistronic transgenes in developing chicken embryos.

    Science.gov (United States)

    Semple-Rowland, Susan L; Berry, Jonathan

    2014-04-01

    The abilities of lentiviral vectors to carry large transgenes (∼8kb) and to efficiently infect and integrate these genes into the genomes of both dividing and non-dividing cells make them ideal candidates for transport of genetic material into cells and tissues. Given the properties of these vectors, it is somewhat surprising that they have seen only limited use in studies of developing tissues and in particular of the developing nervous system. Over the past several years, we have taken advantage of the large capacity of these vectors to explore the expression characteristics of several dual promoter and 2A peptide bicistronic transgenes in developing chick neural retina, with the goal of identifying transgene designs that reliably express multiple proteins in infected cells. Here we summarize the activities of several of these transgenes in neural retina and provide detailed methodologies for packaging lentivirus and delivering the virus into the developing neural tubes of chicken embryos in ovo, procedures that have been optimized over the course of several years of use in our laboratory. Conditions to hatch injected embryos are also discussed. The chicken-specific techniques will be of highest interest to investigators using avian embryos, development and packaging of lentiviral vectors that reliably express multiple proteins in infected cells should be of interest to all investigators whose experiments demand manipulation and expression of multiple proteins in developing cells and tissues. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

    Directory of Open Access Journals (Sweden)

    Rahmberg Andrew R

    2011-05-01

    Full Text Available Abstract Background Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Results Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Conclusions Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

  16. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    Science.gov (United States)

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001. Copyright © 2014, Cai et al.

  17. beta-hexosaminidase lentiviral vectors: transfer into the CNS via systemic administration.

    Science.gov (United States)

    Kyrkanides, Stephanos; Miller, Jennie H; Brouxhon, Sabine M; Olschowka, John A; Federoff, Howard J

    2005-02-18

    Brain inflammation in GM2 gangliosidosis has been recently realized as a key factor in disease development. The aim of this study was to investigate the effects of a FIV beta-hexosaminidase vector in the brain of HexB-deficient (Sandhoff disease) mice following intraperitoneal administration to pups of neonatal age. Since brain inflammation, lysosomal storage and neuromuscular dysfunction are characteristics of HexB deficiency, these parameters were employed as experimental outcomes in our study. The ability of the lentiviral vector FIV(HEX) to infect murine cells was initially demonstrated with success in normal mouse fibroblasts and human Tay-Sachs cells in vitro. Furthermore, systemic transfer of FIV(HEX) to P2 HexB-/- knockout pups lead to transduction of peripheral and central nervous system tissues. Specifically, beta-hexosaminidase expressing cells were immunolocalized in periventricular areas of the cerebrum as well as in the cerebellar cortex. FIV(HEX) neonatal treatment resulted in reduction of GM2 storage along with attenuation of the brain inflammation and amelioration of the attendant neuromuscular deterioration. In conclusion, these results demonstrate the effective transfer of a beta-hexosaminidase lentiviral vector to the brain of Sandhoff mice and resolution of the GM2 gangliosidosis after neonatal intraperitoneal administration.

  18. Cell loss during pseudoislet formation hampers profound improvements in islet lentiviral transduction efficacy for transplantation purposes.

    Science.gov (United States)

    Callewaert, H; Gysemans, C; Cardozo, A K; Elsner, M; Tiedge, M; Eizirik, D L; Mathieu, C

    2007-01-01

    Islet transplantation is a promising treatment in type 1 diabetes, but the need for chronic immunosuppression is a major hurdle to broad applicability. Ex vivo introduction of agents by lentiviral vectors-improving beta-cell resistance against immune attack-is an attractive path to pursue. The aim of this study was to investigate whether dissociation of islets to single cells prior to viral infection and reaggregation before transplantation would improve viral transduction efficacy without cytotoxicity. This procedure improved transduction efficacy with a LV-pWPT-CMV-EGFP construct from 11.2 +/- 4.1% at MOI 50 in whole islets to 80.0 +/- 2.8% at MOI 5. Viability (as measured by Hoechst/PI) and functionality (as measured by glucose challenge) remained high. After transplantation, the transfected pseudoislet aggregates remained EGFP positive for more than 90 days and the expression of EGFP colocalized primarily with the insulin-positive beta-cells. No increased vulnerability to immune attack was observed in vitro or in vivo. These data demonstrate that dispersion of islets prior to lentiviral transfection and reaggregation prior to transplantation is a highly efficient way to introduce genes of interest into islets for transplantation purposes in vitro and in vivo, but the amount of beta-cells needed for normalization of glycemia was more than eightfold higher when using dispersed cell aggregates versus unmanipulated islets. The high price to pay to reach stable and strong transgene expression in islet cells is certainly an important cell loss.

  19. Production of germline transgenic prairie voles (Microtus ochrogaster) using lentiviral vectors.

    Science.gov (United States)

    Donaldson, Zoe R; Yang, Shang-Hsun; Chan, Anthony W S; Young, Larry J

    2009-12-01

    The study of alternative model organisms has yielded tremendous insights into the regulation of behavioral and physiological traits not displayed by more widely used animal models, such as laboratory rats and mice. In particular, comparative approaches often exploit species ideally suited for investigating specific phenomenon. For instance, comparative studies of socially monogamous prairie voles and polygamous meadow voles have been instrumental toward gaining an understanding of the genetic and neurobiological basis of social bonding. However, laboratory studies of less commonly used organisms, such as prairie voles, have been limited by a lack of genetic tools, including the ability to manipulate the genome. Here, we show that lentiviral vector-mediated transgenesis is a rapid and efficient approach for creating germline transgenics in alternative laboratory rodents. Injection of a green fluorescent protein (GFP)-expressing lentiviral vector into the perivitelline space of 23 single-cell embryos yielded three live offspring (13 %), one of which (33%) contained germline integration of a GFP transgene driven by the human ubiquitin-C promoter. In comparison, transfer of 23 uninjected embryos yielded six live offspring (26%). Green fluorescent protein is present in all tissues examined and is expressed widely in the brain. The GFP transgene is heritable and stably expressed until at least the F(2) generation. This technology has the potential to allow investigation of specific gene candidates in prairie voles and provides a general protocol to pursue germline transgenic manipulation in many different rodent species.

  20. Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins

    DEFF Research Database (Denmark)

    Kushon, S A; Jordan, J P; Seifert, J L

    2001-01-01

    The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA...... structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic...

  1. Lentiviral vector-mediated genetic modification of cell substrates for the manufacture of proteins and other biologics.

    Science.gov (United States)

    Baranyi, Lajos; Roy, Andre; Embree, Heather D; Dropulic, Boro

    2010-01-01

    Transduction with Lentiviral vectors has been shown to be the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Lentiviral vectors have been widely used in research and have recently shown success in clinical trials for human gene therapy. In this paper, we describe the use of lentiviral vectors to generate genetically modified cell substrates for the manufacture of proteins and other complex biologics. The use of lentiviral vectors for the generation of genetically modified cell substrates for the production of biologic material has several advantages over other systems: (1) highly productive mammalian cell lines can be rapidly generated without selection or gene amplification; (2) the high number of vector copies are distributed throughout the open chromatin of the genome, resulting in cell lines that are extremely stable for high levels of gene expression and, consequently, protein production; and (3) high levels of protein glycosylation are maintained despite very high levels of protein production. These advantages offer the potential to significantly improve the quality, time-to-market, and manufacturing cost of biologics for human use.

  2. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo

    NARCIS (Netherlands)

    Pfeifer, A.; Brandon, E. P.; Kootstra, N.; Gage, F. H.; Verma, I. M.

    2001-01-01

    The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M

  3. Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

    Directory of Open Access Journals (Sweden)

    Anne Louise Askou

    Full Text Available Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF. Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

  4. A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts

    International Nuclear Information System (INIS)

    Graf, Marcus; Ludwig, Christine; Kehlenbeck, Sylvia; Jungert, Kerstin; Wagner, Ralf

    2006-01-01

    We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5' major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to the cytoplasm following fusion of the 3' Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays

  5. β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor

    Science.gov (United States)

    Mirecka, Ewa A.; Feuerstein, Sophie; Gremer, Lothar; Schröder, Gunnar F.; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2016-01-01

    In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. PMID:27641459

  6. Statistical scale of hairpin packets in the later stage of bypass transition induced by cylinder wake

    Science.gov (United States)

    Tang, Z. Q.; Jiang, N.

    2012-08-01

    The hairpin packet's structure and its statistical scale in the later stage of bypass transition induced by a cylinder wake are investigated by time-resolved particle image velocimetry from the side and top view, respectively. Linear stochastic estimation is used to achieve the conditionally averaged velocity fields. For the side view case, the conditionally averaged structure consists of a series of swirling motions located along a line inclining at a large angle (18°) from the wall and a low-speed region occupied by the cylinder wake appearing right above them. In the ( x, z)-plane at the wall-normal height y/δ = 0.2, the dominant structures are shown to be the large-scale regions of low momentum elongated almost over 3δ along the streamwise. The low-speed regions are consistently bordered by small-scale counter-rotating vortice pairs organized in the streamwise with a statistical spanwise width of 0.55δ. The results suggest that in the later part of the transitional zone, the upward induction of the cylinder wake enhances both the wall-normal and spanwise extent of the hairpin packets.

  7. Coordinate enhancement of transgene transcription and translation in a lentiviral vector

    Directory of Open Access Journals (Sweden)

    Fernandez Soledad

    2006-02-01

    Full Text Available Abstract Background Coordinate enhancement of transgene transcription and translation would be a potent approach to significantly improve protein output in a broad array of viral vectors and nonviral expression systems. Many vector transgenes are complementary DNA (cDNA. The lack of splicing can significantly reduce the efficiency of their translation. Some retroviruses contain a 5' terminal post-transcriptional control element (PCE that facilitates translation of unspliced mRNA. Here we evaluated the potential for spleen necrosis virus PCE to stimulate protein production from HIV-1 based lentiviral vector by: 1 improving translation of the internal transgene transcript; and 2 functionally synergizing with a transcriptional enhancer to achieve coordinate increases in RNA synthesis and translation. Results Derivatives of HIV-1 SIN self-inactivating lentiviral vector were created that contain PCE and cytomegalovirus immediate early enhancer (CMV IE. Results from transfected cells and four different transduced cell types indicate that: 1 PCE enhanced transgene protein synthesis; 2 transcription from the internal promoter is enhanced by CMV IE; 3 PCE and CMV IE functioned synergistically to significantly increase transgene protein yield; 4 the magnitude of translation enhancement by PCE was similar in transfected and transduced cells; 5 differences were observed in steady state level of PCE vector RNA in transfected and transduced cells; 6 the lower steady state was not attributable to reduced RNA stability, but to lower cytoplasmic accumulation in transduced cells. Conclusion PCE is a useful tool to improve post-transcriptional expression of lentiviral vector transgene. Coordinate enhancement of transcription and translation is conferred by the combination of PCE with CMV IE transcriptional enhancer and increased protein yield up to 11 to 17-fold in transfected cells. The incorporation of the vector provirus into chromatin correlated with reduced

  8. Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

    OpenAIRE

    Zargarian, Loussin?; Kanevsky, Igor; Bazzi, Ali; Boynard, Jonathan; Chaminade, Fran?oise; Foss?, Philippe; Mauffret, Olivier

    2009-01-01

    First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop?loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loop...

  9. On the a priori possibility of the formation of hexameric mini-hairpin d(GCGAGC) in solution

    Science.gov (United States)

    Rubin, Yu. V.; Belous, L. F.; Evstigneev, M. P.

    2012-11-01

    Combined high-level (M05-2x, M06-2x functionals of DFT method) and semi-empirical structural and energetic analyses have confirmed the a priori possibility for the formation of the shortest ever known hexameric hairpin d(GCGAGC) with GA loop in aqueous solution. The origin of stabilisation comes from intramolecular H-bonding and hydrophobic interactions and, to lesser extent, from intramolecular van der Waals and electrostatic forces. This result provides a basis for seeking of such mini-hairpins in experiment.

  10. Optimized PEI-based Transfection Method for Transient Transfection and Lentiviral Production.

    Science.gov (United States)

    Yang, Shaozhe; Zhou, Xiaoling; Li, Rongxiang; Fu, Xiuhong; Sun, Pingnan

    2017-09-14

    Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI-based transfection has been optimized to improve its efficiency, reproducibility, and consistency. This protocol outlines the steps for ordinary transient transfection and lentiviral production using PEI. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  11. Lentiviral-mediated administration of IL-25 in the CNS induces alternative activation of microglia

    DEFF Research Database (Denmark)

    Maiorino, C; Khorooshi, R; Ruffini, F

    2013-01-01

    was partly inhibited and the CNS protected from immune-mediated damage. To our knowledge, this is the first example of M2 shift (alternative activation) induced in vivo on CNS-resident myeloid cells by gene therapy, and may constitute a promising strategy to investigate the potential role of protective...... immune system, namely macrophages. We used a lentiviral-mediated gene therapy approach to deliver IL-25 to the central nervous system (CNS) in two mouse models of neuroinflammation, entorhinal cortex lesion and experimental autoimmune encephalomyelitis. In both, we found that IL-25 gene therapy was able...... to modulate CNS myeloid cells, either infiltrating macrophages or resident microglia, towards an anti-inflammatory, tissue-protective phenotype, as testified by the increase in markers such as Arginase-1 (Arg1), Mannose receptor 1 (CD206) and Chitinase 3-like 3 (Ym1). As a consequence, neuroinflammation...

  12. Dynamics of lentiviral infection in vivo in the absence of adaptive immune responses.

    Science.gov (United States)

    Schwartz, Elissa J; Vaidya, Naveen K; Dorman, Karin S; Carpenter, Susan; Mealey, Robert H

    2018-01-01

    Understanding the dynamics of acute viral infection is crucial for developing strategies to prevent and control infection. In this study, lentiviral dynamics in a host without adaptive immunity were examined in order to determine kinetic parameters of infection and quantify the effect of neutralizing antibodies in preventing infection, using mathematical modeling of data from equine infectious anemia virus (EIAV) infection of horses with severe combined immunodeficiency (SCID). Estimated parameters were used to calculate the basic reproductive number and virus doubling time and found that the rate that antibodies neutralized virus was ~18 times greater than the virus clearance rate. These results establish EIAV replication kinetics in SCID horses and the minimal efficacy of antibodies that blocked infection. Furthermore, they indicate that EIAV is at most mildly cytopathic. This study advances our understanding of EIAV infection and may have important implications for the control of other viral infections, including HIV. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy

    Directory of Open Access Journals (Sweden)

    Matthew M Wielgosz

    Full Text Available We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS protein. An insulator element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12–20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells.

  14. Ethical considerations in the use of lentiviral vectors for genetic transfer.

    Science.gov (United States)

    Roy, I

    2001-11-01

    This chapter will outline the various concerns which have been raised in scientific, bioethics, and lay communities about the use of lentiviral vectors for purposes of gene therapy. Many of these concerns are ranged around gene therapy itself; others are concerns particular to using this sort of vector for genetic modification of human cells. These concerns are outlined within the chapter, and arguments are given in favor and against various approaches to these concerns. Lastly, it is noted throughout that at this stage of research into gene therapy, the most practical approach to these dilemmas is to maintain awareness of the ethical problems and provide information to those concerned with all aspects of the development of this set of technologies.

  15. Lentiviral Modulation of Wnt/β-Catenin Signaling Affects In Vivo LTP.

    Science.gov (United States)

    Ivanova, Olga Ya; Dobryakova, Yulia V; Salozhin, Sergey V; Aniol, Viktor A; Onufriev, Mikhail V; Gulyaeva, Natalia V; Markevich, Vladimir A

    2017-10-01

    Wnt signaling is involved in hippocampal development and synaptogenesis. Numerous recent studies have been focused on the role of Wnt ligands in the regulation of synaptic plasticity. Inhibitors and activators of canonical Wnt signaling were demonstrated to decrease or increase, respectively, in vitro long-term potentiation (LTP) maintenance in hippocampal slices (Chen et al. in J Biol Chem 281:11910-11916, 2006; Vargas et al. in J Neurosci 34:2191-2202, 2014, Vargas et al. in Exp Neurol 264:14-25, 2015). Using lentiviral approach to down- and up-regulate the canonical Wnt signaling, we explored whether Wnt/β-catenin signaling is critical for the in vivo LTP. Chronic suppression of Wnt signaling induced an impairment of in vivo LTP expression 14 days after lentiviral suspension injection, while overexpression of Wnt3 was associated with a transient enhancement of in vivo LTP magnitude. Both effects were related to the early phase LTP and did not affect LTP maintenance. A loss-of-function study demonstrated decreased initial paired pulse facilitation ratio, β-catenin, and phGSK-3β levels. A gain-of-function study revealed not only an increase in PSD-95, β-catenin, and Cyclin D1 protein levels, but also a reduced phGSK-3β level and enhanced GSK-3β kinase activity. These results suggest a presynaptic dysfunction predominantly underlying LTP impairment while postsynaptic modifications are primarily involved in transient LTP amplification. This study is the first demonstration of the involvement of Wnt/β-catenin signaling in synaptic plasticity regulation in an in vivo LTP model.

  16. A nonintegrative lentiviral vector-based vaccine provides long-term sterile protection against malaria.

    Directory of Open Access Journals (Sweden)

    Frédéric Coutant

    Full Text Available Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5-62.5 of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice. The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042. Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = -0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia. However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine.

  17. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    International Nuclear Information System (INIS)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

    2007-01-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

  18. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    Energy Technology Data Exchange (ETDEWEB)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application.

  19. Short hairpin RNA targeting of fibroblast activation protein inhibits tumor growth and improves the tumor microenvironment in a mouse model

    Directory of Open Access Journals (Sweden)

    Fan Cai

    2013-05-01

    Full Text Available Fibroblast activation protein (FAP is a specific serine proteaseexpressed in tumor stroma proven to be a stimulatory factor inthe progression of some cancers. The purpose of this studywas to investigate the effects of FAP knockdown on tumorgrowth and the tumor microenvironment. Mice bearing 4T1subcutaneous tumors were treated with liposome-shRNAcomplexes targeting FAP. Tumor volumes and weights weremonitored, and FAP, collagen, microvessel density (MVD,and apoptosis were measured. Our studies showed thatshRNA targeting of FAP in murine breast cancer reduces FAPexpression, inhibits tumor growth, promotes collagenaccumulation (38%, and suppresses angiogenesis (71.7%, aswell as promoting apoptosis (by threefold. We suggest thatFAP plays a role in tumor growth and in altering the tumormicroenvironment. Targeting FAP may therefore represent asupplementary therapy for breast cancer. [BMB Reports 2013;46(5: 252-257

  20. A study of short wave instability on vortex filaments

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hong Yun [Univ. of California, Berkeley, CA (United States)

    1996-12-01

    The numerical stability and accuracy of the vortex method are studied. The effect of the ordinary differential equations (ODE) solver and of the time step on the numerical stability is analyzed. Various ODE solvers are compared and a best performer is chosen. A new constraint on the time step based on numerical stability is proposed and verified in numerical simulations. It is shown through numerical examples that empirical rules for selecting the spatial discretization obtained in simple test problems may not be extended to more general problems. The thin tube vortex filament method is applied to the problem of Widnall's instability on vortex rings. Numerical results different from previous calculations are presented and the source of the discrepancies is explained. The long time behavior of the unstable mode on thin vortex rings is simulated and analyzed. The short wave instability on vortex filaments is investigated both theoretically and numerically. It is shown that the short wave instability always occurs on co-rotating vortex filaments of fixed core structure. Furthermore when they are close to each other, vortex filaments produce short wave unstable modes which lead to wild stretching and folding. However, when the inter-filament distance is large in comparison with the core size of the filaments, unstable modes are bounded by a small fraction of the core size and the vortex filaments do not create hairpins nor wild stretching. These findings may explain the smooth behavior of the superfluid vortices. The formation of hairpin structures on numerical vortex filaments is investigated. It is shown that the formation of hairpin structures is independent of the ODE solver, of the time step and of other numerical parameters. The hairpin structures are primarily caused by short wave instability on co-rotating vortex filaments.

  1. Compact Hairpin Bandpass Filter with Wide Stopband and High Attenuation Using Multilayer Broadside-coupled Stripline

    Directory of Open Access Journals (Sweden)

    Xiaowei GU

    2014-11-01

    Full Text Available This paper presents a compact bandpass filter with stopband and high attenuation by using multilayer folded broadside-coupled quarter-wavelength stripline resonators in a low- temperature co-fired ceramic (LTCC substrate. The proposed bandpass filter centered at 1.1 GHz with a fractional bandwith of 4.5 % shows the first spurious frequency at 3.8 times the center frequency. In comparison to the conventional half-wavelength hairpin bandpass filter with the same passband performance, the proposed bandpass filter shows not only a 50 % size reduction but also a wider stopband from 1.3 GHz -3.8 GHz with a high rejection level up to 60 dB.

  2. Decay of the electron number density in the nitrogen afterglow using a hairpin resonator probe

    International Nuclear Information System (INIS)

    Siefert, Nicholas S.; Ganguly, Biswa N.; Sands, Brian L.; Hebner, Greg A.

    2006-01-01

    A hairpin resonator was used to measure the electron number density in the afterglow of a nitrogen glow discharge (p=0.25-0.75 Torr). Electron number densities were measured using a time-dependent approach similar to the approach used by Spencer et al. [J. Phys. D 20, 923 (1987)]. The decay time of the electron number density was used to determine the electron temperature in the afterglow, assuming a loss of electrons via ambipolar diffusion to the walls. The electron temperature in the near afterglow remained between 0.4 and 0.6 eV, depending on pressure. This confirms the work by Guerra et al. [IEEE Trans. Plasma. Sci. 31, 542 (2003)], who demonstrated experimentally and numerically that the electron temperature stays significantly above room temperature via superelastic collisions with highly vibrationally excited ground state molecules and metastables, such as A 3 Σ u +

  3. One-step isothermal detection of multiple KRAS mutations by forming SNP specific hairpins on a gold nanoshell.

    Science.gov (United States)

    Chung, Chan Ho; Kim, Joong Hyun

    2018-04-24

    We developed a one-step isothermal method for typing multiple KRAS mutations using a designed set of primers to form a hairpin on a gold nanoshell upon being ligated by a SNP specific DNA ligase after binding of targets. As a result, we could detect as low as 20 attomoles of KRAS mutations within 1 h.

  4. Melting of a beta-hairpin peptide using isotope-edited 2D IR spectroscopy and simulations.

    NARCIS (Netherlands)

    Smith, A.W.; Lessing, J.; Ganim, Z.; Peng, C.S.; Tokmakoff, A.; Roy, S.; Jansen, T.L.Th.A.; Knoester, J.

    2010-01-01

    Isotope-edited two-dimensional infrared spectroscopy has been used to characterize the conformational heterogeneity of the beta-hairpin peptide TrpZip2 (TZ2) across its thermal unfolding transition. Four isotopologues were synthesized to probe hydrogen bonding and solvent exposure of the beta-turn

  5. Melting of a beta-Hairpin Peptide Using Isotope-Edited 2D IR Spectroscopy and Simulations

    NARCIS (Netherlands)

    Smith, Adam W.; Lessing, Joshua; Ganim, Ziad; Peng, Chunte Sam; Tokmakoff, Andrei; Roy, Santanu; Jansen, Thomas L. C.; Knoester, Jasper

    2010-01-01

    Isotope-edited two-dimensional infrared spectroscopy has been used! to characterize the conformational heterogeneity of the beta-hairpin peptide TrpZip2 (17.2) across its thermal unfolding transition Four isotopologues were synthesized to probe hydrogen bonding and solvent exposure of the beta-turn

  6. Origins of biological function in DNA and RNA hairpin loop motifs from replica exchange molecular dynamics simulation.

    Science.gov (United States)

    Swadling, Jacob B; Ishii, Kunihiko; Tahara, Tahei; Kitao, Akio

    2018-01-31

    Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have remarkably similar chemical structures, but despite this, they play significantly different roles in modern biology. In this article, we explore the possible conformations of DNA and RNA hairpins to better understand the fundamental differences in structure formation and stability. We use large parallel temperature replica exchange molecular dynamics ensembles to sample the full conformational landscape of these hairpin molecules so that we can identify the stable structures formed by the hairpin sequence. Our simulations show RNA adopts a narrower distribution of folded structures compared to DNA at room temperature, which forms both hairpins and many unfolded conformations. RNA is capable of forming twice as many hydrogen bonds than DNA which results in a higher melting temperature. We see that local chemical differences lead to emergent molecular properties such as increased persistence length in RNA that is weakly temperature dependant. These discoveries provide fundamental insight into how RNA forms complex folded tertiary structures which confer enzymatic-like function in ribozymes, whereas DNA retains structural motifs in order to facilitate function such as translation of sequence.

  7. A New Computational Approach for Mechanical Folding Kinetics of RNA Hairpins

    Science.gov (United States)

    Cao, Song; Chen, Shi-Jie

    2009-01-01

    Based on an ensemble of kinetically accessible conformations, we propose a new analytical model for RNA folding kinetics. The model gives populational kinetics, kinetic rates, transition states, and pathways from the rate matrix. Applications of the new kinetic model to mechanical folding of RNA hairpins such as trans-activation-responsive RNA reveal distinct kinetic behaviors in different force regimes, from zero force to forces much stronger than the critical force for the folding-unfolding transition. In the absence of force or a low force, folding can be initiated (nucleated) at any position by forming the first base stack and there exist many pathways for the folding process. In contrast, for a higher force, the folding/unfolding would predominantly proceed along a single zipping/unzipping pathway. Studies for different hairpin-forming sequences indicate that depending on the nucleotide sequence, a kinetic intermediate can emerge in the low force regime but disappear in high force regime, and a new kinetic intermediate, which is absent in the low and high force regimes, can emerge in the medium force range. Variations of the force lead to changes in folding cooperativity and rate-limiting steps. The predicted network of pathways for trans-activation-responsive RNA suggests two parallel dominant pathways. The rate-limiting folding steps (at f = 8 pN) are the formation of specific basepairs that are 2–4 basepairs away from the loop. At a higher force (f = 11 pN), the folding rate is controlled by the formation of the bulge loop. The predicted rates and transition states are in good agreement with the experimental data for a broad force regime. PMID:19450474

  8. Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study

    Science.gov (United States)

    Pathak, Arup K.; Bandyopadhyay, Tusar

    2017-04-01

    Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

  9. Mechanism of membrane permeation induced by synthetic β-hairpin peptides.

    Science.gov (United States)

    Gupta, Kshitij; Jang, Hyunbum; Harlen, Kevin; Puri, Anu; Nussinov, Ruth; Schneider, Joel P; Blumenthal, Robert

    2013-11-05

    We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form β-hairpin structures under certain conditions, and a control non-β-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ~50% of the liposomes that contain small (R(h) peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Combinatorial Pattern Discovery Approach for the Folding Trajectory Analysis of a beta-Hairpin.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity cinRO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  11. Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia.

    LENUS (Irish Health Repository)

    McGinley, Lisa

    2012-01-31

    INTRODUCTION: A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia. METHODS: Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-alpha-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated. RESULTS: The second generation lentiviral vector rHIV-pWPT-EF1-alpha-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression. CONCLUSIONS: Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate

  12. Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

    LENUS (Irish Health Repository)

    McGinley, Lisa

    2011-03-07

    Abstract Introduction A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia. Methods Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated. Results The second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression. Conclusions Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate

  13. Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU).

    Science.gov (United States)

    Zhang, H; Fountain, M A; Krugh, T R

    2001-08-21

    The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.

  14. Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

    Science.gov (United States)

    Vink, Conrad A; Counsell, John R; Perocheau, Dany P; Karda, Rajvinder; Buckley, Suzanne M K; Brugman, Martijn H; Galla, Melanie; Schambach, Axel; McKay, Tristan R; Waddington, Simon N; Howe, Steven J

    2017-08-02

    Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

    Science.gov (United States)

    Monzani, P S; Sangalli, J R; De Bem, T H C; Bressan, F F; Fantinato-Neto, P; Pimentel, J R V; Birgel-Junior, E H; Fontes, A M; Covas, D T; Meirelles, F V

    2013-02-28

    Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

  16. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    International Nuclear Information System (INIS)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping; Ye, Lihong; Zhang, Xiaodong

    2015-01-01

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro

  17. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2015-04-03

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

  18. A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation

    NARCIS (Netherlands)

    Eekels, J. J. M.; Pasternak, A. O.; Schut, A. M.; Geerts, D.; Jeeninga, R. E.; Berkhout, B.

    2012-01-01

    RNA interference (RNAi) is a sequence-specific gene silencing mechanism with therapeutic potential against many human pathogens. To obtain a durable therapeutic effect, stable transduction of target cells with for instance a lentiviral vector that expresses a short hairpin (shRNA) inducer of the

  19. Intrahippocampal injection of a lentiviral vector expressing neurogranin enhances cognitive function in 5XFAD mice.

    Science.gov (United States)

    Jeon, Seong Gak; Kang, Moonkyung; Kim, Yeon-Soo; Kim, Dong-Hyun; Nam, Dong Woo; Song, Eun Ji; Mook-Jung, Inhee; Moon, Minho

    2018-03-23

    Progressive cognitive declines are the main clinical symptoms of Alzheimer's disease (AD). Cognitive impairment in AD is directly correlated with amyloid beta (Aβ)-mediated synaptic deficits. It is known that upregulation of neurogranin (Ng), a postsynaptic protein, contributes to the enhancement of synaptic plasticity and cognitive function. By contrast, downregulation of Ng expression results in learning and memory impairments. Interestingly, Ng expression is significantly reduced in the parenchyma of brains with AD. However, the pathological role that downregulated Ng plays in the cognitive dysfunctions observed in AD remains unclear. Therefore, the present study examined whether enhancing Ng expression affected cognitive functions in 5XFAD mice, an animal model of AD. We found that the Ng reductions and cognitive decline observed in 5XFAD mice were restored in mice that were intrahippocampally injected with an Ng-expressing lentiviral vector. Furthermore, overexpression of Ng upregulated expression of postsynaptic density protein-95 in the hippocampus of 5XFAD mice. These results suggest that the cause of cognitive decline in AD may be at least partially associated with reduced Ng levels, and thus, supplementation of Ng may be an appropriate therapeutic strategy for individuals with AD.

  20. Mucosal immunization with integrase-defective lentiviral vectors protects against influenza virus challenge in mice.

    Directory of Open Access Journals (Sweden)

    Judith M Fontana

    Full Text Available Recent reports highlight the potential for integrase-defective lentiviral vectors (IDLV to be developed as vaccines due to their ability to elicit cell-mediated and humoral immune responses after intramuscular administration. Differently from their integrase-competent counterpart, whose utility for vaccine development is limited by the potential for insertional mutagenesis, IDLV possess a mutation in their integrase gene that prevents genomic integration. Instead, they are maintained as episomal DNA circles that retain the ability to stably express functional proteins. Despite their favorable profile, it is unknown whether IDLV elicit immune responses after intranasal administration, a route that could be advantageous in the case of infection with a respiratory agent. Using influenza as a model, we constructed IDLV expressing the influenza virus nucleoprotein (IDLV-NP, and tested their ability to generate NP-specific immune responses and protect from challenge in vivo. We found that administration of IDLV-NP elicited NP-specific T cell and antibody responses in BALB/c mice. Importantly, IDLV-NP was protective against homologous and heterosubtypic influenza virus challenge only when given by the intranasal route. This is the first report demonstrating that IDLV can induce protective immunity after intranasal administration, and suggests that IDLV may represent a promising vaccine platform against infectious agents.

  1. DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Siew Ching Ngai

    2015-01-01

    Full Text Available Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP reporter gene driven by cytomegalovirus (CMV promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

  2. Visualization of cortical projection neurons with retrograde TET-off lentiviral vector.

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    Akiya Watakabe

    Full Text Available We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA and the transgene under the TET Responsive Element promoter (TRE on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP or turboFP635 (RFP in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.

  3. Immune modulation by genetic modification of dendritic cells with lentiviral vectors.

    Science.gov (United States)

    Liechtenstein, Therese; Perez-Janices, Noemi; Bricogne, Christopher; Lanna, Alessio; Dufait, Inès; Goyvaerts, Cleo; Laranga, Roberta; Padella, Antonella; Arce, Frederick; Baratchian, Mehdi; Ramirez, Natalia; Lopez, Natalia; Kochan, Grazyna; Blanco-Luquin, Idoia; Guerrero-Setas, David; Breckpot, Karine; Escors, David

    2013-09-01

    Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector

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    Swati Singh

    2017-03-01

    Full Text Available Wiskott-Aldrich syndrome (WAS is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS promoter driving WASp expression. Used as a gene therapeutic in Was−/− mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.

  5. Structure of the antimicrobial beta-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Kaznessis, Yiannis N

    2007-01-01

    -18 to extend perpendicular to the beta-hairpin plane. This bend was driven by a highly persistent hydrogen-bond between the polar peptide side-chain of TYR7 and the unshielded backbone carbonyl oxygen atom of GLY17. The H-bond formation relieves the unfavorable free energy of insertion of polar groups......All atom molecular dynamics simulations of the 18-residue beta-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH(2)) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure......-550]), and to delineate specific peptide-membrane interactions which are responsible for the peptide's membrane binding properties. A novel, previously unknown, "kick" shaped conformation of the peptide was detected, where a bend at the C-terminal beta-strand of the peptide caused the peptide backbone at residues 16...

  6. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

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    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  7. Correlations between structure, material properties and bioproperties in self-assembled beta-hairpin peptide hydrogels.

    Science.gov (United States)

    Hule, Rohan A; Nagarkar, Radhika P; Altunbas, Aysegul; Ramay, Hassna R; Branco, Monica C; Schneider, Joel P; Pochan, Darrin J

    2008-01-01

    A de novo designed beta-hairpin peptide (MAX8), capable of undergoing intramolecular folding and consequent intermolecular self-assembly into a cytocompatible hydrogel, has been studied. A combination of small angle neutron scattering (SANS) and cryogenic-transmission electron microscopy (cryo-TEM) have been used to quantitatively investigate the MAX8 nanofibrillar hydrogel network morphology. A change in the peptide concentration from 0.5 to 2 wt% resulted in a denser fibrillar network as revealed via SANS by a change in the high q (q = (4 pi/lambda) x sin (theta/2), where lambda = wavelength of incident neutrons and theta = scattering angle) mass fractal exponent from 2.5 to 3 and by a decrease in the measured correlation length from 23 to 16 A. A slope of -4 in the USANS regime indicates well-defined gel microporosity, an important characteristic for cellular substrate applications. These changes, both at the network as well as the individual fibril lengthscales, can be directly visualized in situ by cryo-TEM. Fibrillar nanostructures and network properties are directly related to bulk hydrogel stiffness via oscillatory rheology. Preliminary cell viability and anchorage studies at varying hydrogel stiffness confirm cell adhesion at early stages of cell culture within the window of stiffness investigated. Knowledge of the precise structure spanning length scales from the nanoscale up to the microscale can help in the formation of future, specific structure-bioproperty relationships when studying in vitro and in vivo behavior of these new peptide scaffolds.

  8. Comparison of Cytotoxic Activity in Leukemic Lineages Reveals Important Features of β-Hairpin Antimicrobial Peptides.

    Science.gov (United States)

    Buri, Marcus V; Torquato, Heron F Vieira; Barros, Carlos Castilho; Ide, Jaime S; Miranda, Antonio; Paredes-Gamero, Edgar J

    2017-07-01

    Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Biological Applications of Designed Hairpin Peptides: As Antimicrobials and as Inhibitors of Amyloidogenesis

    Science.gov (United States)

    Sivanesam, Kalkena

    More than 40 diseases have been associated with the misfolding of peptides (or proteins) that form fibrils with a very specific morphology. These peptides classified as amyloidogenic peptides have been implicated in the development of Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, Hungtinton's Disease etc. To date, these diseases have no cure, only therapies that can ameliorate the symptoms to a degree. Inhibition of the amyloidogenesis of these peptides has been proposed as a possible treatment option. While small molecules have been heavily tested as inhibitors of amyloidogenesis, peptides have emerged as potential inhibitors. In this work, the ability of a set of designed hairpin peptides to inhibit the amyloidogenesis of two different systems, alpha-synuclein (implicated in Parkinson's Disease) and human amylin (implicated in Type II Diabetes) is tested. Using circular dichroism and thioflavin T fluorescence, the ability of these peptides to inhibit amyloidogenesis is tested. The binding loci of these inhibitors to alpha-synuclein are also explored. The use of peptides as antimicrobials on the other hand is not a novel concept. However, most antimicrobial peptides, both natural and designed, rely heavily on covalent stabilizations in order to maintain secondary structure. In this study, non-covalent stabilizations are applied to a couple of natural as well as designed antimicrobials in order to study the effects of secondary structure stabilization on biological activity.

  10. Optimum Design of Novel UWB Multilayer Microstrip Hairpin Filters with Harmonic Suppression and Impedance Matching

    Directory of Open Access Journals (Sweden)

    Homayoon Oraizi

    2012-01-01

    Full Text Available Optimum design of a novel ultra-wideband multilayer microstrip hairpin filter is presented, providing for harmonic suppression and impedance matching between source and load impedances. The theory of N-coupled transmission lines is employed to obtain an equivalent circuit for development of a design procedure based on the method of least squares. A prototype model of proposed two-layer filter of order 5 is fabricated for 3.1–10.6 GHz. The dimensions of designed filter are 23 mm × 7 mm. The insertion loss in the passband varies from 0.3 dB to 3 dB (in the worst case at the edge of passband and in the stopband is about 30 dB up to 20 GHz. Its group delay in the UWB region is about 0.5 ns. Close agreement among the filter frequency responses as obtained by the proposed method, full-wave computer simulation softwares, and measurement data verify the effectiveness of the proposed filter structure and design methods.

  11. Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme.

    Science.gov (United States)

    White, Neil A; Hoogstraten, Charles G

    2017-09-01

    The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2'-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The Inverse Autotransporter Intimin Exports Its Passenger Domain via a Hairpin Intermediate*

    Science.gov (United States)

    Oberhettinger, Philipp; Leo, Jack C.; Linke, Dirk; Autenrieth, Ingo B.; Schütz, Monika S.

    2015-01-01

    Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation. PMID:25488660

  13. Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression.

    Science.gov (United States)

    Yu, Han; Jiang, Xiaoou; Tan, Kar Tong; Hang, Liting; Patzel, Volker

    2015-10-15

    Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vector/span>s more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression

    Science.gov (United States)

    Yu, Han; Jiang, Xiaoou; Tan, Kar Tong; Hang, Liting; Patzel, Volker

    2015-01-01

    Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system. PMID:26068470

  15. The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer

    Science.gov (United States)

    Sanchez, Jacint G.; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M.; Sundquist, Wesley I.; Pornillos, Owen

    2014-01-01

    Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication. PMID:24550273

  16. Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides.

    Science.gov (United States)

    Oparin, Peter B; Mineev, Konstantin S; Dunaevsky, Yakov E; Arseniev, Alexander S; Belozersky, Mikhail A; Grishin, Eugene V; Egorov, Tsezi A; Vassilevski, Alexander A

    2012-08-15

    A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

  17. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate.

    Science.gov (United States)

    Oberhettinger, Philipp; Leo, Jack C; Linke, Dirk; Autenrieth, Ingo B; Schütz, Monika S

    2015-01-16

    Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  19. Crystal structure of raptor adenovirus 1 fibre head and role of the beta-hairpin in siadenovirus fibre head domains.

    Science.gov (United States)

    Nguyen, Thanh H; Ballmann, Mónika Z; Do, Huyen T; Truong, Hai N; Benkő, Mária; Harrach, Balázs; van Raaij, Mark J

    2016-06-22

    Most adenoviruses recognize their host cells via an interaction of their fibre head domains with a primary receptor. The structural framework of adenovirus fibre heads is conserved between the different adenovirus genera for which crystal structures have been determined (Mastadenovirus, Aviadenovirus, Atadenovirus and Siadenovirus), but genus-specific differences have also been observed. The only known siadenovirus fibre head structure, that of turkey adenovirus 3 (TAdV-3), revealed a twisted beta-sandwich resembling the reovirus fibre head architecture more than that of other adenovirus fibre heads, plus a unique beta-hairpin embracing a neighbouring monomer. The TAdV-3 fibre head was shown to bind sialyllactose. Raptor adenovirus 1 (RAdV-1) fibre head was expressed, crystallized and its structure was solved and refined at 1.5 Å resolution. The structure could be solved by molecular replacement using the TAdV-3 fibre head structure as a search model, despite them sharing a sequence identity of only 19 %. Versions of both the RAdV-1 and TAdV-3 fibre heads with their beta-hairpin arm deleted were prepared and their stabilities were compared with the non-mutated proteins by a thermal unfolding assay. The structure of the RAdV-1 fibre head contains the same twisted ABCJ-GHID beta-sandwich and beta-hairpin arm as the TAdV-3 fibre head. However, while the predicted electro-potential surface charge of the TAdV-3 fibre head is mainly positive, the RAdV-1 fibre head shows positively and negatively charged patches and does not appear to bind sialyllactose. Deletion of the beta-hairpin arm does not affect the structure of the raptor adenovirus 1 fibre head and only affects the stability of the RAdV-1 and TAdV-3 fibre heads slightly. The high-resolution structure of RAdV-1 fibre head is the second known structure of a siadenovirus fibre head domain. The structure shows that the siadenovirus fibre head structure is conserved, but differences in the predicted surface charge

  20. Investigation of the Mesenchymal Stem Cell Compartment by Means of a Lentiviral Barcode Library.

    Science.gov (United States)

    Bigildeev, A E; Cornils, K; Aranyossy, T; Sats, N V; Petinati, N A; Shipounova, I N; Surin, V L; Pshenichnikova, O S; Riecken, K; Fehse, B; Drize, N I

    2016-04-01

    The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

  1. The transduction pattern of IL-12-encoding lentiviral vectors shapes the immunological outcome.

    Science.gov (United States)

    Goyvaerts, Cleo; Broos, Katrijn; Escors, David; Heirman, Carlo; Raes, Geert; De Baetselier, Patrick; Thielemans, Kris; Breckpot, Karine

    2015-12-01

    In situ modification of antigen-presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC-targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL-12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC-targeted LV-based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV-G-LV) with APC-targeted LVs (DC2.1-LV)-encoding enhanced GFP and ovalbumin, or IL-12 and ovalbumin in mice. We show that codelivery of IL-12 by VSV-G-LVs or DC2.1-LVs augments CD4(+) or CD8(+) T-cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL-12 enhances the CD4(+) TH 1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8(+) T cells was only induced upon VSV-G-LV injection. While codelivery of IL-12 by DC2.1-LVs did not enhance CD8(+) T-cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD-1. Finally, the discrepancy between CD4(+) T-cell stimulation with and without functional CD8(+) T-cell stimulation by VSV-G- and DC2.1-LVs is partly explained by the observation that IL-12 relieves CD8(+) T cells from CD4(+) T-cell help, implying that a T(H)1 profile is of minor importance for antitumor immunotherapy if IL-12 is exogenously delivered. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Optical control of retrogradely infected neurons using drug-regulated "TLoop" lentiviral vectors.

    Science.gov (United States)

    Cetin, Ali; Callaway, Edward M

    2014-05-01

    Many approaches that use viral vectors to deliver transgenes have limited transduction efficiency yet require high levels of transgene expression. In particular, infection via axon terminals is relatively inefficient but is a powerful means of achieving infection of specific neuron types. Combining this with optogenetic approaches requires high gene expression levels that are not typically achieved with nontoxic retrogradely infecting vectors. We generated rabies glycoprotein-pseudotyped lentiviral vectors that use a positive feedback loop composed of a Tet promoter driving both its own tetracycline-dependent transcription activator (tTA) ("TLoop") and channelrhodopsin-2-YFP (ChR2YFP). We show that TLoop vectors strongly express proteins in a drug-controllable manner in neurons that project to injection sites within the mouse brain. After initial infection, the virus travels retrogradely, stably integrates into the host genome, and expresses gene products. The expression is robust and allows optogenetic studies of neurons projecting to the location of virus injection, as demonstrated by fluorescence-targeted intracellular recordings. ChR2YFP expression did not cause observable signs of toxicity and continued for up to 6 mo after infection. Expression can be reversibly blocked by administration of doxycycline, if necessary, for expression of gene products that might be more toxic. Overall, we present a system that will allow researchers to achieve high levels of gene expression even in the face of inefficient viral transduction. The particular vectors that we demonstrate may enhance efforts to gain a precise understanding of the contributions of specific types of projection neurons to brain function. Copyright © 2014 the American Physiological Society.

  3. Oviduct-Specific Expression of Human Neutrophil Defensin 4 in Lentivirally Generated Transgenic Chickens

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    Liu, Tongxin; Wu, Hanyu; Cao, Dainan; Li, Qingyuan; Zhang, Yaqiong; Li, Ning; Hu, Xiaoxiang

    2015-01-01

    The expression of oviduct-specific recombinant proteins in transgenic chickens is a promising technology for the production of therapeutic biologics in eggs. In this study, we constructed a lentiviral vector encoding an expression cassette for human neutrophil defensin 4 (HNP4), a compound that displays high activity against Escherichia coli, and produced transgenic chickens that expressed the recombinant HNP4 protein in egg whites. After the antimicrobial activity of the recombinant HNP4 protein was tested at the cellular level, a 2.8-kb ovalbumin promoter was used to drive HNP4 expression specifically in oviduct tissues. From 669 injected eggs, 218 chickens were successfully hatched. Ten G0 roosters, with semens identified as positive for the transgene, were mated with wild-type hens to generate G1 chickens. From 1,274 total offspring, fifteen G1 transgenic chickens were positive for the transgene, which was confirmed by PCR and Southern blotting. The results of the Southern blotting and genome walking indicated that a single copy of the HNP4 gene was integrated into chromosomes 1, 2, 3, 4, 6 and 24 of the chickens. As expected, HNP4 expression was restricted to the oviduct tissues, and the levels of both transcriptional and translational HNP4 expression varied greatly in transgenic chickens with different transgene insertion sites. The amount of HNP4 protein expressed in the eggs of G1 and G2 heterozygous transgenic chickens ranged from 1.65 μg/ml to 10.18 μg/ml. These results indicated that the production of transgenic chickens that expressed HNP4 protein in egg whites was successful. PMID:26020529

  4. Improving expression of reporter transgene in stem cell by construction of different lentiviral vectors

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    Tae, Seong Ho; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of); Le, Uyenchi N.; Padmanabhan, Parasuraman [Singapore Bio-Imaging Imaging Consortium, Singapore (Singapore)

    2007-07-01

    For stem cell trafficking applications, it is imperative to express transgenes at desired and stable levels. In recent years, lentivirus-mediated gene transfer was shown to be an efficient method to stably introduce genetic modifications in target cells, even if these are in proliferative or nonproliferative states. Moreover, transgene expression levels can be controlled by using different promoters. The present study was designed to compare the potency of various promoters regulating expression of imaging reporter genes in embryonic H9c2 cardiomyoblasts derived from rat heart. Lentiviral vector was produced by the transient transfection of plasmids carrying required genes and those encoding for virus coating proteins into 293T cells. Harvested viral constructs were incubated with Hela and H9c2 cells, respectively. Transgene expressions were detected by several imaging modalities and evaluated by enzymatic assays. Results - We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. Here we show that lentivirus-mediated gene transfer allows efficient and stable transgene expression in embryonic cardiomyoblasts in vitro and that transgene expression levels can be varied by using different well-characterized gene promoters. In vivo trials about gene expression will probably further determine the potential of long-term trafficking stem cells using lentivirus.

  5. Improving expression of reporter transgene in stem cell by construction of different lentiviral vectors

    International Nuclear Information System (INIS)

    Tae, Seong Ho; Min, Jung Joon; Le, Uyenchi N.; Padmanabhan, Parasuraman

    2007-01-01

    For stem cell trafficking applications, it is imperative to express transgenes at desired and stable levels. In recent years, lentivirus-mediated gene transfer was shown to be an efficient method to stably introduce genetic modifications in target cells, even if these are in proliferative or nonproliferative states. Moreover, transgene expression levels can be controlled by using different promoters. The present study was designed to compare the potency of various promoters regulating expression of imaging reporter genes in embryonic H9c2 cardiomyoblasts derived from rat heart. Lentiviral vector was produced by the transient transfection of plasmids carrying required genes and those encoding for virus coating proteins into 293T cells. Harvested viral constructs were incubated with Hela and H9c2 cells, respectively. Transgene expressions were detected by several imaging modalities and evaluated by enzymatic assays. Results - We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. Here we show that lentivirus-mediated gene transfer allows efficient and stable transgene expression in embryonic cardiomyoblasts in vitro and that transgene expression levels can be varied by using different well-characterized gene promoters. In vivo trials about gene expression will probably further determine the potential of long-term trafficking stem cells using lentivirus

  6. Prior Virus Exposure Alters the Long-Term Landscape of Viral Replication during Feline Lentiviral Infection

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    Sue VandeWoude

    2011-10-01

    Full Text Available We developed a feline model of lentiviral cross-species transmission using a puma lentivirus (PLV or FIVPco which infects domestic cats but does not cause disease. Infection with PLV protects cats from CD4+ T-cell decline caused by subsequent infection with virulent feline immunodeficiency virus (FIV. Previous studies implicate innate immune and/or cellular restriction mechanisms for FIV disease attenuation in PLV-infected cats. In this study, we evaluated viral infection and cytokine mRNA transcription in 12 different tissue reservoirs approximately five months post infection. We quantitated tissue proviral load, viral mRNA load and relative transcription of IL-10, IL-12p40 and IFNγ from tissues of cats exposed to FIV, PLV or both viruses and analyzed these parameters using a multivariate statistical approach. The distribution and intensity of FIV infection and IFNγ transcription differed between single and co-infected cats, characterized by higher FIV proviral loads and IFNγ expression in co-infected cat tissues. Variability in FIV mRNA load and IFNγ was significantly more constrained in co-infected versus singly infected cat tissues. Single-infected:co-infected ratios of FIV mRNA load compared to FIV proviral load indicated that active viral transcription was apparently inhibited during co-infection. These results indicate that previous PLV infection increases activation of tissue innate immunity and constrains the ability of FIV to productively infect tissue reservoirs of infection for months, independent of FIV proviral load, supporting a model in which innate immunity and/or modulation of target cell susceptibility play a key role in PLV-induced protection from FIV disease.

  7. Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

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    Gideon Hen

    Full Text Available The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV, into the chorioallantoic membrane (CAM of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP or recombinant alpha-melanocyte-stimulating hormone (α-MSH genes, driven by the cytomegalovirus (CMV promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP nick end labeling (TUNEL assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA, and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

  8. Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

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    Cheng, Lei; Liu, Yi; Zhao, Hua; Zhang, Wen; Guo, Ying-Jun; Nie, Lin, E-mail: chengleiyx@126.com

    2013-10-18

    Highlights: •CDNF was successfully transfected by a lentiviral vector into the distal sciatic nerve. •CDNF improved S-100, NF200 expression and nerve regeneration after sciatic injury. •CDNF improved the remyelination and thickness of the regenerated sciatic nerve. •CDNF improved gastrocnemius muscle weight and sciatic functional recovery. -- Abstract: Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a

  9. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

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    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  10. Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology.

    Science.gov (United States)

    Reichenbach, Myriam; Lim, Tiongti; Reichenbach, Horst-Dieter; Guengoer, Tuna; Habermann, Felix A; Matthiesen, Marieke; Hofmann, Andreas; Weber, Frank; Zerbe, Holm; Grupp, Thomas; Sinowatz, Fred; Pfeifer, Alexander; Wolf, Eckhard

    2010-08-01

    Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.

  11. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    Science.gov (United States)

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  12. Correlations between structure, material properties and bioproperties in self-assembled β-hairpin peptide hydrogels

    Science.gov (United States)

    Hule, Rohan A.; Nagarkar, Radhika P.; Altunbas, Aysegul; Ramay, Hassna R.; Branco, Monica C.; Schneider, Joel P.; Pochan, Darrin J.

    2009-01-01

    A de novo designed β-hairpin peptide (MAX8), capable of undergoing intramolecular folding and consequent intermolecular self-assembly into a cytocompatible hydrogel, has been studied. A combination of small angle neutron scattering (SANS) and cryogenic-transmission electron microscopy (cryo-TEM) have been used to quantitatively investigate the MAX8 nanofibrillar hydrogel network morphology. A change in the peptide concentration from 0.5 to 2 wt% resulted in a denser fibrillar network as revealed via SANS by a change in the high q (q = (4π/λ) × sin (θ/2), where λ = wavelength of incident neutrons and θ = scattering angle) mass fractal exponent from 2.5 to 3 and by a decrease in the measured correlation length from 23 to 16 A. Å slope of −4 in the USANS regime indicates well-defined gel microporosity, an important characteristic for cellular substrate applications. These changes, both at the network as well as the individual fibril lengthscales, can be directly visualized in situ by cryo-TEM. Fibrillar nanostructures and network properties are directly related to bulk hydrogel stiffness via oscillatory rheology. Preliminary cell viability and anchorage studies at varying hydrogel stiffness confirm cell adhesion at early stages of cell culture within the window of stiffness investigated. Knowledge of the precise structure spanning length scales from the nanoscale up to the microscale can help in the formation of future, specific structure-bioproperty relationships when studying in vitro and in vivo behavior of these new peptide scaffolds. PMID:19048999

  13. Function and anatomy of plant siRNA pools derived from hairpin transgenes

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    Lee Kevin AW

    2007-11-01

    Full Text Available Abstract Background RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs. Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants. Results Testing the gene silencing capacity of plant-derived siRNAs in mammalian cells proved to be very challenging and required partial siRNA purification and design of a highly sensitive assay. Using the above assay we found that plant-derived siRNAs are ineffective for gene silencing in mammalian cells. Plant-derived siRNAs are almost exclusively double-stranded and most likely comprise a mixture of bona fide siRNAs and aberrant partially complementary duplexes. We also provide indirect evidence that plant-derived siRNAs may contain a hitherto undetected physiological modification, distinct from 3' terminal 2-O-methylation. Conclusion siRNAs produced from plant hairpin transgenes and extracted from plants are ineffective for gene silencing in mammalian cells. Thus our findings establish that a previous claim that transgenic plants offer a cost-effective, scalable and sustainable source of siRNAs is unwarranted. Our results also indicate that the presence of aberrant siRNA duplexes and possibly a plant-specific siRNA modification, compromises the gene silencing capacity of plant-derived siRNAs in mammalian cells.

  14. Lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells.

    Science.gov (United States)

    Palii, Carmen G; Pasha, Roya; Brand, Marjorie

    2011-07-16

    the erythroid lineage. Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

  15. Lentiviral Reprogramming of A-T Patient Fibroblasts to Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Nayler, Sam; Kozlov, Sergei V; Lavin, Martin F; Wolvetang, Ernst

    2017-01-01

    Reprogramming of cells enables generation of pluripotent stem cells and resulting progeny through directed differentiation, making this technology an invaluable tool for the study of human development and disease. Reprogramming occurs with a wide range of efficiency, a culmination of intrinsic and extrinsic factors including the tissue of origin, the passage number and culture history of the target cells. Another major factor affecting reprogramming is the methodology used and the quality of the reprogramming process itself, including for conventional viral-based approaches viral titer and subsequent viral transduction efficiency, including downstream transgene insertion and stoichiometry. Genetic background is an important parameter affecting the efficiency of the reprogramming process with reports that cells from individuals harboring specific mutations are more difficult to reprogram than control counterparts.Ataxia-Telangiectasia (A-T) fibroblasts underwent reprogramming at reduced efficiency in contrast to their controls. To optimize reprogramming of fibroblasts from patients with A-T, we examined the response of A-T cells to various cell culture conditions after lentiviral transduction with reprogramming factors Oc4/Sox2 (pSIN4-EF2-O2S) and Klf4/c-Myc (pSIN4-CMV-K2M). Parameters included media type (KSR or serum-containing DMEM), treatment with a p53 inhibitor (small-molecule cyclic pifithrin-α), and either a low or high concentration of bFGF. Post-transduction, equivalent numbers of cells from heterozygote and homozygote patients were plated and assessed at regular intervals for survival and proliferation. Our findings indicate that A-T cells responded favorably to the addition of FCS and gradual weaning away from their native media into KSR-containing stem cell media that produced suitable conditions for their reprogramming. We examined a range of properties to identify and isolate good quality iPSCs including the expression status of important stem cell

  16. Integrase Defective Lentiviral Vector as a Vaccine Platform for Delivering Influenza Antigens

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    Alessandra Gallinaro

    2018-02-01

    Full Text Available Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA and nucleoprotein (NP were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively. Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate

  17. Natural host genetic resistance to lentiviral CNS disease: a neuroprotective MHC class I allele in SIV-infected macaques.

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    Joseph L Mankowski

    Full Text Available Human immunodeficiency virus (HIV infection frequently causes neurologic disease even with anti-retroviral treatment. Although associations between MHC class I alleles and acquired immunodeficiency syndrome (AIDS have been reported, the role MHC class I alleles play in restricting development of HIV-induced organ-specific diseases, including neurologic disease, has not been characterized. This study examined the relationship between expression of the MHC class I allele Mane-A*10 and development of lentiviral-induced central nervous system (CNS disease using a well-characterized simian immunodeficiency (SIV/pigtailed macaque model. The risk of developing CNS disease (SIV encephalitis was 2.5 times higher for animals that did not express the MHC class I allele Mane-A*10 (P = 0.002; RR = 2.5. Animals expressing the Mane-A*10 allele had significantly lower amounts of activated macrophages, SIV RNA, and neuronal dysfunction in the CNS than Mane-A*10 negative animals (P<0.001. Mane-A*10 positive animals with the highest CNS viral burdens contained SIV gag escape mutants at the Mane-A*10-restricted KP9 epitope in the CNS whereas wild type KP9 sequences dominated in the brain of Mane-A*10 negative animals with comparable CNS viral burdens. These concordant findings demonstrate that particular MHC class I alleles play major neuroprotective roles in lentiviral-induced CNS disease.

  18. Immune Modulatory Cell Therapy for Hemophilia B Based on CD20-Targeted Lentiviral Gene Transfer to Primary B Cells

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    Xiaomei Wang

    2017-06-01

    Full Text Available Gene-modified B cells expressing immunoglobulin G (IgG fusion proteins have been shown to induce tolerance in several autoimmune and other disease models. However, lack of a vector suitable for gene transfer to human B cells has been an obstacle for translation of this approach. To overcome this hurdle, we developed an IgG-human factor IX (hFIX lentiviral fusion construct that was targeted to specifically transduce cells expressing human CD20 (hCD20. Receptor-specific retargeting by mutating envelope glycoproteins of measles virus (MV-lentiviral vector (LV and addition of a single-chain variable fragment specific for hCD20 resulted in gene delivery into primary human and transgenic hCD20 mouse B cells with high specificity. Notably, this protocol neither required nor induced activation of the B cells, as confirmed by minimal activation of inflammatory cytokines. Using this strategy, we were able to demonstrate induction of humoral tolerance, resulting in suppression of antibody formation against hFIX in a mouse model of hemophilia B (HB. In conclusion, transduction of receptor-specific retargeted LV into resting B cells is a promising method to develop B cell therapies for antigen-specific tolerance induction in human disease.

  19. An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs.

    Science.gov (United States)

    Shan, Zhixin; Lin, Qiuxiong; Deng, Chunyu; Li, Xiaohong; Huang, Wei; Tan, Honghong; Fu, Yongheng; Yang, Min; Yu, Xi-Yong

    2009-07-01

    Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293 cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins are a good strategy for gene silencing.

  20. The mitochondrial genomes of sponges provide evidence for multiple invasions by Repetitive Hairpin-forming Elements (RHE

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    Lavrov Dennis V

    2009-12-01

    Full Text Available Abstract Background The mitochondrial (mt genomes of sponges possess a variety of features, which appear to be intermediate between those of Eumetazoa and non-metazoan opisthokonts. Among these features is the presence of long intergenic regions, which are common in other eukaryotes, but generally absent in Eumetazoa. Here we analyse poriferan mitochondrial intergenic regions, paying particular attention to repetitive sequences within them. In this context we introduce the mitochondrial genome of Ircinia strobilina (Lamarck, 1816; Demospongiae: Dictyoceratida and compare it with mtDNA of other sponges. Results Mt genomes of dictyoceratid sponges are identical in gene order and content but display major differences in size and organization of intergenic regions. An even higher degree of diversity in the structure of intergenic regions was found among different orders of demosponges. One interesting observation made from such comparisons was of what appears to be recurrent invasions of sponge mitochondrial genomes by repetitive hairpin-forming elements, which cause large genome size differences even among closely related taxa. These repetitive hairpin-forming elements are structurally and compositionally divergent and display a scattered distribution throughout various groups of demosponges. Conclusion Large intergenic regions of poriferan mt genomes are targets for insertions of repetitive hairpin- forming elements, similar to the ones found in non-metazoan opisthokonts. Such elements were likely present in some lineages early in animal mitochondrial genome evolution but were subsequently lost during the reduction of intergenic regions, which occurred in the Eumetazoa lineage after the split of Porifera. Porifera acquired their elements in several independent events. Patterns of their intra-genomic dispersal can be seen in the mt genome of Vaceletia sp.

  1. Stabilization of the beta-hairpin in Mason-Pfizer monkey virus capsid protein- a critical step for infectivity

    Czech Academy of Sciences Publication Activity Database

    Obr, M.; Hadravová, Romana; Doležal, Michal; Křížová, Ivana; Papoušková, V.; Žídek, L.; Hrabal, R.; Ruml, T.; Rumlová, Michaela

    2014-01-01

    Roč. 11, Oct 30 (2014), 94/1-94/14 ISSN 1742-4690 R&D Projects: GA ČR(CZ) GA14-15326S; GA MŠk LO1302 Grant - others:GA MŠk(CZ) ED1.1.00/02.0068; Seventh Framework Programme of the European Union(XE) FP7-261863 Program:ED Institutional support: RVO:61388963 Keywords : retrovirus * assembly * M-PMV * capsid protein * maturation * beta-hairpin Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/94

  2. Structure of 23S rRNA hairpin 35 and its interaction with the tylosin-resistance methyltransferase RlmAII

    Science.gov (United States)

    Lebars, Isabelle; Yoshizawa, Satoko; Stenholm, Anne R.; Guittet, Eric; Douthwaite, Stephen; Fourmy, Dominique

    2003-01-01

    The bacterial rRNA methyltransferase RlmAII (formerly TlrB) contributes to resistance against tylosin-like 16-membered ring macrolide antibiotics. RlmAII was originally discovered in the tylosin-producer Streptomyces fradiae, and members of this subclass of methyltransferases have subsequently been found in other Gram-positive bacteria, including Streptococcus pneumoniae. In all cases, RlmAII methylates 23S rRNA at nucleotide G748, which is situated in a stem–loop (hairpin 35) at the macrolide binding site of the ribosome. The conformation of hairpin 35 recognized by RlmAII is shown here by NMR spectroscopy to resemble the anticodon loop of tRNA. The loop folds independently of the rest of the 23S rRNA, and is stabilized by a non-canonical G–A pair and a U-turn motif, rendering G748 accessible. Binding of S.pneumoniae RlmAII induces changes in NMR signals at specific nucleotides that are involved in the methyltransferase–RNA interaction. The conformation of hairpin 35 that interacts with RlmAII is radically different from the structure this hairpin adopts within the 50S subunit. This indicates that the hairpin undergoes major structural rearrangement upon interaction with ribosomal proteins during 50S assembly. PMID:12514124

  3. Short philtrum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003302.htm Short philtrum To use the sharing features on this page, please enable JavaScript. A short philtrum is a shorter than normal distance between ...

  4. In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

    DEFF Research Database (Denmark)

    Holmgaard, Andreas; Askou, Anne Louise; Benckendorff, Josephine Natalia Esther

    2017-01-01

    Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp) Cas9, delivered by lentiviral vectors (LVs), can be used...

  5. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2011-01-01

    Full Text Available Abstract Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV movement protein (MP gene and the partial Cucumber mosaic virus (CMV replication protein (Rep gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58 immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32 immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃ did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

  6. HTS dual-band bandpass filters using stub-loaded hair-pin resonators for mobile communication systems

    Energy Technology Data Exchange (ETDEWEB)

    Sekiya, N., E-mail: nsekiya@yamanashi.ac.jp; Sugiyama, S.

    2014-09-15

    Highlights: • We have developed a HTS five-pole dual-band bandpass filter using stub-loaded hair-pin resonators. • The proposed dual-band BPF can independently control of the center frequency. • Flexibly adjustment of the bandwidth can be achieved by the H-shaped waveguide. • The proposed BPF is evaluated by simulation and measurement with good agreement. - Abstract: A HTS dual-band bandpass filter is developed to obtain sharp-cut off characteristics for mobile communication systems. The filter is composed of five stub-loaded hair-pin resonators with H-shaped waveguides between them. The main advantage of the proposed filter is to allow independent control of the center frequency of the first and second bands. The bandwidths can be flexibly adjusted using the H-shaped waveguide. An electromagnetic simulator was used to design and analyze the filter, which have a 3.5-GHz center frequency and a 70-MHz (2%) bandwidth for the first band and a 5.0-GHz center frequency and a 100-MHz (2%) bandwidth for the second band. The filter was fabricated using YBa{sub 2}Cu{sub 3}O{sub y} thin film on an Al{sub 2}O{sub 3} substrate. Ground plane was fabricated using Au thin film. The measured frequency responses of the filter tally well with the simulated ones.

  7. Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

    Science.gov (United States)

    Zargarian, Loussiné; Kanevsky, Igor; Bazzi, Ali; Boynard, Jonathan; Chaminade, Françoise; Fossé, Philippe; Mauffret, Olivier

    2009-01-01

    First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction. PMID:19417069

  8. Folding topology of a bimolecular DNA quadruplex containing a stable mini-hairpin motif within the diagonal loop.

    Science.gov (United States)

    Balkwill, Graham D; Garner, Thomas P; Williams, Huw E L; Searle, Mark S

    2009-02-06

    We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G(3)ACGTAGTG(3))(2) containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G(3)T(4)G(3))(2) dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T(4)-loop sequence. A truncated version of the bimolecular quadruplex d(G(2)ACGTAGTG(2))(2), with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.

  9. Structure of HIV-1 reverse transcriptase bound to a novel 38-mer hairpin template-primer DNA aptamer.

    Science.gov (United States)

    Miller, Matthew T; Tuske, Steve; Das, Kalyan; DeStefano, Jeffrey J; Arnold, Eddy

    2016-01-01

    The development of a modified DNA aptamer that binds HIV-1 reverse transcriptase (RT) with ultra-high affinity has enabled the X-ray structure determination of an HIV-1 RT-DNA complex to 2.3 Å resolution without the need for an antibody Fab fragment or RT-DNA cross-linking. The 38-mer hairpin-DNA aptamer has a 15 base-pair duplex, a three-deoxythymidine hairpin loop, and a five-nucleotide 5'-overhang. The aptamer binds RT in a template-primer configuration with the 3'-end positioned at the polymerase active site and has 2'-O-methyl modifications at the second and fourth duplex template nucleotides that interact with the p66 fingers and palm subdomains. This structure represents the highest resolution RT-nucleic acid structure to date. The RT-aptamer complex is catalytically active and can serve as a platform for studying fundamental RT mechanisms and for development of anti-HIV inhibitors through fragment screening and other approaches. Additionally, the structure allows for a detailed look at a unique aptamer design and provides the molecular basis for its remarkably high affinity for RT. © 2015 The Protein Society.

  10. Selective inhibition of HIV-1 reverse transcriptase (HIV-1 RT) RNase H by small RNA hairpins and dumbbells.

    Science.gov (United States)

    Hannoush, Rami N; Carriero, Sandra; Min, Kyung-Lyum; Damha, Masad J

    2004-04-02

    We present here the design of a novel class of RNA inhibitors of the RNase H domain of HIV-1 RT, a ribonuclease activity that is essential for viral replication in vivo. Specifically, we show that small RNA hairpins and dumbbells can selectively inhibit the RNase H activity of HIV-1 RT without affecting other cellular RNases H (e.g., E. coli and human RNase H). These results suggest that the inhibitors do not interact with the nucleic acid binding site of RT RNase H, as this region should be well conserved among the various enzymes. The most potent inhibitors displayed IC50 values in the 3-8 microM range. Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by the hairpin and dumbbell aptamers, a property not previously observed for any nucleic acid aptamer directed against RT RNase H. The results described here suggest a noncompetitive binding mechanism, as outlined in the differential inhibitory characteristics of each of the nucleic acid aptamers against the bacterial, human, and viral RNase H homologues.

  11. Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme.

    Science.gov (United States)

    Wilson, T J; Zhao, Z Y; Maxwell, K; Kontogiannis, L; Lilley, D M

    2001-02-20

    The hairpin ribozyme in its natural context consists of two loops in RNA duplexes that are connected as arms of a four-way helical junction. Magnesium ions induce folding into the active conformation in which the two loops are in proximity. In this study, we have investigated nucleotides that are important to this folding process. We have analyzed the folding in terms of the cooperativity and apparent affinity for magnesium ions as a function of changes in base sequence and functional groups, using fluorescence resonance energy transfer. Our results suggest that the interaction between the loops is the sum of a number of component interactions. Some sequence variants such as A10U, G+1A, and C25U exhibit loss of cooperativity and reduced affinity of apparent magnesium ion binding. These variants are also very impaired in ribozyme cleavage activity. Nucleotides A10, G+1, and C25 thus appear to be essential in creating the conformational environment necessary for ion binding. The double variant G+1A/C25U exhibits a marked recovery of both folding and catalytic activity compared to either individual variant, consistent with the proposal of a triple-base interaction among A9, G+1, and C25 [Pinard, R., Lambert, D., Walter, N. G., Heckman, J. E., Major, F., and Burke, J. M. (1999) Biochemistry 38, 16035-16039]. However, substitution of A9 leads to relatively small changes in folding properties and cleavage activity, and the double variant G+1DAP/C25U (DAP is 2,6-diaminopurine), which could form an isosteric triple-base interaction, exhibits folding and cleavage activities that are both very impaired compared to those of the natural sequence. Our results indicate an important role for a Watson--Crick base pair between G+1 and C25; this may be buttressed by an interaction with A9, but the loss of this has less significant consequences for folding. 2'-Deoxyribose substitution leads to folding with reduced magnesium ion affinity in the following order: unmodified RNA > dA9

  12. Construction of a single lentiviral vector containing tetracycline-inducible Alb-uPA for transduction of uPA expression in murine hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jiasi Bai

    Full Text Available The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1]. But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2]. In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under "Tet-on/off" system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.

  13. Extraordinarily stable mini-hairpins: electrophoretical and thermal properties of the various sequence variants of d(GCGAAAGC) and their effect on DNA sequencing.

    OpenAIRE

    Hirao, I; Nishimura, Y; Tagawa, Y; Watanabe, K; Miura, K

    1992-01-01

    A small DNA fragment having a characteristic sequence d(GCGAAAGC) has been shown to form an extraordinarily stable mini-hairpin structure and to have an unusually rapid mobility in polyacrylamide gel electrophoresis, even when containing 7M urea. Here, we have studied the stability of the various sequence variants of d(GCGAAAGC) and the corresponding RNA fragments. Many such sequence variants form stable mini-hairpins in a similar manner to the d(GCGAAAGC) sequence. The RNA fragment, r(GCGAAA...

  14. Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.

    Science.gov (United States)

    Farley, Daniel C; Bannister, Richard; Leroux-Carlucci, Marie A; Evans, Nerys E; Miskin, James E; Mitrophanous, Kyriacos A

    2012-10-01

    The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.

  15. Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice

    Directory of Open Access Journals (Sweden)

    Sytwu Huey-Kang

    2009-08-01

    Full Text Available Abstract Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD mice. Methods Islets were isolated from NOD mice and transduced with lentivirus carrying TRX (Lt-TRX or enhanced green fluorescence protein (Lt-eGFP, respectively. Transduced islets were transplanted under the left kidney capsule of female diabetic NOD mice, and blood glucose concentration was monitored daily after transplantation. The histology of the islet graft was assessed at the end of the study. The protective effect of TRX on islets was investigated. Results The lentiviral vector effectively transduced islets without altering the glucose-stimulating insulin-secretory function of islets. Overexpression of TRX in islets reduced hydrogen peroxide-induced cytotoxicity in vitro. After transplantation into diabetic NOD mice, euglycemia was maintained for significantly longer in Lt-TRX-transduced islets than in Lt-eGFP-transduced islets; the mean graft survival was 18 vs. 6.5 days (n = 9 and 10, respectively, p Conclusion We successfully transduced the TRX gene into islets and demonstrated that these genetically modified grafts are resistant to inflammatory insult and survived longer in diabetic recipients. Our results further support the concept that the reactive oxygen species (ROS scavenger and antiapoptotic functions of TRX are critical to islet survival after

  16. 3' self-inactivating long terminal repeat inserts for the modulation of transgene expression from lentiviral vectors.

    Science.gov (United States)

    Manic, Gwenola; Maurin-Marlin, Aurélie; Galluzzi, Lorenzo; Subra, Frédéric; Mouscadet, Jean-François; Bury-Moné, Stéphanie

    2012-04-01

    Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.

  17. Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation.

    Science.gov (United States)

    Papanikolaou, Eleni; Kontostathi, Georgia; Drakopoulou, Ekati; Georgomanoli, Maria; Stamateris, Evangelos; Vougas, Kostas; Vlahou, Antonia; Maloy, Andrew; Ware, Mark; Anagnou, Nicholas P

    2013-07-01

    Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudotyped lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6×10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy. Copyright © 2013 Elsevier B.V. All

  18. A Cell-Permeable Hairpin Peptide Inhibits Hepatitis C Viral Nonstructural Protein 5A Mediated Translation and Virus Production

    Science.gov (United States)

    Khachatoorian, Ronik; Arumugaswami, Vaithilingaraja; Ruchala, Piotr; Raychaudhuri, Santanu; Maloney, Eden M.; Miao, Edna; Dasgupta, Asim; French, Samuel W.

    2012-01-01

    NS5A is a key regulator of hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown NS5A to augment HCV IRES-mediated translation. Further, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also co-immunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) to be the interaction site. Further, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, while controls did not. Triple-alanine scan mutagenesis identified an exposed beta-sheet hairpin in C34 to be primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10 amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A

  19. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    International Nuclear Information System (INIS)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-01-01

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R h ) and reduced thermal stability in the mutant complex. Taken together

  20. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  1. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

    Science.gov (United States)

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

    2014-10-01

    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

  2. Short stature

    Science.gov (United States)

    ... as her parents. Providers call this "constitutional growth delay." If one or both parents are short, your ... urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. follows ...

  3. SHORT COMMUNICATION

    African Journals Online (AJOL)

    PROF P.T. KAYE

    . SHORT COMMUNICATION. Formation and Structural Analysis of Novel Dibornyl Ethers. Perry T. Kaye*, Andrew R. Duggan, Joseph M. Matjila, Warner E. Molema, and. Swarnam S. Ravindran. Department of Chemistry, Rhodes University, Grahamstown, ...

  4. Differential Effects of Strategies to Improve the Transduction Efficiency of Lentiviral Vector that Conveys an Anti-HIV Protein, Nullbasic, in Human T Cells.

    Science.gov (United States)

    Rustanti, Lina; Jin, Hongping; Li, Dongsheng; Lor, Mary; Sivakumaran, Haran; Harrich, David

    2018-03-14

    Nullbasic is a mutant form of HIV-1 Tat that has strong ability to protect cells from HIV-1 replication by inhibiting three different steps of viral replication: reverse transcription, Rev export of viral mRNA from the nucleus to the cytoplasm and transcription of viral mRNA by RNA polymerase II. We previously showed that Nullbasic inhibits transduction of human cells including T cells by HIV-1-based lentiviral vectors. Here we investigated whether the Nullbasic antagonists huTat2 (a Tat targeting intrabody), HIV-1 Tat or Rev proteins or cellular DDX1 protein could improve transduction by a HIV-1 lentiviral vector conveying Nullbasic-ZsGreen1 to human T cells. We show that overexpression of huTat2, Tat-FLAG and DDX1-HA in virus-like particle (VLP) producer cells significantly improved transduction efficiency of VLPs that convey Nullbasic in Jurkat cells. Specifically, co-expression of Tat-FLAG and DDX1-HA in the VLP producer cell improved transduction efficiency better than if used individually. Transduction efficiencies could be further improved by including a spinoculation step. However, the same optimised protocol and using the same VLPs failed to transduce primary human CD4 + T cells. The results imply that the effects of Nullbasic on VLPs on early HIV-1 replication are robust in human CD4 + T cells. Given this significant block to lentiviral vector transduction by Nullbasic in primary CD4 + T cells, our data indicate that gammaretroviral, but not lentiviral, vectors are suitable for delivering Nullbasic to primary human T cells.

  5. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

    Science.gov (United States)

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan

    2016-09-01

    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  6. In vivo knockdown of antisense non-coding mitochondrial RNAs by a lentiviral-encoded shRNA inhibits melanoma tumor growth and lung colonization.

    Science.gov (United States)

    Varas-Godoy, Manuel; Lladser, Alvaro; Farfan, Nicole; Villota, Claudio; Villegas, Jaime; Tapia, Julio C; Burzio, Luis O; Burzio, Veronica A; Valenzuela, Pablo D T

    2018-01-01

    The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

    Science.gov (United States)

    Nam, Bo Young; Kim, Dong Ki; Park, Jung Tak; Kang, Hye-Young; Paeng, Jisun; Kim, Seonghun; Park, Jimin; Um, Jae Eun; Oh, Hyung Jung; Han, Seung Hyeok; Yoo, Tae-Hyun; Kang, Shin-Wook

    2015-12-15

    In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species. Copyright © 2015 the American Physiological Society.

  8. Short Stature

    DEFF Research Database (Denmark)

    Christesen, Henrik Boye Thybo; Pedersen, Birgitte Tønnes; Pournara, Effie

    2016-01-01

    The use of appropriate growth standards/references is of significant clinical importance in assessing the height of children with short stature as it may determine eligibility for appropriate therapy. The aim of this study was to determine the impact of using World Health Organization (WHO) instead...... of national growth standards/references on height assessment in short children. Data were collected from routine clinical practice (1998-2014) from nine European countries that have available national growth references and were enrolled in NordiNet® International Outcome Study (IOS) (NCT00960128), a large......-scale, non-interventional, multinational study. The patient cohort consisted of 5996 short pediatric patients diagnosed with growth hormone deficiency (GHD), Turner syndrome (TS) or born small for gestational age (SGA). The proportions of children with baseline height standard deviation score (SDS) below...

  9. Receptor-targeted lentiviral vectors are exceptionally sensitive toward the biophysical properties of the displayed single-chain Fv.

    Science.gov (United States)

    Friedel, Thorsten; Hanisch, Lydia J; Muth, Anke; Honegger, Annemarie; Abken, Hinrich; Plückthun, Andreas; Buchholz, Christian J; Schneider, Irene C

    2015-04-01

    An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into CD30-positive cells. Fused to the C-terminus of the type-II transmembrane protein hemagglutinin (H) of measles virus and expressed in LV packaging cells, gene transfer mediated by the released LV particles was inefficient. A series of point mutations in the scFv framework regions addressing its biophysical properties, which substantially improved production and increased the melting temperature without impairing its kinetic binding behavior to CD30, also improved the performance of LV particles. Gene transfer into CD30-positive cells increased ∼100-fold due to improved transport of the H-scFv protein to the plasma membrane. Concomitantly, LV particle aggregation and syncytia formation in packaging cells were substantially reduced. The data suggest that syncytia formation can be triggered by trans-cellular dimerization of H-scFv proteins displayed on adjacent cells. Taken together, we show that the biophysical properties of the targeting ligand have a decisive role for the gene transfer efficiency of receptor-targeted LVs. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. IL10 Released by a New Inflammation-regulated Lentiviral System Efficiently Attenuates Zymosan-induced Arthritis

    Science.gov (United States)

    Garaulet, Guillermo; Alfranca, Arántzazu; Torrente, María; Escolano, Amelia; López-Fontal, Raquel; Hortelano, Sonsoles; Redondo, Juan M; Rodríguez, Antonio

    2013-01-01

    Administration of anti-inflammatory cytokines is a common therapeutic strategy in chronic inflammatory diseases. Gene therapy is an efficient method for delivering therapeutic molecules to target cells. Expression of the cell adhesion molecule E-selectin (ESEL), which is expressed in the early stages of inflammation, is controlled by proinflammatory cytokines, making its promoter a good candidate for the design of inflammation-regulated gene therapy vectors. This study describes an ESEL promoter (ESELp)-based lentiviral vector (LV) that drives localized transgene expression during inflammation. Mouse matrigel plug assays with ESELp-transduced endothelial cells showed that systemic lipopolysaccharide (LPS) administration selectively induces ESELp-controlled luciferase expression in vivo. Inflammation-specific induction was confirmed in a mouse model of arthritis, showing that this LV is repeatedly induced early in acute inflammation episodes and is downregulated during remission. Moreover, the local acute inflammatory response in this animal model was efficiently blocked by expression of the anti-inflammatory cytokine interleukin-10 (IL10) driven by our LV system. This inflammation-regulated expression system has potential application in the design of new strategies for the local treatment of chronic inflammatory diseases such as cardiovascular and autoimmune diseases. PMID:22760540

  11. A stable producer cell line for the manufacture of a lentiviral vector for gene therapy of Parkinson's disease.

    Science.gov (United States)

    Stewart, Hannah J; Fong-Wong, Liang; Strickland, Iain; Chipchase, Daniel; Kelleher, Michelle; Stevenson, Laura; Thoree, Vinay; McCarthy, Janine; Ralph, G Scott; Mitrophanous, Kyriacos A; Radcliffe, Pippa A

    2011-03-01

    ProSavin is an equine infectious anemia virus vector-based gene therapy for Parkinson's disease for which inducible HEK293T-based producer cell lines (PCLs) have been developed. These cell lines demonstrate stringent tetracycline-regulated expression of the packaging components and yield titers comparable to the established transient production system. A prerequisite for the use of PCL-derived lentiviral vectors (LVs) in clinical applications is the thorough characterization of both the LV and respective PCL with regard to identity and genetic stability. We describe the detailed characterization of two ProSavin PCLs (PS5.8 and PS46.2) and resultant ProSavin vector. The two cell lines demonstrate stable production of vector over a time period sufficient to allow generation of master and working cell banks, and subsequent large-scale vector production. ProSavin generated from the PCLs performs comparably in vivo to that produced by the standard transient transfection process with respect to transduction efficiency and immunogenicity. The development of ProSavin PCLs, and the detailed characterization described here, will aid the advancement of ProSavin for clinical application.

  12. The Comparative Value of Feline Virology Research: Can Findings from the Feline Lentiviral Vaccine Be Translated to Humans?

    Science.gov (United States)

    Hosie, Margaret J; Techakriengkrai, Navapon; Bęczkowski, Paweł M; Harris, Matthew; Logan, Nicola; Willett, Brian J

    2017-01-28

    Feline immunodeficiency virus (FIV) is a lentivirus of domestic cats that shares several similarities with its human counterpart, human immunodeficiency virus (HIV). Their analogies include genomic organization, lymphocyte tropism, viral persistence and induction of immunodeficiency. FIV is the only lentivirus for which a commercial vaccine is registered for prevention in either human or veterinary medicine. This provides a unique opportunity to investigate the mechanisms of protection induced by lentivirus vaccines at the population level and might contribute to the development of efficacious HIV vaccines. As well as having comparative value for vaccine studies, FIV research has shed some light on the relationship between lentiviral tropism and pathogenesis. Recent studies in our laboratory demonstrated that the interaction between FIV and its primary receptor changes as disease progresses, reminiscent of the receptor switch observed as disease progresses in HIV infected individuals. Here we summarise findings illustrating that, in addition to its veterinary significance, FIV has comparative value, providing a useful model to explore lentivirus-host interactions and to examine potential immune correlates of protection against HIV infection.

  13. Dendritic cell-targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation.

    Science.gov (United States)

    Kim, Jocelyn T; Liu, Yarong; Kulkarni, Rajan P; Lee, Kevin K; Dai, Bingbing; Lovely, Geoffrey; Ouyang, Yong; Wang, Pin; Yang, Lili; Baltimore, David

    2017-07-21

    Dendritic cell (DC) activation and antigen presentation are critical for efficient priming of T cell responses. Here, we study how lentiviral vectors (LVs) deliver antigen and activate DCs to generate T cell immunization in vivo. We report that antigenic proteins delivered in vector particles via pseudotransduction were sufficient to stimulate an antigen-specific immune response. The delivery of the viral genome encoding the antigen increased the magnitude of this response in vivo but was irrelevant in vitro. Activation of DCs by LVs was independent of MyD88, TRIF, and MAVS, ruling out an involvement of Toll-like receptor or RIG-I-like receptor signaling. Cellular DNA packaged in LV preparations induced DC activation by the host STING (stimulator of interferon genes) and cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase) pathway. Envelope-mediated viral fusion also activated DCs in a phosphoinositide 3-kinase-dependent but STING-independent process. Pseudotransduction, transduction, viral fusion, and delivery of cellular DNA collaborate to make the DC-targeted LV preparation an effective immunogen. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  14. Efficient transfer of HTLV-1 tax gene in various primary and immortalized cells using a flap lentiviral vector.

    Science.gov (United States)

    Royer-Leveau, Christelle; Mordelet, Elodie; Delebecque, Frédéric; Gessain, Antoine; Charneau, Pierre; Ozden, Simona

    2002-08-01

    Human T cell leukemia virus type 1 (HTLV-1) causes two major diseases: adult T-cell leukemia-lymphoma and tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). In order to understand the involvement of Tax protein in HTLV-1 pathogenesis, we constructed a HIV-1 based lentiviral vector containing the central DNA flap sequence and either the green fluorescent protein (GFP) or the HTLV-1 tax genes. Using these vectors, GFP and tax genes were introduced in several primary and immortalized cells of endothelial, lymphoid, astrocytic or macrophagic origin. As assessed by GFP expression, up to 100% efficiency of transduction was obtained for all cell types tested. Tax expression was detected by Western blot and immuno-fluorescence in the transduced cells. After transduction, the Tax transcriptional activity was confirmed by the transactivation of HTLV-1 LTR-lacZ or HTLV-1 LTR-GFP reporter genes. Increased CD25 and HLA DR expression was observed in human peripheral blood lymphocytes transduced with the Tax vector. These results indicate that both pathways of Tax transactivation, CREB (viral LTR) and NF-kappa B (CD25 and HLA DR), are functional after transduction by TRIP Tax vector. Therefore, this vector provides a useful tool for investigating the role of the Tax viral protein in the pathogenesis of diseases linked to HTLV-1 infection.

  15. An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

    Directory of Open Access Journals (Sweden)

    Vreugdenhil Erno

    2010-07-01

    Full Text Available Abstract Background This study compared the transduction efficiencies of an adeno-associated viral (AAV vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP, with a lentiviral (LV vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed, to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 108 or 1 × 109 genomic copies of AAV1-GFP and 1 × 105 transducing units of LV-dsRed. Results Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. Conclusions This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

  16. Establishment of clonal MIN-O transplant lines for molecular imaging via lentiviral transduction & in vitro culture.

    Directory of Open Access Journals (Sweden)

    David L Boucher

    Full Text Available As the field of molecular imaging evolves and increasingly is asked to fill the discovery and validation space between basic science and clinical applications, careful consideration should be given to the models in which studies are conducted. The MIN-O mouse model series is an established in vivo model of human mammary precancer ductal carcinoma in situ with progression to invasive carcinoma. This series of transplant lines is propagated in vivo and experiments utilizing this model can be completed in non-engineered immune intact FVB/n wild type mice thereby modeling the tumor microenvironment with biological relevance superior to traditional tumor cell xenografts. Unfortunately, the same qualities that make this and many other transplant lines more biologically relevant than standard cell lines for molecular imaging studies present a significant obstacle as somatic genetic re-engineering modifications common to many imaging applications can be technically challenging. Here, we describe a protocol for the efficient lentiviral transduction of cell slurries derived from precancerous MIN-O lesions, in vitro culture of "MIN-O-spheres" derived from single cell clones, and the subsequent transplantation of these spheres to produce transduced sublines suitable for optical imaging applications. These lines retain the physiologic and pathologic properties, including multilineage differentiation, and complex microanatomic interaction with the host stroma characteristic of the MIN-O model. We also present the in vivo imaging and immunohistochemical analysis of serial transplantation of one such subline and detail the progressive multifocal loss of the transgene in successive generations.

  17. Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis)

    Science.gov (United States)

    Franklin, S.P.; Troyer, J.L.; TerWee, J.A.; Lyren, L.M.; Kays, R.W.; Riley, S.P.D.; Boyce, W.M.; Crooks, K.R.; VandeWoude, S.

    2007-01-01

    Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide ap adequate alternative for screening of some species and is more easily adapted to field conditions. ?? Wildlife Disease Association 2007.

  18. Induction of broadly neutralising HCV antibodies in mice by integration-deficient lentiviral vector-based pseudotyped particles.

    Directory of Open Access Journals (Sweden)

    Yao Deng

    Full Text Available INTRODUCTION: Integration-deficient lentiviral vectors (IDLVs are a promising platform for immunisation to elicit both humoral immunity and cellular mediated immunity (CMI. Here, we compared the specific immunity in mice immunised via different regimens (homologous and cocktail with IDLV-based HCV pseudoparticles (HCVpps carrying pseudotyped glycoproteins E1E2 and bearing the HCV NS3 gene. Humoral and cell-mediated immune responses were also evaluated after IDLV-HCVpp immunisation combined with heterologous rAd5-CE1E2 priming protocols. Sera from the mice effectively elicited anti-E1, -E2, and -NS3 antibody responses, and neutralised various HCVpp subtypes (1a, 1b, 2a, 3a and 5a. No significant CMI was detected in the groups immunised with IDLV-based HCVpps. In contrast, the combination of rAd5-CE1E2 priming and IDLV-based HCVpp boosting induced significant CMI against multiple antigens (E1, E2, and NS3. CONCLUSION: IDLV-based HCVpps are a promising vaccination platform and the combination of rAd5-CE1E2 and IDLV-based HCVpp prime-boost strategy should be further explored for the development of a cross-protective HCV vaccine.

  19. Sindbis Virus-Pseudotyped Lentiviral Vectors Carrying VEGFR2-Specific Nanobody for Potential Transductional Targeting of Tumor Vasculature.

    Science.gov (United States)

    Ahani, Roshank; Roohvand, Farzin; Cohan, Reza Ahangari; Etemadzadeh, Mohammad Hossein; Mohajel, Nasir; Behdani, Mahdi; Shahosseini, Zahra; Madani, Navid; Azadmanesh, Kayhan

    2016-11-01

    Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s). For this purpose, either sequence of a VEGFR2-specific nanobody or its natural ligand (VEGF 121 ) was inserted into the binding site of sindbis virus E2 glycoprotein. In silico modeling data suggested that the inserted targeting motifs were exposed in the context of cSVE2s. Western blot analysis of LVs indicated the incorporation of cSVE2s into viral particles. Capture ELISA demonstrated the specificity/functionality of the incorporated cSVE2s. Transduction of 293/KDR (expressing VEGFR2) or 293T cells (negative control) by constructed LVs followed by fluorescent microscopy and flow cytometric analyses indicated selective transduction of 293/KDR cells (30 %) by both targeting motifs compared to 293T control cells (1-2 %). These results implied similar targeting properties of VEGFR2-specific nanobody compared to the VEGF 121 and indicated the potential for transductional targeting of tumor vasculature by the nanobody displaying LVs.

  20. Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Michelle Millington

    2009-07-01

    Full Text Available Hematopoietic stem cells (HSC, in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34(+ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.Using commercially available G-CSF mobilized peripheral blood (PB CD34(+ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI, transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV carrying enhanced green fluorescent protein (GFP was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+ cells.

  1. Short communication

    African Journals Online (AJOL)

    Personal

    Short communication. A PRELIMINARY SURVEY OF ERYTHROCEBUS PATAS IN ANBESA CHAKA,. BAMBESI WOREDA OF BENISHANGUL-GUMUZ REGION, WESTERN ETHIOPIA. Solomon Yirga 1, Manyingerew Shenkut 1, .... the guard or watch dog of the group whereas the females are group leaders that initiate ...

  2. Short communication

    African Journals Online (AJOL)

    UPuser

    South African Society for Animal Science. 27. Short communication. Polymorphisms of the CAST gene in the Meishan and five other pig populations in China. Q.S. Wang. 1. , Y.C. Pan. 1#. , L.B. Sun. 2 and H. Meng. 1. 1 Department of Animal Science, School of Agriculture and Biology, Shanghai Jiaotong University, ...

  3. Short Report

    African Journals Online (AJOL)

    2016-10-02

    Oct 2, 2016 ... Short Report. Identifying educational strategies to assess and address the health needs of communities is a high priority, ... using different learning strategies and activities. One such setting is the community, as .... Using the IDEA framework in an interprofessional didactic elective course to facilitate positive ...

  4. Recognition of a 10 base pair sequence of DNA and stereochemical control of the binding affinity of chiral hairpin polyamide-Hoechst 33258 conjugates.

    Science.gov (United States)

    Reddy, Putta Mallikarjuna; Toporowski, Joseph W; Kahane, Alexandra L; Bruice, Thomas C

    2005-12-15

    Chiral hairpin polyamides linked to a Hoechst 33258 analogue at the alpha-position of the hairpin turn amino acid (1,2) were synthesized on solid phase by adopting Fmoc and ivDde techniques. The DNA-binding properties of enantiomeric conjugates 1 and 2, and N-terminal linked conjugate 3 for 8-14bp sequences were determined by spectrofluorometric and thermal melting studies. Conjugates 1 and 2 recognize a 10bp sequence, while conjugate 3 recognizes a 9bp sequence. Interestingly, R-enantiomer 1 exhibited 10- to 30-fold higher binding affinities than S-enantiomer 2 for the DNA sequences studied. These binding differences were accounted for by molecular modeling studies, which revealed that the amide proton nearest to the chiral center in R-conjugate 1 is better positioned to form hydrogen bonds to the DNA bases, while S-conjugate 2 does not.

  5. Short ROSE-like RNA thermometers control IbpA synthesis in Pseudomonas species.

    Directory of Open Access Journals (Sweden)

    Stefanie S Krajewski

    Full Text Available The bacterial small heat shock protein IbpA protects client proteins from aggregation. Due to redundancy in the cellular chaperone network, deletion of the ibpA gene often leads to only a mild or no phenotypic defect. In this study, we show that a Pseudomonas putida ibpA deletion mutant has a severe growth defect under heat stress conditions and reduced survival during recovery revealing a critical role of IbpA in heat tolerance. Transcription of the ibpA gene depends on the alternative heat shock sigma factor σ(32. Production of IbpA protein only at heat shock temperatures suggested additional translational control. We conducted a comprehensive structural and functional analysis of the 5' untranslated regions of the ibpA genes from P. putida and Pseudomonas aeruginosa. Both contain a ROSE-type RNA thermometer that is substantially shorter and simpler than previously reported ROSE elements. Comprised of two hairpin structures only, they inhibit translation at low temperature and permit translation initiation after a temperature upshift. Both elements regulate reporter gene expression in Escherichia coli and ribosome binding in vitro in a temperature-dependent manner. Structure probing revealed local melting of the second hairpin whereas the first hairpin remained unaffected. High sequence and structure conservation of pseudomonal ibpA untranslated regions and their ability to confer thermoregulation in vivo suggest that short ROSE-like thermometers are commonly used to control IbpA synthesis in Pseudomonas species.

  6. Short ROSE-Like RNA Thermometers Control IbpA Synthesis in Pseudomonas Species

    Science.gov (United States)

    Krajewski, Stefanie S.; Nagel, Miriam; Narberhaus, Franz

    2013-01-01

    The bacterial small heat shock protein IbpA protects client proteins from aggregation. Due to redundancy in the cellular chaperone network, deletion of the ibpA gene often leads to only a mild or no phenotypic defect. In this study, we show that a Pseudomonas putida ibpA deletion mutant has a severe growth defect under heat stress conditions and reduced survival during recovery revealing a critical role of IbpA in heat tolerance. Transcription of the ibpA gene depends on the alternative heat shock sigma factor σ32. Production of IbpA protein only at heat shock temperatures suggested additional translational control. We conducted a comprehensive structural and functional analysis of the 5′ untranslated regions of the ibpA genes from P. putida and Pseudomonas aeruginosa. Both contain a ROSE-type RNA thermometer that is substantially shorter and simpler than previously reported ROSE elements. Comprised of two hairpin structures only, they inhibit translation at low temperature and permit translation initiation after a temperature upshift. Both elements regulate reporter gene expression in Escherichia coli and ribosome binding in vitro in a temperature-dependent manner. Structure probing revealed local melting of the second hairpin whereas the first hairpin remained unaffected. High sequence and structure conservation of pseudomonal ibpA untranslated regions and their ability to confer thermoregulation in vivo suggest that short ROSE-like thermometers are commonly used to control IbpA synthesis in Pseudomonas species. PMID:23741480

  7. Design of hybrid β-hairpin peptides with enhanced cell specificity and potent anti-inflammatory activity.

    Science.gov (United States)

    Liu, YiFan; Xia, Xi; Xu, Liang; Wang, YiZhen

    2013-01-01

    Antimicrobial peptides (AMPs) have attracted considerable attention for their broad-spectrum antimicrobial activity and reduced tendency to cause bacterial resistance. Emerging concerns over the host cytotoxicity of AMPs, however, may ultimately compromise their development as pharmaceuticals. In order to optimize AMPs with potent cell specificity and anti-inflammatory activity, we designed β-hairpin hybrid peptides based upon progetrin-1, bovine lactoferricin and cecropin A. The synthetic hybrid peptides LB-PG and CA-PG demonstrated high selectivity over a wide range of microbes from Gram-positive and Gram-negative bacteria in porcine red blood cells. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) show that these peptides kill microbial cells by penetrating the cell membrane and damaging the membrane envelope. Gel retardation demonstrates that the peptides have a high affinity for DNA, indicating an additional possible intracellular bactericidal mechanism. Moreover, the hybrid peptides inhibit the expression of LPS-induced proinflammatory cytokines and chemokines, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein 1(MCP-1), following LPS stimulation in RAW264.7 cells. Our results indicate that these hybrid peptides have considerable potential for future development as antimicrobial and anti-inflammatory agents. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Enzyme-free amplification for sensitive electrochemical detection of DNA via target-catalyzed hairpin assembly assisted current change.

    Science.gov (United States)

    Qian, Yong; Wang, Chunyan; Gao, Fenglei

    2014-12-01

    An isothermal, enzyme-free and sensitive method for electrochemical detection of DNA is proposed based on target catalyzed hairpin assembly and for signal amplification. Molecular beacon 1 (MB1) contains a ferrocene (Fc) tag, which was immobilized on the gold electrode as recognition probe to hybridize with target DNA. Then, molecular beacon 2 hybridized with the opened MB1, allowing the target to be displaced. The displaced target again triggered the next round of strand exchange reaction resulting in many Fc far away from the GE to achieve signal amplification for sensitive DNA detection. The current signal amplification strategy is relatively simple and inexpensive owing to avoid the use of any kind of enzyme or sophisticated equipment. It can achieve a sensitivity of 42 fM with a wide linear dynamic range from 10(-13) to 10(-9)M and discriminate mismatched DNA from perfect matched target DNA with a high selectivity. The proposed method showed excellent specificity, high sensitivity and low detection limit, and could be applied in analysis of real samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Fast and quantitative differentiation of single-base mismatched DNA by initial reaction rate of catalytic hairpin assembly.

    Science.gov (United States)

    Li, Chenxi; Li, Yixin; Xu, Xiao; Wang, Xinyi; Chen, Yang; Yang, Xiaoda; Liu, Feng; Li, Na

    2014-10-15

    The widely used catalytic hairpin assembly (CHA) amplification strategy generally needs several hours to accomplish one measurement based on the prevailingly used maximum intensity detection mode, making it less practical for assays where high throughput or speed is desired. To make the best use of the kinetic specificity of toehold domain for circuit reaction initiation, we developed a mathematical model and proposed an initial reaction rate detection mode to quantitatively differentiate the single-base mismatch. Using the kinetic mode, assay time can be reduced substantially to 10 min for one measurement with the comparable sensitivity and single-base mismatch differentiating ability as were obtained by the maximum intensity detection mode. This initial reaction rate based approach not only provided a fast and quantitative differentiation of single-base mismatch, but also helped in-depth understanding of the CHA system, which will be beneficial to the design of highly sensitive and specific toehold-mediated hybridization reactions. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. The hairpin structure of the 6F11F22F2 fragment from human fibronectin enhances gelatin binding

    Science.gov (United States)

    Pickford, Andrew R.; Smith, Steven P.; Staunton, David; Boyd, Jonathan; Campbell, Iain D.

    2001-01-01

    The solution structure of the 6F11F22F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous 6F1 and 2F2 modules. The buried surface area between 6F1 and 2F2 (∼870 Å2) is the largest intermodule interface seen in fibronectin to date. The dissection of 6F11F22F2 into the 6F11F2 pair and 2F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of 6F11F22F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear ‘string of beads’. PMID:11285216

  11. The influence of junction conformation on RNA cleavage by the hairpin ribozyme in its natural junction form.

    Science.gov (United States)

    Thomson, J B; Lilley, D M

    1999-01-01

    In the natural form of the hairpin ribozyme the two loop-carrying duplexes that comprise the majority of essential bases for activity form two adjacent helical arms of a four-way RNA junction. In the present work we have manipulated the sequence around the junction in a way known to perturb the global folding properties. We find that replacement of the junction by a different sequence that has the same conformational properties as the natural sequence gives closely similar reaction rate and Arrhenius activation energy for the substrate cleavage reaction. By comparison, rotation of the natural sequence in order to alter the three-dimensional folding of the ribozyme leads to a tenfold reduction in the kinetics of cleavage. Replacement with the U1 four-way junction that is resistant to rotation into the antiparallel structure required to allow interaction between the loops also gives a tenfold reduction in cleavage rate. The results indicate that the conformation of the junction has a major influence on the catalytic activity of the ribozyme. The results are all consistent with a role for the junction in the provision of a framework by which the loops are presented for interaction in order to create the active form of the ribozyme. PMID:10024170

  12. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    International Nuclear Information System (INIS)

    Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

    2012-01-01

    Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  13. Effects of short-hairpin RNA-inhibited {beta}-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Wenqing, E-mail: liangwenqing_1234@126.com [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Yang, Chengwei [Department of Spinal Surgery, Lanzhou General Hospital, Lanzhou Military Area Command, 333 Nanbinhe Road, Lanzhou 730050 (China); Qian, Yu [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Fu, Qiang, E-mail: chyygklwq@hotmail.com [Department of Orthopaedics, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433 (China)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.

  14. Target-induced structure switching of hairpin aptamers for label-free and sensitive fluorescent detection of ATP via exonuclease-catalyzed target recycling amplification.

    Science.gov (United States)

    Xu, Yunying; Xu, Jin; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-01-15

    In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a "mix-and-detect" fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules. © 2013 Elsevier B.V. All rights reserved.

  15. Natural abundance heteronuclear NMR studies of the T3 mini-loop hairpin in the terminal repeat of the adenoassociated virus 2.

    Science.gov (United States)

    Chou, S H; Tseng, Y Y; Chu, B Y

    2000-05-01

    A DNA hairpin containing a T3 loop, as occurs in the terminal repeat of a popular gene therapy vector (Adenoassociated Virus 2, AAV2), has been extensively studied using homo- and heteronuclear NMR experiments. Almost complete assignment of the proton and carbon resonances, including H5'(Pro-S) and H5'(Pro-R) protons, has been accomplished at natural abundance. NOESY spectra in H2O and D2O have revealed many unusual NOEs, which, when combined with the epsilon, beta, gamma, and chi torsion angles determined from heteronuclear 1H-13C, 1H-31P, and 13C-31P coupling constants, have allowed for a more detailed picture of the T3 mini-loop hairpin. The three loop thymidines are all unpaired, yet are highly structured when bracketed by a 5'-GC...GC-3' stem sequence. The structure determined in this manuscript is considerably different from several other structures reported so far. Contrary to an RNA oligomer with a central U3 sequence that has the tendency to form a duplex with three U*U mismatches, the d(GAAGC-TTT-GCTTC) sequence exists mostly as a hairpin under millimolar NMR conditions. Since T3 triloop was found to be an essential element for the site-specific non-homologous integration of the AAV2 virus, and modification of the T3 loop residue abolishes such capability, the structure we report here may be of biological significance.

  16. Transduction of Photoreceptors With Equine Infectious Anemia Virus Lentiviral Vectors: Safety and Biodistribution of StarGen for Stargardt Disease

    Science.gov (United States)

    Binley, Katie; Widdowson, Peter; Loader, Julie; Kelleher, Michelle; Iqball, Sharifah; Ferrige, Georgina; de Belin, Jackie; Carlucci, Marie; Angell-Manning, Diana; Hurst, Felicity; Ellis, Scott; Miskin, James; Fernandes, Alcides; Wong, Paul; Allikmets, Rando; Bergstrom, Christopher; Aaberg, Thomas; Yan, Jiong; Kong, Jian; Gouras, Peter; Prefontaine, Annick; Vezina, Mark; Bussieres, Martin; Naylor, Stuart; Mitrophanous, Kyriacos A.

    2013-01-01

    Purpose. StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. Methods. Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. Results. Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. Conclusions. In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration. PMID:23620430

  17. Development of B-lineage predominant lentiviral vectors for use in genetic therapies for B cell disorders.

    Science.gov (United States)

    Sather, Blythe D; Ryu, Byoung Y; Stirling, Brigid V; Garibov, Mikhail; Kerns, Hannah M; Humblet-Baron, Stéphanie; Astrakhan, Alexander; Rawlings, David J

    2011-03-01

    Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin β (Igβ) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.

  18. Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Yang, Hee-Jong; Chwae, Yong-Joon; Kim, Kyongmin; Park, Sun; Shin, Ho-Joon

    2013-07-01

    Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

  19. Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography

    Directory of Open Access Journals (Sweden)

    Marino Michael P

    2009-02-01

    Full Text Available Abstract Background During the past twelve years, lentiviral (LV vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly. Methods Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography. Results Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 1010 TU were recovered from a single HYPERFlask vessel. Conclusion The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.

  20. Retroviral and Lentiviral Safety Analysis of Gene-Modified T Cell Products and Infused HIV and Oncology Patients.

    Science.gov (United States)

    Marcucci, Katherine T; Jadlowsky, Julie K; Hwang, Wei-Ting; Suhoski-Davis, Megan; Gonzalez, Vanessa E; Kulikovskaya, Irina; Gupta, Minnal; Lacey, Simon F; Plesa, Gabriela; Chew, Anne; Melenhorst, J Joseph; Levine, Bruce L; June, Carl H

    2018-01-03

    Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks with integrating viruses in gene-modified cell therapies. As such, the Food and Drug Administration guidances outline RCR/L-monitoring methods throughout the entire gene therapy treatment cycle. We present data for 17 vector lots, 375 manufactured T cell products, and 308 patients post-infusion across both HIV and oncology indications, showing no evidence of RCR/L. Given our data, a Poisson probability model estimates that a single patient, or a group of patients, would need to be followed for at least 52.8 years to observe one positive RCR/L event, highlighting the unlikelihood of RCR/L development. Additionally, we estimate the median time for lentivirus-modified T cell products to fall below the 1% vector sequence threshold in peripheral or whole blood that would trigger vector integration site analysis. These estimated times are 1.4 months in hematologic malignancies, 0.66 month in solid tumors, and 0.92 month in HIV. Based on these considerable safety data in HIV and oncology and recent Biologics License Applications filed for lentiviral-modified T cell products for hematologic malignancies, this may be an opportune time to re-evaluate the current guidelines for T cell gene therapy product testing and long-term patient monitoring. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  1. Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Kasahara Noriyuki

    2010-05-01

    Full Text Available Abstract Background Gene transfer to the gastrointestinal (GI mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD, GI infections, and cancer. Methods We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G-pseudotyped lentiviral vectors (LV for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal or firefly-luciferase (LV-fLuc reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining. Results Imaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP. Conclusions We have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

  2. Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography

    Science.gov (United States)

    Kutner, Robert H; Puthli, Sharon; Marino, Michael P; Reiser, Jakob

    2009-01-01

    Background During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly. Methods Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography. Results Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 1010 TU were recovered from a single HYPERFlask vessel. Conclusion The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols. PMID:19220915

  3. Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

    Science.gov (United States)

    Yang, Xiangdong; Niu, Lu; Zhang, Wei; He, Hongli; Yang, Jing; Xing, Guojie; Guo, Dongquan; Du, Qian; Qian, Xueyan; Yao, Yao; Li, Qiyun; Dong, Yingshan

    2017-10-01

    Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

  4. Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient

    Directory of Open Access Journals (Sweden)

    Yohei Sato

    Full Text Available Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA. Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

  5. Optimal construction and delivery of dual-functioning lentiviral vectors for type I collagen-suppressed chondrogenesis in synovium-derived mesenchymal stem cells.

    Science.gov (United States)

    Zhang, Feng; Yao, Yongchang; Zhou, Ruijie; Su, Kai; Citra, Fudiman; Wang, Dong-An

    2011-06-01

    This study aims to deliver both transforming growth factor β3 (TGF-β3) and shRNA targeting type I collagen (Col I) by optimal construction and application of various dual-functioning lentiviral vectors to induce Col I-suppressed chondrogenesis in synovium-derived mesenchymal stem cells (SMSCs). We constructed four lentiviral vectors (LV-1, LV-2, LV-3 and LV-4) with various arrangements of the two expression cassettes in different positions and orientations. Col I inhibition efficiency and chondrogenic markers were assessed with qPCR, ELISA and staining techniques. Among the four vectors, LV-1 has two distant and reversely oriented cassettes, LV-2 has two distant and same-oriented cassettes, LV-3 has two proximal and reversely oriented cassettes, and LV-4 has two proximal and same-oriented cassettes. Col I and chondrogenic markers, including type II collagen (Col II), aggrecan and glycosaminoglycan (GAG), were examined in SMSCs cultured in 3-D alginate hydrogel. All of the four vectors showed distinct effects in Col I level as well as diverse inductive efficiencies in upregulation of the cartilaginous markers. Based on real-time PCR results, LV-1 was optimal towards Col I-suppressed chondrogenesis. LV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.

  6. Induction of SerpinB2 and Th1/Th2 modulation by SerpinB2 during lentiviral infections in vivo.

    Directory of Open Access Journals (Sweden)

    Lee D Major

    Full Text Available SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic SIVmac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic SHIVmn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2(-/- mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2(-/- mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2(+/+ mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.

  7. The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

    Science.gov (United States)

    Papanikolaou, Eleni; Georgomanoli, Maria; Stamateris, Evangelos; Panetsos, Fottes; Karagiorga, Markisia; Tsaftaridis, Panagiotis; Graphakos, Stelios

    2012-01-01

    Abstract To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the Aγ-globin gene with the −117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of Aγ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro. PMID:21875313

  8. Combination of grafted Schwann cells and lentiviral-mediated prevention of glial scar formation improve recovery of spinal cord injured rats.

    Science.gov (United States)

    Do-Thi, Anh; Perrin, Florence E; Desclaux, Mathieu; Saillour, Paulette; Amar, Lahouari; Privat, Alain; Mallet, Jacques

    2016-10-01

    The present study was intended to combine three therapeutic approaches in a well-defined rat model of spinal cord injury, a lateral hemisection at thoracic level. A guidance channel was implanted at the lesion site. This channel was seeded with native Schwann cells or Schwann cells that had been previously transduced with a lentiviral vector carrying the GDNF gene. Thereafter, these experiences were reproduced in animals injected with lentiviral vectors carrying a shRNA for GFAP (Lv-shGFAP), which has recently been shown to block glial scar formation. Functional evaluations showed that Lv-shGFAP induced a significant improvement in recovery in animals grafted with Schwann cells. Histological studies demonstrated the outgrowth of axons in the guidance channel containing Schwann cells transduced or not with GDNF. This axonal growth was enhanced in rats receiving Lv-shGFAP vector. Also, a significant increase of serotonergic innervation of the injured hemicord, distal to the lesion, was found only in animals treated with Lv-shGFAP vectors. Importantly, this study confirms that glial scar formation is a major impediment for axonal sprouting after spinal cord injury, and emphasizes the importance of serotonergic innervation for locomotor function. Moreover we show a significant additive effect of a combinatorial approach to axonal regeneration in the injured spinal cord. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    Science.gov (United States)

    Wang, Weiming; Ye, Chaobaihui; Liu, Jingjing; Zhang, Di; Kimata, Jason T; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  10. Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

    Science.gov (United States)

    Liu, Yang; Reeves, Dara; Kropachev, Konstantin; Cai, Yuqin; Ding, Shuang; Kolbanovskiy, Marina; Kolbanovskiy, Alexander; Bolton, Judith L.; Broyde, Suse; Van Houten, Bennett; Geacintov, Nicholas E.

    2011-01-01

    Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend towards similar relative NER incision efficiencies for ~ 65% of these substrates; the other cases deviate mostly by ~ 30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER. PMID:21741328

  11. Conformation-Specific Infrared and Ultraviolet Spectroscopy of Cold [YAPAA+H]^{+} and [YGPAA+H]^{+} Ions: a Stereochemical "twist" on the β-HAIRPIN Turn

    Science.gov (United States)

    DeBlase, Andrew F.; Harrilal, Christopher P.; Lawler, John T.; Burke, Nicole L.; McLuckey, Scott A.; Zwier, Timothy S.

    2017-06-01

    Incorporation of the unnatural D-proline (^{D}P) stereoisomer into a polypeptide sequence is a typical strategy to encourage formation of β-hairpin loops because natural sequences are often unstructured in solution. Using conformation-specific IR and UV spectroscopy of cold (10 K) gas-phase ions, we probe the inherent conformational preferences of the ^{D}P and ^{L}P diastereomers in the protonated peptide [YAPAA+H]^{+}, where only intramolecular interactions are possible. Consistent with the solution phase studies, one of the conformers of [YADPAA+H]^{+} is folded into a charge-stabilized β-hairpin turn. However, a second predominant conformer family containing two sequential γ-turns is also identified, with similar energetic stability. A single conformational isomer of the ^{L}P diastereomer, [YALPAA+H]^{+}, is found and assigned to a structure that is not the anticipated "mirror image" β-turn. Instead, the ^{L}P stereo center promotes a cis alanine-proline amide bond. The assigned structures contain clues that the preference of the ^{D}P diastereomer to support a trans-amide bond and the proclivity of ^{L}P for a cis-amide bond is sterically driven and can be reversed by substituting glycine for alanine in position 2, forming [YGLPAA+H]^{+}. These results provide a basis for understanding the residue-specific and stereo-specific alterations in the potential energy surface that underlie these changing preferences, providing insights to the origin of β-hairpin formation.

  12. Metschnikowia Species Share a Pool of Diverse rRNA Genes Differing in Regions That Determine Hairpin-Loop Structures and Evolve by Reticulation.

    Directory of Open Access Journals (Sweden)

    Matthias Sipiczki

    Full Text Available Modern taxonomy of yeasts is mainly based on phylogenetic analysis of conserved DNA and protein sequences. By far the most frequently used sequences are those of the repeats of the chromosomal rDNA array. It is generally accepted that the rDNA repeats of a genome have identical sequences due to the phenomenon of sequence homogenisation and can thus be used for identification and barcoding of species. Here we show that the rDNA arrays of the type strains of Metschnikowia andauensis and M. fructicola are not homogenised. Both have arrays consisting of diverse repeats that differ from each other in the D1/D2 domains by up to 18 and 25 substitutions. The variable sites are concentrated in two regions that correspond to back-folding stretches of hairpin loops in the predicted secondary structure of the RNA molecules. The substitutions do not alter significantly the overall hairpin-loop structure due to wobble base pairing at sites of C-T transitions and compensatory mutations in the complementary strand of the hairpin stem. The phylogenetic and network analyses of the cloned sequences revealed that the repeats had not evolved in a vertical tree-like way but reticulation might have shaped the rDNA arrays of both strains. The neighbour-net analysis of all cloned sequences of the type strains and the database sequences of different strains further showed that these species share a continuous pool of diverse repeats that appear to evolve by reticulate evolution.

  13. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

    Directory of Open Access Journals (Sweden)

    Kenneth A Matreyek

    Full Text Available Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA protein mediated the dependency on cellular nucleoporin (NUP 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG-repeat enriched NUP153 C-terminal domain (NUP153(C. NUP153(C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C potently restricted HIV-1, providing an intracellular readout for the NUP153(C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV bound NUP153(C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74 inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6 protein, with Asn57 (Asp58 in EIAV playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors

  14. Effect of hairpin loop structure on reactivity, sequence preference and adduct orientation of a DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumour agent.

    Science.gov (United States)

    Thurston, David E; Vassoler, Higia; Jackson, Paul J M; James, Colin H; Rahman, Khondaker M

    2015-04-07

    The pyrrolobenzodiazepines (PBDs) are a family of covalent-binding DNA-interactive minor-groove binding agents with a thermodynamic preference for binding to 5'-Pu-G-Pu-3' sequences (Pu = Purine) but a kinetic preference for 5'-Py-G-Py-3' (Py = Pyrimidine). Using HPLC/MS methodology and a range of designed hairpin-forming oligonucleotides, the kinetics of reaction of a C8-bis-pyrrole pyrrolobenzodiazepine (PBD) conjugate (GWL-78, 2) with sixteen isomeric oligonucleotides has been evaluated, each containing a single PBD binding site in one of two locations. The PBD-binding base-pair triplets were designed to include every possible combination of A and T bases adjacent to the covalently-reacting guanine, with the set of hairpins consisting of isomeric pairs containing the same sequence in the hairpin stem but with either hexaethylene glycol (HEG) or TTT loops. The PBD 2 reacted most rapidly with TGT and TGA sequences, with the possibility that adducts might form in both the 3'- and 5'-directions with some sequences according to modelling studies. A faster reaction rate was observed for all hairpins containing the HEG loop except one (Seq 10) when the PBD binding triplets were located either near the loop or adjacent to the 5'-end. Modelling studies have suggested that this difference in reactivity could be due to the structural flexibility of the HEG loop allowing both A-ring-3' and A-ring-5' adducts to form, while a TTT loop should favour only A-ring-5' adducts due to steric considerations. These findings contrast with the results reported by Nguyen and Wilson for the interaction of non-covalent DNA-binding molecules with DNA hairpins, where the loop structure was found to have little effect on interaction in the main stem of the hairpin.

  15. Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals

    Science.gov (United States)

    Pridans, Clare; Lillico, Simon; Whitelaw, Bruce; Hume, David A

    2014-01-01

    The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large “cargo” for applications in gene therapy and vaccine delivery. PMID:26015955

  16. Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing.

    Science.gov (United States)

    Heider, Susanne; Muzard, Julien; Zaruba, Marianne; Metzner, Christoph

    2017-07-01

    Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.

  17. Immortalization and Characterization of Porcine Macrophages That Had Been Transduced with Lentiviral Vectors Encoding the SV40 Large T Antigen and Porcine Telomerase Reverse Transcriptase

    Directory of Open Access Journals (Sweden)

    Takato Takenouchi

    2017-08-01

    Full Text Available The domestic pig is an important agricultural animal, and thus, infectious diseases that affect pigs can cause severe economic losses in the global swine industry. Various porcine pathogens target macrophages, which are classical innate immune cells. Although macrophages basically protect the host from pathogens, they also seem to contribute to infectious processes. Therefore, cultured macrophages can be used to develop in vitro models for studying not only genes associated with porcine innate immunity but also the infectious processes of porcine pathogens. However, the availability of porcine macrophage cell lines is limited. In this study, we describe a novel immortalized porcine kidney-derived macrophage (IPKM cell line, which was generated by transferring the SV40 large T antigen (SV40LT and porcine telomerase reverse transcriptase (pTERT genes into primary porcine kidney-derived macrophages using lentiviral vectors. The IPKM displayed a typical macrophage morphology and was routinely passaged (doubling time: about 4 days. These cells were immunostained for macrophage markers. In addition, they exhibited substantial phagocytosis of polystyrene microbeads and released inflammatory cytokines upon lipopolysaccharide (LPS stimulation. Furthermore, the maturation and secretion of interleukin-1β were observed after nigericin-induced inflammasome activation in LPS-primed IPKM. These findings suggest that IPKM exhibit the typical inflammatory characteristics of macrophages. By transferring the SV40LT and pTERT genes using lentiviral vectors, we also successfully immortalized macrophages derived from the peripheral blood of a low-density lipoprotein receptor-deficient pig. These results suggest that the co-expression of SV40LT and pTERT is an effective way of immortalizing porcine macrophages.

  18. Targeting both viral and host determinants of human immunodeficiency virus entry, using a new lentiviral vector coexpressing the T20 fusion inhibitor and a selective CCL5 intrakine.

    Science.gov (United States)

    Petit, Nicolas; Dorgham, Karim; Levacher, Béatrice; Burlion, Aude; Gorochov, Guy; Marodon, Gilles

    2014-08-01

    Numerous strategies targeting early and late steps of the HIV life cycle have been proposed for gene therapy. However, targeting viral and host determinants of HIV entry is the only strategy that would prevent viral DNA-mediated CD4(+) cell death while diminishing the possibility for the virus to escape. To this end, we devised a bicistronic lentiviral vector expressing the membrane-bound form of the T20 fusion inhibitor, referred to as the C46 peptide, and a CCR5 superagonist, modified to sequester CCR5 away from the cell surface, referred to as the P2-CCL5 intrakine. We tested the effects of the vector on HIV infection and replication, using the human CEMR5 cell line expressing CD4 and CCR5, and primary human T cells. Transduced cells expressed the C46 peptide, detected with the 2F5 monoclonal antibody by flow cytometry. Expression of the P2-CCL5 intrakine correlates with lower levels of cell surface CCR5. Complete protection against HIV infection could be observed in cells expressing the protective transgenes. Importantly, we show that the combination of the transgenes was more potent than either transgene alone, showing the interest of expressing two entry inhibitors to inhibit HIV infection. Last, genetically modified cells possessed a selective advantage over nonmodified cells on HIV challenge in vitro, showing that modified cells were protected from HIV-induced cell death. Our results demonstrate that lentiviral vectors coexpressing the T20 fusion inhibitor and the P2-CCL5 intrakine represent promising tools for HIV gene therapy.

  19. Proximity hybridization-regulated catalytic DNA hairpin assembly for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Fuyi [School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou 221116 (China); Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, 221004, Xuzhou (China); Yao, Yao; Luo, Jianjun; Zhang, Xing; Zhang, Yu; Yin, Dengyang [Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, 221004, Xuzhou (China); Gao, Fenglei, E-mail: jsxzgfl@sina.com [Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical College, 221004, Xuzhou (China); Wang, Po, E-mail: wangpo@jsnu.edu.cn [School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou 221116 (China)

    2017-05-29

    Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH{sub 4} oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10{sup −15} to 10{sup −11} g mL{sup −1} and a detection limit of 0.43 × 10{sup −15} g mL{sup −1}. Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10{sup −16} g mL{sup −1}. And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. - Graphical abstract: A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA. - Highlights: • A novel enzyme-free electrochemical immunosensor was developed for detection of CEA. • The signal amplification was based on catalytic DNA hairpin assembly and DNA-template-synthesized Pd nanoparticles. • The biosensor could detect CEA down to 0.52 × 10{sup −16} g mL{sup −1} level with a dynamic range spanning 5 orders of magnitude.

  20. Eroding dipoles and vorticity growth for Euler flows in {{{R}}}^{3}: the hairpin geometry as a model for finite-time blowup

    Science.gov (United States)

    Childress, Stephen; Gilbert, Andrew D.

    2018-02-01

    A theory of an eroding ‘hairpin’ vortex dipole structure in three-dimensions is developed, extending our previous study of an axisymmetric eroding dipole without swirl. The axisymmetric toroidal dipole was found to lead to maximal growth of vorticity, as {t}4/3. The hairpin is here similarly proposed as a model to produce large ‘self-stretching’ of vorticity, with the possibility of finite-time blow-up. We derive a system of partial differential equations of ‘generalized’ form, involving contour averaging of a locally two-dimensional Euler flow. We do not attempt here to solve the system exactly, but point out that non-existence of physically acceptable solutions would most probably be a result of the axial flow. Because of the axial flow the vorticity distribution within the dipole eddies is no longer of the simple Sadovskii type (vorticity constant over a cross-section) obtained in the axisymmetric problem. Thus the solution of the system depends upon the existence of a larger class of propagating two-dimensional dipoles. The hairpin model is obtained by formal asymptotic analysis. As in the axisymmetric problem a local transformation to ‘shrinking’ coordinates is introduced, but now in a self-similar form appropriate to the study of a possible finite-time singularity. We discuss some properties of the model, including a study of the helicity and a first step in iterating toward a solution from the Sadovskii structure. We also present examples of two-dimensional propagating dipoles not previously studied, which have a vorticity profile consistent with our model. Although no rigorous results can be given, and analysis of the system is only partial, the formal calculations are consistent with the possibility of a finite time blowup of vorticity at a point of vanishing circulation of the dipole eddies, but depending upon the existence of the necessary two-dimensional propagating dipole. Our results also suggest that conservation of kinetic energy as

  1. A Dual-Band Band-Pass Filter with Overlap Step-Impedance and Capacitively Loaded Hairpin Resonators for Wireless LAN Systems

    Directory of Open Access Journals (Sweden)

    P. Chomtong

    2011-01-01

    Full Text Available This paper presents a dual-band band-pass filter using modified cross-coupled step-impedance and capacitively loaded hairpin resonators for WLAN systems. The proposed filter has been designed to operate at a fundamental frequency of 2.4 GHz and the first harmonics frequency of 5.2 GHz. The techniques of step impedance and load capacitor are combined in the design of the proposed filter. In particular, the techniques of modified cross-coupling and overlap resonators are applied to improve the response of insertion losses 21 at the first harmonic frequency of 5.2 GHz. The simulated and experimental results of insertion losses and return losses are better than 3 dB and 20 dB, respectively, at the operating frequencies.

  2. Design and Simulation of Microstrip Hairpin Bandpass Filter with Open Stub and Defected Ground Structure (DGS) at X-Band Frequency

    Science.gov (United States)

    Hariyadi, T.; Mulyasari, S.; Mukhidin

    2018-02-01

    In this paper we have designed and simulated a Band Pass Filter (BPF) at X-band frequency. This filter is designed for X-band weather radar application with 9500 MHz center frequency and bandwidth -3 dB is 120 MHz. The filter design was performed using a hairpin microstrip combined with an open stub and defected ground structure (DGS). The substrate used is Rogers RT5880 with a dielectric constant of 2.2 and a thickness of 1.575 mm. Based on the simulation results, it is found that the filter works on frequency 9,44 - 9,56 GHz with insertion loss value at pass band is -1,57 dB.

  3. Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms

    DEFF Research Database (Denmark)

    Galli, Andrea; Lai, Alessia; Corvasce, Stefano

    2008-01-01

    throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation......Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly...... with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six...

  4. Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli)*

    Science.gov (United States)

    Nolde, Svetlana B.; Vassilevski, Alexander A.; Rogozhin, Eugene A.; Barinov, Nikolay A.; Balashova, Tamara A.; Samsonova, Olga V.; Baranov, Yuri V.; Feofanov, Alexey V.; Egorov, Tsezi A.; Arseniev, Alexander S.; Grishin, Eugene V.

    2011-01-01

    This study presents purification, activity characterization, and 1H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold. PMID:21561864

  5. Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli).

    Science.gov (United States)

    Nolde, Svetlana B; Vassilevski, Alexander A; Rogozhin, Eugene A; Barinov, Nikolay A; Balashova, Tamara A; Samsonova, Olga V; Baranov, Yuri V; Feofanov, Alexey V; Egorov, Tsezi A; Arseniev, Alexander S; Grishin, Eugene V

    2011-07-15

    This study presents purification, activity characterization, and (1)H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.

  6. Natural short sleeper

    Science.gov (United States)

    Sleep - natural short sleeper ... 7 to 9 hours of sleep each night. Short sleepers sleep less than 75% of what is normal for their age. Natural short sleepers are different from people who chronically do ...

  7. Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD/SCIDγc-/-mice.

    Science.gov (United States)

    Levy, C; Fusil, F; Amirache, F; Costa, C; Girard-Gagnepain, A; Negre, D; Bernadin, O; Garaulet, G; Rodriguez, A; Nair, N; Vandendriessche, T; Chuah, M; Cosset, F-L; Verhoeyen, E

    2016-12-01

    Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc -/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc -/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein

  8. Hepatic lentiviral gene transfer prevents the long-term onset of hepatic tumours of glycogen storage disease type 1a in mice.

    Science.gov (United States)

    Clar, Julie; Mutel, Elodie; Gri, Blandine; Creneguy, Alison; Stefanutti, Anne; Gaillard, Sophie; Ferry, Nicolas; Beuf, Olivier; Mithieux, Gilles; Nguyen, Tuan Huy; Rajas, Fabienne

    2015-04-15

    Glycogen storage disease type 1a (GSD1a) is a rare disease due to the deficiency in the glucose-6-phosphatase (G6Pase) catalytic subunit (encoded by G6pc), which is essential for endogenous glucose production. Despite strict diet control to maintain blood glucose, patients with GSD1a develop hepatomegaly, steatosis and then hepatocellular adenomas (HCA), which can undergo malignant transformation. Recently, gene therapy has attracted attention as a potential treatment for GSD1a. In order to maintain long-term transgene expression, we developed an HIV-based vector, which allowed us to specifically express the human G6PC cDNA in the liver. We analysed the efficiency of this lentiviral vector in the prevention of the development of the hepatic disease in an original GSD1a mouse model, which exhibits G6Pase deficiency exclusively in the liver (L-G6pc(-/-) mice). Recombinant lentivirus were injected in B6.G6pc(ex3lox/ex3lox). SA(creERT2/w) neonates and G6pc deletion was induced by tamoxifen treatment at weaning. Magnetic resonance imaging was then performed to follow up the development of hepatic tumours. Lentiviral gene therapy restored glucose-6 phosphatase activity sufficient to correct fasting hypoglycaemia during 9 months. Moreover, lentivirus-treated L-G6pc(-/-) mice presented normal hepatic triglyceride levels, whereas untreated mice developed steatosis. Glycogen stores were also decreased although liver weight remained high. Interestingly, lentivirus-treated L-G6pc(-/-) mice were protected against the development of hepatic tumours after 9 months of gene therapy while most of untreated L-G6pc(-/-) mice developed millimetric HCA. Thus the treatment of newborns by recombinant lentivirus appears as an attractive approach to protect the liver from the development of steatosis and hepatic tumours associated to GSD1a pathology. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

    Science.gov (United States)

    Azzouz, Mimoun; Martin-Rendon, Enca; Barber, Robert D; Mitrophanous, Kyriacos A; Carter, Emma E; Rohll, Jonathan B; Kingsman, Susan M; Kingsman, Alan J; Mazarakis, Nicholas D

    2002-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.

  10. Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4⁺ T-Cells.

    Science.gov (United States)

    Urusov, Felix; Glazkova, Dina; Omelchenko, Denis; Bogoslovskaya, Elena; Tsyganova, Galina; Kersting, Katerina; Shipulin, German; Pokrovsky, Vadim

    2018-02-04

    C-C chemokine receptor type 5 (CCR5) is utilized by human immunodeficiency virus (HIV) as a co-receptor for cell entry. Suppression of the CCR5 gene by artificial microRNAs (amiRNAs) could confer cell resistance. In previous work, we created a lentivector that encoded the polycistron of two identical amiRNAs that could effectively suppress CCR5. However, tandem repeats in lentiviral vectors led to deletions of the repeated sequences during reverse transcription of the vector RNA. To solve this problem, we have created a new amiRNA against CCR5, mic1002, which has a different microRNA scaffold and targets a different sequence. Replacing one of the two identical tandem amiRNAs in the polycistron with the mic1002 amiRNA increased the accuracy of its lentiviral vector transfer while retaining its ability to effectively suppress CCR5. A lentiviral vector containing two heterogenic amiRNAs significantly inhibited HIV replication in a vector-transduced human CD4⁺ lymphocyte culture.

  11. Modified Lentiviral LTRs Allow Flp Recombinase–mediated Cassette Exchange and In Vivo Tracing of “Factor-free” Induced Pluripotent Stem Cells

    Science.gov (United States)

    Kuehle, Johannes; Turan, Soeren; Cantz, Tobias; Hoffmann, Dirk; Suerth, Julia D; Maetzig, Tobias; Zychlinski, Daniela; Klein, Christoph; Steinemann, Doris; Baum, Christopher; Bode, Juergen; Schambach, Axel

    2014-01-01

    Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for “safe harbor” integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy. PMID:24434935

  12. Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Hiroko Kita-Matsuo

    Full Text Available Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

  13. LV305, a dendritic cell-targeting integration-deficient ZVex(TM)-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response.

    Science.gov (United States)

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; Ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1.

  14. LV305, a dendritic cell-targeting integration-deficient ZVexTM-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response

    Science.gov (United States)

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1. PMID:27626061

  15. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

    Science.gov (United States)

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-04-19

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  16. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Benjamin Geiling

    Full Text Available The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG and retroviral (pREG vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs. In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

  17. A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN vectors and multi-colour analyses

    Directory of Open Access Journals (Sweden)

    Prokofjeva Maria M

    2013-01-01

    Full Text Available Abstract Background Despite progress in the development of combined antiretroviral therapies (cART, HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. Methods We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN LeGO vector technology. Results We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. Conclusions The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.

  18. In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

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    Andreas Holmgaard

    2017-12-01

    Full Text Available Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp Cas9, delivered by lentiviral vectors (LVs, can be used in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs induce in vitro indel formation at high frequencies (44%–93%. A single unilateral subretinal injection facilitates RPE-specific localization of the vector and disruption of Vegfa in isolated eGFP+ RPE cells obtained from mice five weeks after LV administration. Notably, sgRNA delivery results in the disruption of Vegfa with an in vivo indel formation efficacy of up to 84%. Sequencing of Vegfa-specific amplicons reveals formation of indels, including 4-bp deletions and 2-bp insertions. Taken together, our data demonstrate the capacity of lentivirus-delivered SpCas9 and sgRNAs as a developing therapeutic path in the treatment of ocular diseases, including age-related macular degeneration.

  19. Inhibiting PHD2 in bone marrow mesenchymal stem cells via lentiviral vector-mediated RNA interference facilitates the repair of periodontal tissue defects in SD rats

    Science.gov (United States)

    Chen, Changxing; Li, Houxuan; Jiang, Jun; Zhang, Qian; Yan, Fuhua

    2017-01-01

    Hypoxia-inducible factors (HIFs) play an important role in angiogenesis, and they can activate the expression of several downstream angiogenic factors. HIF-1 is a major transcriptor of HIFs, composed of α and β subunits. Prolyl hydroxylase domain-containing protein 2 (PHD2) is the main catabolic enzyme for HIF-1α, and it can accelerate its degradation under normoxic conditions. PHD2 expression in bone marrow mesenchymal stem cells (BMMSCs) of SD rats was down-regulated under normoxic conditions in this study by utilizing lentiviral vector-mediated RNA interference to promote HIF-1α accumulation, thus enhancing the expression of angiogenic factors. A tissue-engineered compound was constructed using the composite collagen membrane of BMMSCs after PHD2 gene silencing to repair periodontal fenestration defects in SD rats. The results of this study indicated that, after PHD2 gene silencing, the osteogenic differentiation of BMMSCs was enhanced in vitro, the resistance of cells to oxidative stress was also validated in vitro, thereby illustrating the promotion of the repair of artificially constructed periodontal tissue defects in rats. The results of this study provide a reference and guidance for future applications of RNA interference in periodontal tissue engineering and serve as a basis for improving the survival of seed cells in recipient tissues. PMID:29069818

  20. Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

    Directory of Open Access Journals (Sweden)

    X. Joann You

    2011-01-01

    Full Text Available Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs. Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein—before and after differentiation—all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

  1. A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

    International Nuclear Information System (INIS)

    Strauss, Bryan E.; Patricio, Juliana Rotelli; Vieira de Carvalho, Anna Carolina; Bajgelman, Marcio C.

    2006-01-01

    We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for this factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis

  2. Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

    Science.gov (United States)

    Huston, Marshall W; Riegman, Adriaan R A; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P; Wagemaker, Gerard

    2014-10-01

    Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning.

  3. Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection.

    Science.gov (United States)

    Pandolfini, Tiziana; Molesini, Barbara; Avesani, Linda; Spena, Angelo; Polverari, Annalisa

    2003-06-25

    Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in

  4. Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

    Directory of Open Access Journals (Sweden)

    Spena Angelo

    2003-06-01

    Full Text Available Abstract Background Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS, is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana. Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. Results In the ihprolC-PP197 gene (intron hair pin rolC PPV 197, a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330 was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80% transgenic plants are virus free and symptomless. Some plants (20% contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation

  5. High-throughput sequencing as an effective approach in profiling small RNAs derived from a hairpin RNA expression vector in woody plants.

    Science.gov (United States)

    Zhao, Dongyan; Song, Guo-Qing

    2014-11-01

    Hairpin RNA (hpRNA)-mediated gene silencing has proved to be an efficient approach to develop virus-resistant transgenic plants. To characterize small RNA molecules (sRNAs) derived from an hpRNA expression vector in transgenic cherry rootstock plants, we conducted small RNA sequencing of (1) a transgenic rootstock containing an inverted repeat of the partial coat protein of Prunus necrotic ring spot virus (PNRSV-hpRNA); (2) a nontransgenic rootstock; and (3) a PNRSV-infected sweet cherry plant. Analysis of the PNRSV sRNA pools indicated that 24-nt (nucleotide) small interfering RNAs (siRNAs) were the most prevalent sRNAs in the transgenic rootstock whereas the most abundant sRNAs in the PNRSV-infected nontransgenic rootstock were 21-nt siRNAs. In addition, the 24-nt siRNAs of the PNRSV-hpRNA were more abundant on the sense strand than those on the antisense strand in the transgenic rootstock. In contrast, preference in generating PNRSV sRNAs, ranging from 19-nt to 30-nt for sense and antisense strands, was not distinct in the PNRSV-infected nontransgenic sweet cherry. Taken together, this is the first report on profiling hpRNA-derived sRNAs in woody plants using high-throughput sequencing technology, which is an efficient way to verify the presence/absence, the abundance, and the sequence features of certain sRNAs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator.

    Science.gov (United States)

    Ramezani, Ali; Hawley, Teresa S; Hawley, Robert G

    2003-06-15

    Retroviral vectors are the most efficient means of stable gene delivery to hematopoietic stem cells (HSCs). However, transgene expression from retroviral vectors is frequently subject to the negative influence of chromosomal sequences flanking the site of integration. Toward the development of autonomous transgene expression cassettes, we inserted the human interferon-beta scaffold attachment region (IFN-SAR) and the chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator both separately and together into a series of self-inactivating (SIN) lentiviral vector backbones. Transduced cells of the human CD34+ hematopoietic progenitor line KG1a-pooled populations as well as individual clones harboring single integrants--were analyzed for reporter expression during culture periods of up to 4 months. Vectors carrying both the 5'HS4 insulator and the IFN-SAR consistently outperformed control vectors without inserts as well as vectors carrying either element alone. The performance of a set of vectors containing the murine stem cell virus long terminal repeat as an internal promoter was subsequently assessed during in vitro monocytic differentiation of transduced primary human CD34+ cord blood cells. Similar to what was observed in the KG1a hematopoietic progenitor cell model, optimal reporter expression in primary monocytes was obtained with the vector bearing both regulatory elements. These findings indicate that the 5'HS4/IFN-SAR combination is particularly effective at maintaining open chromatin domains permissive for high-level transgene expression at early and late stages of hematopoietic development, and thus could be of utility in HSC-directed retroviral vector-mediated gene transfer applications.

  7. Lentiviral Transfer of γ-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine β-Thalassemia.

    Science.gov (United States)

    Zhao, Hui Fen; Abraham, Allistair; Kim, Yoon-Sang; Wang, Yong-Dong; Pestina, Tamara; Zhan, Jun; Humphries, Keith; Nienhuis, Arthur W; Persons, Derek A

    2017-03-01

    Recently, an engineered Homeobox-nucleoporin fusion gene, NUP98-HOXA10HD or NA10HD, was reported to expand and maintain murine hematopoietic stem cells (HSCs). We postulated that NA10HD would increase the number of human γ-globin-expressing cells to therapeutic levels. We developed a double gene lentiviral vector encoding both human γ-globin and NA10HD, which was used to transduce human peripheral blood CD34 + cells and increased engraftment 2- to 2.5-fold at 15 weeks post-transplantation in immunodeficient mice. In β-thalassemic mice transplanted with β-thalassemic HSCs transduced with the γ-globin/NA10HD vector, the number of fetal hemoglobin (HbF)-expressing cells was significantly increased after 3 months, leading to resolution of the anemia. Furthermore, the increases in HbF were maintained at 6 months and persisted after secondary transplantation. In addition, NA10HD enrichment of transduced HSCs led to HbF increases without affecting homeostasis of the white blood cell lineages. Our results suggest that NA10HD increases the number of γ-globin-transduced HSCs that engraft, leading to an elevated number of fetal hemoglobin-containing red cells. These effects of NA10HD provide an improved platform for testing of the therapeutic efficacy of novel globin vectors and provide further impetus to develop safe and effective methods for selective expansion of genetically modified cells. Copyright © 2017. Published by Elsevier Inc.

  8. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Sablitzky Fred

    2004-01-01

    Full Text Available Abstract Background Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV and lentiviral (LV vectors into discrete regions of the forebrain. Results Recombinant AAV-Cre, AAV-GFP (green fluorescent protein and LV-Cre-EGFP (enhanced GFP were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. Conclusion AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

  9. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach

    Directory of Open Access Journals (Sweden)

    Francesca Spanevello

    2016-01-01

    Full Text Available Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1 were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4+ T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  10. Striatal modulation of BDNF expression using microRNA124a-expressing lentiviral vectors impairs ethanol-induced conditioned-place preference and voluntary alcohol consumption.

    Science.gov (United States)

    Bahi, Amine; Dreyer, Jean-Luc

    2013-07-01

    Alcohol abuse is a major health, economic and social concern in modern societies, but the exact molecular mechanisms underlying ethanol addiction remain elusive. Recent findings show that small non-coding microRNA (miRNA) signaling contributes to complex behavioral disorders including drug addiction. However, the role of miRNAs in ethanol-induced conditioned-place preference (CPP) and voluntary alcohol consumption has not yet been directly addressed. Here, we assessed the expression profile of miR124a in the dorsal striatum of rats upon ethanol intake. The results show that miR124a was downregulated in the dorso-lateral striatum (DLS) following alcohol drinking. Then, we identified brain-derived neurotrophic factor (BDNF) as a direct target of miR124a. In fact, BDNF mRNA was upregulated following ethanol drinking. We used lentiviral vector (LV) gene transfer technology to further address the role of miR124a and its direct target BDNF in ethanol-induced CPP and alcohol consumption. Results reveal that stereotaxic injection of LV-miR124a in the DLS enhances ethanol-induced CPP as well as voluntary alcohol consumption in a two-bottle choice drinking paradigm. Moreover, miR124a-silencer (LV-siR124a) as well as LV-BDNF infusion in the DLS attenuates ethanol-induced CPP as well as voluntary alcohol consumption. Importantly, LV-miR124a, LV-siR124a and LV-BDNF have no effect on saccharin and quinine intake. Our findings indicate that striatal miR124a and BDNF signaling have crucial roles in alcohol consumption and ethanol conditioned reward. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  11. Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV Derived Sequences and Transgenic Resistance Development in a Model Rice Plant

    Directory of Open Access Journals (Sweden)

    Sehrish Akbar

    2017-01-01

    Full Text Available Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA, targeting simultaneously CP (Coat Protein and Hc-Pro (helper component-proteinase genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine and KITC (Lycine-Isoleucine-Threonine-Cysteine motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

  12. Increased electrocatalyzed performance through hairpin oligonucleotide aptamer-functionalized gold nanorods labels and graphene-streptavidin nanomatrix: Highly selective and sensitive electrochemical biosensor of carcinoembryonic antigen.

    Science.gov (United States)

    Wen, Wei; Huang, Jing-Yi; Bao, Ting; Zhou, Jun; Xia, Hong-Xing; Zhang, Xiu-Hua; Wang, Sheng-Fu; Zhao, Yuan-Di

    2016-09-15

    We report a triplex signal amplification strategy for sensitive biosensing of cancer biomarker by taking advantage of hairpin-shaped oligonucleotide-functionalized gold nanorods (HO-GNRs), graphene and the avidin-biotin reation. The strategy expands electrochemical detection of carcinoembryonic antigen (CEA) by using an aptamer as biosensor's recognition element and HO-GNRs as signal enhancer. To construct this biosensor, the GNR was used as a carrier of horseradish peroxidase (HRP) and HO aptamer with a biotin at the 3'-end and a thiol at the 5'-end, which amplified the electrochemical response because of a large molar ratio of HRP to HO. In the presence of target CEA, the binding reactions of CEA with the loop portions of the HOs caused HOs' loop-stem structure opened and exposed the biotins, and then HRP-GNRs-HO conjugates were captured on graphene and streptavidin modified electrodes via the reaction between the exposed biotins and preimmobilized streptavidins. The accumulation of HRP effectively catalyzed the hydrogen peroxide-mediated oxidation of o-phenylenediamine to generate an electrochemical reduction current for CEA detection. Under optimal conditions, the electrochemical biosensor exhibited a wide dynamic range of 5pgmL(-1) and 50ngmL(-1) toward CEA standards with a low detection limit of 1.5pgmL(-1) (signal-to-noise ratio of 3). The proposed biosensor accurately detected CEA concentration in 8 human serum samples from patients with lung diseases, showing excellent correlations with standard chemiluminescence immunoassay. Furthermore, these results of target DNA detection made it abundantly clear that the proposed strategy can also be extended for detection of other relative biomarkers using different functional DNA structures, which shows great prospects in single-nucleotide polymorphisms analysis, biomedical sensing and application for accurate clinical diseases diagnostic. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Effective relief of neuropathic pain by adeno-associated virus-mediated expression of a small hairpin RNA against GTP cyclohydrolase 1

    Directory of Open Access Journals (Sweden)

    Chang Jin

    2009-11-01

    Full Text Available Abstract Background Recent studies show that transcriptional activation of GTP cyclohydrolase I (GCH1 in dorsal root ganglia (DRG is significantly involved in the development and persistency of pain symptoms. We thus hypothesize that neuropathic pain may be attenuated by down-regulation of GCH1 expression, and propose a gene silencing system for this purpose. Results To interrupt GCH1 synthesis, we designed a bidirectional recombinant adeno-associated virus encoding both a small hairpin RNA against GCH1 and a GFP reporter gene (rAAV-shGCH1. After rAAV-shGCH1 was introduced into the sciatic nerve prior to or following pain-inducing surgery, therapeutic efficacy and the underlying mechanisms were subsequently validated in animal models. The GFP expression data indicates that rAAV effectively delivered transgenes to DRG. Subsequently reduced GCH1 expression was evident from immunohistochemistry and western-blotting analysis. Along with the down-regulation of GCH1, the von Frey test correspondingly indicated a sharp decline in pain symptoms upon both pre- and post-treatment with rAAV-shGCH1. Interestingly, GCH1 down-regulation additionally led to decreased microglial activation in the dorsal horn, implying an association between pain attenuation and reduced inflammation. Conclusion Therefore, the data suggests that GCH1 levels can be reduced by introducing rAAV-shGCH1, leading to pain relief. Based on the results, we propose that GCH1 modulation may be developed as a clinically applicable gene therapy strategy to treat neuropathic pain.

  14. Idiopathic short stature

    Directory of Open Access Journals (Sweden)

    Vlaški Jovan

    2013-01-01

    Full Text Available Growth is a complex process and the basic characteristic of child- hood growth monitoring provides insight into the physiological and pathological events in the body. Statistically, the short stature means departure from the values of height for age and sex (in a particular environment, which is below -2 standard deviation score, or less than -2 standard deviation, i.e. below the third percentile. Advances in molecular genetics have contributed to the improvement of diagnostics in endocrinology. Analysis of patients’ genotypes should not be performed before taking a classical history, detailed clinical examination and appropriate tests. In patients with idiopathic short stature specific causes are excluded, such as growth hormone deficiency, Turner syndrome, short stature due to low birth weight, intrauterine growth retardation, small for gestational age, dysmorphology syndromes and chronic childhood diseases. The exclusion of abovementioned conditions leaves a large number of children with short stature whose etiology includes patients with genetic short stature or familial short stature and those who are low in relation to genetic potential, and who could also have some unrecognized endocrine defect. Idiopathic short stature represents a short stature of unknown cause of heterogeneous etiology, and is characterized by a normal response of growth hormone during stimulation tests (>10 ng/ml or 20 mJ/l, without other disorders, of normal body mass and length at birth. In idiopathic short stature standard deviation score rates <-2.25 (-2 to -3 or <1.2 percentile. These are also criteria for the initiation of growth hormone therapy. In children with short stature there is also the presence of psychological and social suffering. Goals of treatment with growth hormone involve achieving normal height and normal growth rate during childhood.

  15. STAT3/NF-κB-Regulated Lentiviral TK/GCV Suicide Gene Therapy for Cisplatin-Resistant Triple-Negative Breast Cancer.

    Science.gov (United States)

    Kuo, Wei-Ying; Hwu, Luen; Wu, Chun-Yi; Lee, Jhih-Shian; Chang, Chi-Wei; Liu, Ren-Shyan

    2017-01-01

    Triple-negative breast cancer (TNBC) represents approximately 20% of all breast cancers and appears resistance to conventional cytotoxic chemotherapy, demonstrating a particularly poor prognosis and a significantly worse clinical outcome than other types of cancer. Suicide gene therapy has been used for the in vivo treatment of various solid tumors in recent clinical trials. In tumor microenvironment, STAT3/NF-κB pathways are constitutively activated in stromal cells as well as in cancer stem cells (CSCs). In this study, we have cloned a novel STAT3/NF-κB-based reporter system to drive the expression of herpes simplex virus thymidine kinase (HSV-TK) against breast cancer. Lentiviral vector expressing HSV-TK under the regulation of STAT3/NF-κB fused response element was developed. In this setting, we exploited the constitutive STAT3/NF-κB activation in tumors to achieve higher transgene expression than that driven by a constitutively active CMV promotor in vivo . An orthotropic MDA-MB-231 triple negative breast cancer mouse model was used for evaluating the feasibility of STAT3-NF-κB-TK/GCV suicide gene therapy system. The basal promoter activity of Lenti-CMV-TK and Lenti-STAT3-NF-κB-TK in MDA-MB-231 cells was compared by 3 H-FEAU uptake assay. The Lenti-CMV-TK showed ~5 fold higher 3 H-FEAU uptake then Lenti -STAT3-NF-κB-TK. In clonogenic assay, cells expressing Lenti-CMV-TK were 2-fold more sensitive to GCV than Lenti-STAT3-NF-κB-TK transduced cells. In vitro effect of STAT3-NF-κB-induced transgene expression was determined by 10ng/mL TNF-α induction and confirmed by western blot analysis and DsRedm fluorescent microscopy. In vivo evaluation of therapeutic effect by bioluminescence and [ 18 F]FHBG microPET imaging indicated that Lenti-STAT3-NF-κB-TK showed more tumor growth retardation than Lenti-CMV-TK when GCV (20 mg/kg) was administered. The invasiveness and expression of cancer stem cell markers were both decreased after STAT3/NF-κB-regulated HSV

  16. Comparison of human sodium iodide symporter (hNIS) gene expression between lentiviral and adenoviral vectors in rat mesenchymal stem cell

    International Nuclear Information System (INIS)

    Park, So Yeon; Lee, Won Woo; Kim, Sung Jin; Lee, Heui Ran; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun

    2007-01-01

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been done. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated stably hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning the hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and Rad-hNIS transduced rMSC (adeno-hNIS-rMSC) was evaluated for the hNIS expression 48 hours post infection at MOI 1, 5, 20, 50, and 100. The hNIS expression in lenti-hNIS-rMSC or adeno-hNIS-rMSC was assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry using mono-clonal anti-hNIS antibody revealed that intensity of hNIS immunoreactivity in lenti-hNIS-rMSC was greater than that in adeno-hNIS-rMSC at MOl 20 but lower than that at MOl 50. Western blot analysis also showed that lenti-hNIS-rMSC was intermediate between adeno-hNIS-rMSCs at MOl 20 and 50 in hNIS expression. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (297046659 picomole/106 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (61682134 picomole/106 cells). These results suggest that lentivirus mediated hNIS expression is greater in terms of hNIS function but lower in terms of hNIS protein amount than adenovirus mediated hNIS expression 48 hours post infection. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative viral efficiency of transgene expression

  17. A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Christian eBarbato

    2014-02-01

    Full Text Available Neurodegeneration associated with amyloid β (Aβ peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD. Numerous microRNAs regulate amyloid precursor protein (APP expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3’ untranslated region (UTR of the mRNA encoding Ran-binding protein 9 (RanBP9. RanBP9 increases APP processing by β-amyloid converting enzyme 1 (BACE1, secretion of soluble APPβ (sAPPβ, and generation of Aβ. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3'-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs, located downstream of the enhanced green fluorescence protein (EGFP open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPβ levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPβ levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on

  18. Imaging in short stature

    Directory of Open Access Journals (Sweden)

    Vikas Chaudhary

    2012-01-01

    Full Text Available Short stature can be a sign of disease, disability, and social stigma causing psychological stress. It is important to have an early diagnosis and treatment. Short stature may result from skeletal dysplasias, endocrine disorders, may be familial, or may be the result of malnutrition and chronic illnesses. A team effort of the healthcare professionals like pediatricians, endocrinologists, radiologists, and pathologists is required to diagnose, treat and monitor various pathological conditions associated with growth abnormality. In this review, we have discussed the role of imaging in diagnosing and characterizing various pathological conditions associated with short stature.

  19. A short note on short pants

    OpenAIRE

    Parlier, Hugo

    2015-01-01

    It is a theorem of Bers that any closed hyperbolic surface admits a pants decomposition consisting of curves of bounded length where the bound only depends on the topology of the surface. The question of the quantification of the optimal constants has been well studied and the best upper bounds to date are linear in genus, a theorem of Buser and Sepp\\"al\\"a. The goal of this note is to give a short proof of an linear upper bound which slightly improves the best known bounds.

  20. Short communication: Effect of inhibition of fatty acid synthase on triglyceride accumulation and effect on lipid metabolism genes in goat mammary epithelial cells.

    Science.gov (United States)

    Zhu, J J; Luo, J; Sun, Y T; Shi, H B; Li, J; Wu, M; Yu, K; Haile, A B; Loor, J J

    2015-05-01

    The role of fatty acid synthase (FASN) on de novo fatty acid synthesis has been well established. In monogastrics, unlike acetyl-coenzyme A carboxylase, FASN is primarily controlled at the transcriptional level. However, no data exist on ruminant mammary cells evaluating effects of FASN knockdown on mRNA expression of lipogenic genes. Inhibition of FASN in mammary cells by C75-mediated interference, a synthetic inhibitor of FASN activity, and short hairpin RNA-mediated interference markedly reduced cellular triglyceride content at least in part by decreasing the expression of genes related to triglyceride synthesis (GPAT, AGPAT6, and DGAT2) and enhancing the expression of lipolysis-related genes (ATGL and HSL). Consistent with the markedly lower expression of genes related to lipid droplet formation and secretion (TIP47, ADFP, BTN1A1, and XDH), cellular lipid droplets also were reduced sharply after incubation with C75 or adenovirus-short-hairpin-RNA. The results underscored the essential role of FASN in the overall process of milk-fat formation in goat mammary epithelial cells. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Shortness of Breath

    Science.gov (United States)

    ... enough air. Shortness of breath — known medically as dyspnea — is often described as an intense tightening in ... properly. Schwartzstein RM. Approach to the patient with dyspnea. http://www.uptodate.com/home. Accessed Feb. 4, ...

  2. Short bowel syndrome

    International Nuclear Information System (INIS)

    Engels, L.G.J.B.

    1983-01-01

    This thesis describes some aspects of short bowel syndrome. When approximately 1 m or less small bowel is retained after extensive resection, a condition called short bowel syndrome is present. Since the advent of parenteral nutrition, the prognosis of patients with a very short bowel has dramatically improved. Patients with 40 to 100 cm remaining jejunum and/or ileum can generally be maintained with oral nutrition due to increased absorption of the small bowel remnant as result of intestinal adaptation. This study reports clinical, biochemical and nutritional aspects of short bowel patients on oral or parenteral nutrition, emphasizing data on absorption of various nutrients and on bone metabolism. Furthermore, some technical apsects concerning long-term parenteral nutrition are discussed. (Auth.)

  3. Short-cut math

    CERN Document Server

    Kelly, Gerard W

    1984-01-01

    Clear, concise compendium of about 150 time-saving math short-cuts features faster, easier ways to add, subtract, multiply, and divide. Each problem includes an explanation of the method. No special math ability needed.

  4. Catapults fall short

    Science.gov (United States)

    Gibson, Marcus

    2018-01-01

    In reply to the news story "UK Catapults fall short, claims review of technology centres", which describes an independent review that criticized the management of the UK's network of technology innovation centres.

  5. Structural Changes of Zn(IIbleomycin Complexes When Bound to DNA Hairpins Containing the 5′-GT-3′ and 5′-GC-3′ Binding Sites, Studied through NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Shelby E. Follett

    2017-12-01

    Full Text Available We have previously investigated the diverse levels of disruption caused by Zn(IIBLMs with different C-termini to DNA hairpins containing 5′-GC-3′ and 5′-GT-3′ binding sites. The results of this investigation indicated that both the DNA-binding site and the bleomycin C-termini have an impact on the final conformation of the aforementioned hairpins in the drug-target complexes, as suggested by the different sets of intramolecular NOEs displayed by both oligonucleotides when bound to each Zn(IIBLM. The NMR signals elicited by 1H nuclei in the oligonucleotide bases and sugar moieties were also affected differently (shifted upfield or downfield in various patterns depending on the BLM C-termini and the binding site in the oligonucleotides. The overall conclusion derived from the precedent research is that the spatial conformation of target DNA segments in DNA-Zn(IIBLM complexes could be forged by interactions between drug and DNA that are guided by the DNA binding site and the BLM C-termini. The present study focuses on the structural alterations exhibited by Zn(IIbleomycin-A2, -B2, -A5 and Zn(IIpeplomycin molecules upon binding to the previously studied hairpins. Our main goal is to determine if different spatial conformations of the drugs in their DNA-bound forms are found in drug-DNA complexes that differ in the oligonucleotide binding site and BLM C-termini. Evidence that suggest that each Zn(IIbleomycin is structurally affected depending these two factors, as indicated by different sets of intramolecular NOE connectivities between drug protons and diverse patterns of shifting of their 1H-NMR signals, is provided.

  6. Functional conservation of the pre-sensor one beta-finger hairpin (PS1-hp) structures in mini-chromosome maintenance proteins of Saccharomyces cerevisiae and archaea.

    Science.gov (United States)

    Ramey, Christopher J; Sclafani, Robert A

    2014-05-28

    Mini-chromosome maintenance (MCM) proteins form complexes that are required for DNA replication and are highly conserved throughout evolution. The replicative helicase of eukaryotic organisms is composed of the six paralogs MCM2-7, which form a heterohexameric ring structure. In contrast, the structure of the archaean replicative MCM helicase is a single Mcm protein that forms a homohexameric complex. Atomic structures of archaeal MCMs have identified multiple beta-finger structures in Mcm proteins whose in vivo function is unknown. In the present study, we have investigated the physiological role of the pre-sensor 1 beta-hairpin (PS1-hp) beta-fingers of Saccharomyces cerevisiae Mcm4p and Mcm5p in DNA replication initiation and elongation in vivo. The PS1-hp beta-finger mutant of Mcm5p (mcm5-HAT K506A::URA3) has a growth defect at both 18° and 37°. Mutation of the Mcm4p PS1-hp beta-finger (mcm4-HA K658A::TRP1) does not have a growth defect, indicating different functional contributions of the PS1-hp beta-finger structures of different MCM helicase subunits. Both Mcm4p and Mcm5p PS1-hp beta-finger mutants can coimmunoprecipitate Mcm2p, indicating the formation of the hexameric MCM helicase complex. Both PS1-hp beta-finger mutants have a plasmid loss phenotype that is suppressible by origin dosage, indicating a defective replication initiation. Surprisingly, a defect in the binding of PS1-hp MCM mutants to origins of DNA replication was not found by chromatin immunoprecipitation, suggesting a novel interpretation in which the defect is in a subsequent step of DNA strand separation by the MCM helicase. The double mutant mcm4-HA K658A::TRP1 mcm5-HAT K506A::URA3 is lethal, displaying a terminal MCM mutant phenotype of large budded cells. Copyright © 2014 Ramey and Sclafani.

  7. Post-transcriptional modulation of protein phosphatase PPP2CA and tumor suppressor PTEN by endogenous siRNA cleaved from hairpin within PTEN mRNA 3'UTR in human liver cells.

    Science.gov (United States)

    Gao, Yu-En; Wang, Yuan; Chen, Fu-Quan; Feng, Jin-Yan; Yang, Guang; Feng, Guo-Xing; Yang, Zhe; Ye, Li-Hong; Zhang, Xiao-Dong

    2016-07-01

    Increasing evidence shows that mRNAs exert regulatory function along with coding proteins. Recently we report that a hairpin within YAP mRNA 3'UTR can modulate the Hippo signaling pathway. PTEN is a tumor suppressor, and is mutated in human cancers. In this study we examined whether PTEN mRNA 3'UTR contained a hairpin structure that could regulate gene regulation at the post-transcriptional level. The secondary structure of PTEN mRNA 3'UTR was analyzed using RNAdraw and RNAstructure. Function of hairpin structure derived from the PTEN mRNA 3'UTR was examined using luciferase reporter assay, RT-PCR and Western blotting. RNA-immunoprecipitation (RIP) assay was used to analyze the interaction between PTEN mRNA and microprocessor Drosha and DGCR8. Endogenous siRNA (esiRNA) derived from PTEN mRNA 3'UTR was identified by RT-PCR and rt-PCR, and its target genes were predicted using RNAhybrid. A bioinformatics analysis revealed that PTEN mRNA contained a hairpin structure (termed PTEN-sh) within 3'UTR, which markedly increased the reporter activities of AP-1 and NF-κB in 293T cells. Moreover, treatment with PTEN-sh (1 and 2 μg) dose-dependently inhibited the expression of PTEN in human liver L-O2 cells. RIP assay demonstrated that the microprocessor Drosha and DGCR8 was bound to PTEN-sh in L-O2 cells, leading to the cleavage of PTEN-sh from PTEN mRNA 3'UTR. In addition, microprocessor Dicer was involved in the processing of PTEN-sh. Interestingly, esiRNA (termed PTEN-sh-3p21) cleaved from PTEN-sh was identified in 293T cells and human liver tissues, which was found to target the mRNA 3'UTRs of protein phosphatase PPP2CA and PTEN in L-O2 cells. Treatment of L-O2 or Chang liver cells with PTEN-sh-3p21 (50, 100 nmol/L) promoted the cell proliferation in dose- and time-dependent manners. The endogenous siRNA (PTEN-sh-3p21) cleaved from PTEN-sh within PTEN mRNA 3'UTR modulates PPP2CA and PTEN at the post-transcriptional level in liver cells.

  8. Post-transcriptional modulation of protein phosphatase PPP2CA and tumor suppressor PTEN by endogenous siRNA cleaved from hairpin within PTEN mRNA 3′UTR in human liver cells

    Science.gov (United States)

    Gao, Yu-en; Wang, Yuan; Chen, Fu-quan; Feng, Jin-yan; Yang, Guang; Feng, Guo-xing; Yang, Zhe; Ye, Li-hong; Zhang, Xiao-dong

    2016-01-01

    Aim: Increasing evidence shows that mRNAs exert regulatory function along with coding proteins. Recently we report that a hairpin within YAP mRNA 3′UTR can modulate the Hippo signaling pathway. PTEN is a tumor suppressor, and is mutated in human cancers. In this study we examined whether PTEN mRNA 3′UTR contained a hairpin structure that could regulate gene regulation at the post-transcriptional level. Methods: The secondary structure of PTEN mRNA 3′UTR was analyzed using RNAdraw and RNAstructure. Function of hairpin structure derived from the PTEN mRNA 3′UTR was examined using luciferase reporter assay, RT-PCR and Western blotting. RNA-immunoprecipitation (RIP) assay was used to analyze the interaction between PTEN mRNA and microprocessor Drosha and DGCR8. Endogenous siRNA (esiRNA) derived from PTEN mRNA 3′UTR was identified by RT-PCR and rt-PCR, and its target genes were predicted using RNAhybrid. Results: A bioinformatics analysis revealed that PTEN mRNA contained a hairpin structure (termed PTEN-sh) within 3′UTR, which markedly increased the reporter activities of AP-1 and NF-κB in 293T cells. Moreover, treatment with PTEN-sh (1 and 2 μg) dose-dependently inhibited the expression of PTEN in human liver L-O2 cells. RIP assay demonstrated that the microprocessor Drosha and DGCR8 was bound to PTEN-sh in L-O2 cells, leading to the cleavage of PTEN-sh from PTEN mRNA 3′UTR. In addition, microprocessor Dicer was involved in the processing of PTEN-sh. Interestingly, esiRNA (termed PTEN-sh-3p21) cleaved from PTEN-sh was identified in 293T cells and human liver tissues, which was found to target the mRNA 3′UTRs of protein phosphatase PPP2CA and PTEN in L-O2 cells. Treatment of L-O2 or Chang liver cells with PTEN-sh-3p21 (50, 100 nmol/L) promoted the cell proliferation in dose- and time-dependent manners. Conclusion: The endogenous siRNA (PTEN-sh-3p21) cleaved from PTEN-sh within PTEN mRNA 3′UTR modulates PPP2CA and PTEN at the post

  9. Measurement of short bunches

    International Nuclear Information System (INIS)

    Wang, D.X.

    1996-01-01

    In recent years, there has been increasing interest in short electron bunches for different applications such as short wavelength FELs, linear colliders, and advanced accelerators such as laser or plasma wakefield accelerators. One would like to meet various requirements such as high peak current, low momentum spread, high luminosity, small ratio of bunch length to plasma wavelength, and accurate timing. Meanwhile, recent development and advances in RF photoinjectors and various bunching schemes make it possible to generate very short electron bunches. Measuring the longitudinal profile and monitoring bunch length are critical to understand the bunching process and longitudinal beam dynamics, and to commission and operate such short bunch machines. In this paper, several commonly used measurement techniques for subpicosecond bunches and their relative advantages and disadvantages are discussed. As examples, bunch length related measurements at Jefferson lab are presented. At Jefferson Lab, bunch lengths s short as 84 fs have been systematically measured using a zero-phasing technique. A highly sensitive Coherent Synchrotron Radiation (CSR) detector has been developed to noninvasively monitor bunch length for low charge bunches. Phase transfer function measurements provide a means of correcting RF phase drifts and reproducing RF phases to within a couple of tenths of a degree. The measurement results are in excellent agreement with simulations. A comprehensive bunch length control scheme is presented

  10. Measurement of short bunches

    International Nuclear Information System (INIS)

    Wang, D.X.

    1996-01-01

    In recent years, there has been increasing interest in short electron bunches for different applications such as short wavelength FELs, linear colliders, and advanced accelerators such as laser or plasma wakefield accelerators. One would like to meet various requirements such as high peak current, low momentum spread, high luminosity, small ratio of bunch length to plasma wavelength, and accurate timing. Meanwhile, recent development and advances in RF photoinjectors and various bunching schemes make it possible to generate very short electron bunches. Measuring the longitudinal profile and monitoring bunch length are critical to understand the bunching process and longitudinal beam dynamics, and to commission and operate such short bunch machines. In this paper, several commonly used measurement techniques for subpicosecond bunches and their relative advantages and disadvantages are discussed. As examples, bunch length related measurements at Jefferson Lab are presented. At Jefferson Lab, bunch lengths as short as 84 fs have been systematically measured using a zero-phasing technique. A highly sensitive Coherent Synchrotron Radiation (CSR) detector has been developed to noninvasively monitor bunch length for low charge bunches. Phase transfer function measurements provide a means of correcting RF phase drifts and reproducing RF phases to within a couple of tenths of a degree. The measurement results are in excellent agreement with simulations. A comprehensive bunch length control scheme is presented. (author)

  11. Congenital Short QT Syndrome

    Directory of Open Access Journals (Sweden)

    Lia Crotti

    2010-02-01

    Full Text Available The Short QT Syndrome is a recently described new genetic disorder, characterized by abnormally short QT interval, paroxysmal atrial fibrillation and life threatening ventricular arrhythmias. This autosomal dominant syndrome can afflict infants, children, or young adults; often a remarkable family background of cardiac sudden death is elucidated. At electrophysiological study, short atrial and ventricular refractory periods are found, with atrial fibrillation and polymorphic ventricular tachycardia easily induced by programmed electrical stimulation. Gain of function mutations in three genes encoding K+ channels have been identified, explaining the abbreviated repolarization seen in this condition: KCNH2 for Ikr (SQT1, KCNQ1 for Iks (SQT2 and KCNJ2 for Ik1 (SQT3. The currently suggested therapeutic strategy is an ICD implantation, although many concerns exist for asymptomatic patients, especially in pediatric age. Pharmacological treatment is still under evaluation; quinidine has shown to prolong QT and reduce the inducibility of ventricular arrhythmias, but awaits additional confirmatory clinical data.

  12. Short notice inspections

    International Nuclear Information System (INIS)

    Pouchkarev, V.

    1998-01-01

    For 30 years the IAEA safeguards system have evolved and have been strengthened by the regular introduction of new methods and techniques, improving both its effectiveness and efficiency. The member States of the IAEA have indicated their willingness to accept new obligations and associated technical measure that greatly strengthen the nuclear safeguards system. One element of this is the extent to which the IAEA inspectors have physical access to relevant locations for the purpose of providing independent verification of the exclusively peaceful intent of a State nuclear program. The Protocol to Safeguards granted new legal authority with respect to information on, and short notice inspector access to, all buildings on a nuclear site and administrative agreements that improve the process of designating inspectors and IAEA access to modern means of communication. This report is a short description of unannounced or short notice inspections as measures on which the new strengthened and cost efficient system will be based

  13. Short wavelength FELS

    International Nuclear Information System (INIS)

    Sheffield, R.L.

    1991-01-01

    The generation of coherent ultraviolet and shorter wavelength light is presently limited to synchrotron sources. The recent progress in the development of brighter electron beams enables the use of much lower energy electron rf linacs to reach short-wavelengths than previously considered possible. This paper will summarize the present results obtained with synchrotron sources, review proposed short- wavelength FEL designs and then present a new design which is capable of over an order of magnitude higher power to the extreme ultraviolet. 17 refs., 10 figs

  14. Short wavelength FELS

    Energy Technology Data Exchange (ETDEWEB)

    Sheffield, R.L.

    1991-01-01

    The generation of coherent ultraviolet and shorter wavelength light is presently limited to synchrotron sources. The recent progress in the development of brighter electron beams enables the use of much lower energy electron rf linacs to reach short-wavelengths than previously considered possible. This paper will summarize the present results obtained with synchrotron sources, review proposed short- wavelength FEL designs and then present a new design which is capable of over an order of magnitude higher power to the extreme ultraviolet. 17 refs., 10 figs.

  15. Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

    Science.gov (United States)

    Follett, Shelby E; Ingersoll, Azure D; Murray, Sally A; Reilly, Teresa M; Lehmann, Teresa E

    2017-10-01

    Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A 2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

  16. without intersecting short cycles

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... ... Lecture Workshops · Refresher Courses · Symposia. Home; Journals; Proceedings – Mathematical Sciences; Volume 126; Issue 2. (3, 1)*-Choosability of graphs of nonnegative characteristic without intersecting short cycles. Haihui Zhang. Research Article Volume 126 Issue 2 May 2016 pp 159-165 ...

  17. Troubling Practices: Short Responses

    Science.gov (United States)

    Anderson, Gary; Simic, Lena; Haley, David; Svendsen, Zoe; Neal, Lucy; Samba, Emelda Ngufor

    2012-01-01

    In this "RiDE" themed edition on environmentalism, some short pieces are chosen where practitioners describe their own specific environmental practices. Zoe Svendsen and Lucy Neal point to the positives in two commissioned works ("The Trashcatchers' Carnival" and "3rd Ring Out"), underlining the importance of…

  18. Congenital Short QT Syndrome

    Directory of Open Access Journals (Sweden)

    Charles Antzelevitch

    2004-04-01

    Full Text Available Long QT intervals in the ECG have long been associated with sudden cardiac death. The congenital long QT syndrome was first described in individuals with structurally normal hearts in 1957.1 Little was known about the significance of a short QT interval. In 1993, after analyzing 6693 consecutive Holter recordings Algra et al concluded that an increased risk of sudden death was present not only in patients with long QT interval, but also in patients with short QT interval (<400 ms.2 Because this was a retrospective analysis, further evaluation of the data was not possible. It was not until 2000 that a short-QT syndrome (SQTS was proposed as a new inherited clinical syndrome by Gussak et al.3 The initial report was of two siblings and their mother all of whom displayed persistently short QT interval. The youngest was a 17 year old female presenting with several episodes of paroxysmal atrial fibrillation requiring electrical cardioversion.3 Her QT interval measured 280 msec at a heart rate of 69. Her 21 year old brother displayed a QT interval of 272 msec at a heart rate of 58, whereas the 51 year old mother showed a QT of 260 msec at a heart rate of 74. The authors also noted similar ECG findings in another unrelated 37 year old patient associated with sudden cardiac death.

  19. Minimizing Shortness of Breath

    Science.gov (United States)

    ... is also placed on proper use of the abdominal muscles to better control episodes of shortness of breath, limit overuse of the accessory muscles and manage respiratory symptoms. Monitor Breathing During an activity, it is important to pause frequently to check ...

  20. Short pulse neutron generator

    Science.gov (United States)

    Elizondo-Decanini, Juan M.

    2016-08-02

    Short pulse neutron generators are described herein. In a general embodiment, the short pulse neutron generator includes a Blumlein structure. The Blumlein structure includes a first conductive plate, a second conductive plate, a third conductive plate, at least one of an inductor or a resistor, a switch, and a dielectric material. The first conductive plate is positioned relative to the second conductive plate such that a gap separates these plates. A vacuum chamber is positioned in the gap, and an ion source is positioned to emit ions in the vacuum chamber. The third conductive plate is electrically grounded, and the switch is operable to electrically connect and disconnect the second conductive plate and the third conductive plate. The at least one of the resistor or the inductor is coupled to the first conductive plate and the second conductive plate.

  1. Short-term memory

    Science.gov (United States)

    Toulouse, G.

    This is a rather bold attempt to bridge the gap between neuron structure and psychological data. We try to answer the question: Is there a relation between the neuronal connectivity in the human cortex (around 5,000) and the short-term memory capacity (7±2)? Our starting point is the Hopfield model (Hopfield 1982), presented in this volume by D.J. Amit.

  2. Short QT syndrome

    Directory of Open Access Journals (Sweden)

    Fiorenzo Gaita

    2011-12-01

    Full Text Available The short QT syndrome (SQTS is a recently described genetic arrhythmogenic disorder, characterized by abnormally short QT intervals on surface electrocardiogram (ECG and a high incidence of sudden death (SD during life, including the first months of life. The inheritance of SQTS is autosomal dominant, with genetic heterogeneity. Gain-of-function mutations in 3 genes encoding potassium channels have been associated to the disease: KCNH2 encoding IKr (SQT1, KCNQ1 encoding IKs (SQT2, and KCNJ2 encoding IK1 (SQT3. Loss-of-function mutations in 3 genes encoding the cardiac L-type calcium channel, CACNA1C, CACNB2b and CACNA2D1 may underlie a mixed phenotype of Brugada pattern ECG (or non-specific repolarization changes in case of CACNA2D1 and shorter than normal QT intervals. Clinical presentation is often severe, as cardiac arrest represents the first clinical presentation in most subjects. Moreover, often a noticeable family history of cardiac SD is present. Atrial fibrillation may be observed, also in young individuals. At electrophysiological study, short atrial and ventricular refractory periods are found, and atrial and ventricular fibrillation are easily induced by programmed electrical stimulation. The outcome of patients with SQTS becomes relatively safe when they are identified and treated. Currently, the suggested therapeutic strategy is an implantable cardioverter- defibrillator (ICD in patients with personal history of aborted SD or syncope. In asymptomatic adult patients from highly symptomatic families and in newborn children pharmacological treatment with hydroquinidine, which has been shown to prolong the QT interval and reduce the inducibility of ventricular arrhythmias, may be proposed.

  3. Short bowel syndrome.

    LENUS (Irish Health Repository)

    Donohoe, Claire L

    2012-02-01

    The short bowel syndrome (SBS) is a state of malabsorption following intestinal resection where there is less than 200 cm of intestinal length. The management of short bowel syndrome can be challenging and is best managed by a specialised multidisciplinary team. A good understanding of the pathophysiological consequences of resection of different portions of the small intestine is necessary to anticipate and prevent, where possible, consequences of SBS. Nutrient absorption and fluid and electrolyte management in the initial stages are critical to stabilisation of the patient and to facilitate the process of adaptation. Pharmacological adjuncts to promote adaptation are in the early stages of development. Primary restoration of bowel continuity, if possible, is the principle mode of surgical treatment. Surgical procedures to increase the surface area of the small intestine or improve its function may be of benefit in experienced hands, particularly in the paediatric population. Intestinal transplant is indicated at present for patients who have failed to tolerate long-term parenteral nutrition but with increasing experience, there may be a potentially expanded role for its use in the future.

  4. What Is a Short Story?

    Science.gov (United States)

    Singer, Marilyn

    2000-01-01

    Presents 12 brief answers by editors and authors to the question of what a short story is. Includes a 23-item annotated bibliography of the best new (published between 1997 and 2000) short story collections for young adults. (RS)

  5. Short duration gamma ray bursts

    Indian Academy of Sciences (India)

    Abstract. After a short review of gamma ray bursts (GRBs), we discuss the physical implications of strong statistical correlations seen among some of the parameters of short duration bursts (90 < 2 s). Finally, we conclude with a brief sketch of a new unified model for long and short GRBs.

  6. Short apraxia screening test.

    Science.gov (United States)

    Leiguarda, Ramon; Clarens, Florencia; Amengual, Alejandra; Drucaroff, Lucas; Hallett, Mark

    2014-01-01

    Limb apraxia comprises many different and common disorders, which are largely unrecognized essentially because there is no easy-to-use screening test sensitive enough to identify all types of limb praxis deficits. We evaluated 70 right-handed patients with limb apraxia due to a single focal lesion of the left hemisphere and 40 normal controls, using a new apraxia screening test. The test covered 12 items including: intransitive gestures, transitive gestures elicited under verbal, visual, and tactile modalities, imitation of meaningful and meaningless postures and movements, and a multiple object test. Interrater reliability was maximum for a cutoff of >2 positive items identifying apraxia on the short battery (Cohen's kappa .918, p 3 items (Cohen's kappa .768, p 2 was higher, indicating greater apraxia diagnosis agreement between raters at this cutoff value. The screening test proved to have high specificity and sensitivity to diagnose every type of upper limb praxis deficit, thus showing advantages over previously published tests.

  7. First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient.

    Science.gov (United States)

    Pollack, Seth M; Lu, Hailing; Gnjatic, Sacha; Somaiah, Neeta; O'Malley, Ryan B; Jones, Robin L; Hsu, Frank J; Ter Meulen, Jan

    2017-10-01

    Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.

  8. Newnes short wave listening handbook

    CERN Document Server

    Pritchard, Joe

    2013-01-01

    Newnes Short Wave Listening Handbook is a guide for starting up in short wave listening (SWL). The book is comprised of 15 chapters that discuss the basics and fundamental concepts of short wave radio listening. The coverage of the text includes electrical principles; types of signals that can be heard in the radio spectrum; and using computers in SWL. The book also covers SWL equipment, such as receivers, converters, and circuits. The text will be of great use to individuals who want to get into short wave listening.

  9. Enhancing Discussion through Short Stories.

    Science.gov (United States)

    Marcus, Sybil

    A teacher of English in a college-level intensive English language program describes a method for stimulating speech in high-intermediate and advanced students, using short stories. It is argued that in short stories, the themes are universal, and even shy students are willing to discuss this form of literature in class. Criteria for selecting…

  10. Short duration gamma ray bursts

    Indian Academy of Sciences (India)

    This suggests either larger bulk Lorentz factors or spatially closer locations for the short GRBs [13]. From luminosity function studies, the local space density of the short GRBs is likely to be lower than that of the long ones by a factor of ~3 [14]. While analysing time tagged event (TTE) data for 156 category A type bursts.

  11. Studying of cellular interaction of hairpin-like peptide EcAMP1 from barnyard grass (Echinochloa crusgalli L.) seeds with plant pathogenic fungus Fusarium solani using microscopy techniques.

    Science.gov (United States)

    Vasilchenko, Alexey S; Yuryev, Mikhail; Ryazantsev, Dmitry Yu; Zavriev, Sergey K; Feofanov, Alexey V; Grishin, Eugene V; Rogozhin, Eugene A

    2016-11-01

    An interaction of recombinant hairpin-like cationic peptide EcAMP1 with conidia of plant pathogenic fungus Fusarium solani at the cellular level was studied by a combination of microscopic methods. EcAMP1 is from barnyard grass (Echinochloa crusgalli L.), and obtained by heterologous expression in Escherichia coli system. As a result, a direct relationship between hyphal growth inhibition and increasing active peptide concentration, time of incubation and fungal physiological condition has been determined. Dynamics of accumulation and redistribution of the peptide studied on fungal cellular cover and inside the conidia cells has been shown. The dynamics are dependent on time of coupling, as well as, a dissimilarity of EcAMP1 binding with cover of fungal conidia and its stepwise accumulation and diffuse localization in the cytoplasm. Correlation between structural disruption of fungal conidia and the presence of morphological changes has also been found. The correlation was found under the influence of peptide high concentrations at concentrations above 32 μM. The results indicate the presence of a binding of EcAMP1 with the surface of fungal conidia, thus, demonstrating a main specificity for its antifungal action at the cellular level. These results, however, cannot exclude the existence of attendant EcAMP1 action based on its intracellular localization on some specific targets. SCANNING 38:591-598, 2016. © 2016 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  12. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  13. Superconducting cyclotron magnet coil short

    International Nuclear Information System (INIS)

    Mallory, M.L.; Blosser, H.G.; Clark, D.J.; Launer, H.; Lawton, D.; Miller, P.; Resmini, F.

    1982-01-01

    In February 1981, a short circuit appeared in the superconducting coil of the K500 cyclotron. The short is resistive in character and therefore has no effect on steady state operation of the magnet. The resistance of the short varies, sometimes being below threshold of detection as a heat load on the cooling system and sometimes being significant. The resistance under certain conditions shows approximately cyclic phenomena with time constants in the range of seconds and other approximately cyclic phenomena which correlate with gross operating parameters of the magnet (shifting current from one coil to another at high field and lowering and raising the liquid helium level). A number of diagnostic studies of the short have been made, using 1) an array of flux sensing loops to sense the magnetic effect of the short, 2) voltage comparisons between upper and lower sections of the coil, 3) comparisons of forces in the nine member coil support system and 4) the effect of the short on the thermal charactersitics of the coil. Insulation failure or a metal chip shorting out turns have been explored in some detail but a convincing determination of the exact cause of the short may never be available, (even the extreme step of unwinding the coil having a significant probability that an imperfection with the observed characteristics would pass unnoticed). Analysis of the characteristics of the short indicated that the most serious consequence would be failure of the coils mechanical support system in the event that the magnet was quickly discharged, as in a dump or quench. To deal with this hazard, the support system has been modified by installing solid supports which prevent the coil from moving by an amount sufficient to damage the support system. We have also reexamined the data and calculations used in the original coil design and have made some additional measurements of the properties of the materials (yield strength, friction coefficient, Young's modulus) used in the

  14. Intestinal Failure (Short Bowel Syndrome)

    Science.gov (United States)

    Intestinal Failure (Short Bowel Syndrome) What is intestinal failure? Intestinal failure occurs when a significant portion of the small ... intestine does. Who is at risk for intestinal failure? N Babies (usually premature) who have had surgery ...

  15. SHORT COMMUNICATION ENVIRONMENTAL IMPACTS OF ...

    African Journals Online (AJOL)

    Preferred Customer

    2009 Chemical Society of Ethiopia. ______. *Corresponding author. E-mail: abrishgk@yahoo.com. SHORT COMMUNICATION. ENVIRONMENTAL IMPACTS OF SHEBA TANNERY (ETHIOPIA) EFFLUENTS ON. THE SURROUNDING WATER BODIES. Abraha Gebrekidan*, Gebrekidan Gebresellasie and Afework Mulugeta.

  16. Response of a simian immunodeficiency virus (SIVmac251 to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Greenhouse Jack

    2010-03-01

    Full Text Available Abstract Background In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI class. Results In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC and tenofovir (PMPA were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy. Conclusions In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body.

  17. Short template switch events explain mutation clusters in the human genome.

    Science.gov (United States)

    Löytynoja, Ari; Goldman, Nick

    2017-06-01

    Resequencing efforts are uncovering the extent of genetic variation in humans and provide data to study the evolutionary processes shaping our genome. One recurring puzzle in both intra- and inter-species studies is the high frequency of complex mutations comprising multiple nearby base substitutions or insertion-deletions. We devised a generalized mutation model of template switching during replication that extends existing models of genome rearrangement and used this to study the role of template switch events in the origin of short mutation clusters. Applied to the human genome, our model detects thousands of template switch events during the evolution of human and chimp from their common ancestor and hundreds of events between two independently sequenced human genomes. Although many of these are consistent with a template switch mechanism previously proposed for bacteria, our model also identifies new types of mutations that create short inversions, some flanked by paired inverted repeats. The local template switch process can create numerous complex mutation patterns, including hairpin loop structures, and explains multinucleotide mutations and compensatory substitutions without invoking positive selection, speculative mechanisms, or implausible coincidence. Clustered sequence differences are challenging for current mapping and variant calling methods, and we show that many erroneous variant annotations exist in human reference data. Local template switch events may have been neglected as an explanation for complex mutations because of biases in commonly used analyses. Incorporation of our model into reference-based analysis pipelines and comparisons of de novo assembled genomes will lead to improved understanding of genome variation and evolution. © 2017 Löytynoja and Goldman; Published by Cold Spring Harbor Laboratory Press.

  18. Short implants: A systematic review

    Science.gov (United States)

    Karthikeyan, I.; Desai, Shrikar R.; Singh, Rika

    2012-01-01

    Background: Short implants are manufactured for use in atrophic regions of the jaws. Although many studies report on short implants as ≤10 mm length with considerable success, the literature regarding survival rate of ≤7 mm is sparse. Purpose: The purpose of this study was to systematically evaluate the publications concerning short dental implants defined as an implant with a length of ≤7 mm placed in the maxilla or in the mandible. Materials and Methods: A Medline and manual search was conducted to identify studies concerning short dental implants of length ≤7 mm published between 1991 and 2011. The articles included in this study report data on implant length ≤7 mm, such as demographic variables, implant type, location in jaws, observation time, prostheses and complications. Results: The 28 included studies represent one randomized controlled trial, 12 prospective studies and 10 retrospective studies. The survival rate of short implant was found to be increased from 80% to 90% gradually, with recent articles showing 100%. Conclusion: When severe atrophy of jaws was encountered, short and wide implants can be placed successfully. PMID:23162320

  19. The novel as short story

    Directory of Open Access Journals (Sweden)

    Kirk Schlueter

    2013-06-01

    Full Text Available In recent history, the novel has been thought of and defined primarily as a long prose narrative. However, this has not been the case historically, as the original meaning of "novel" was for "a piece of news" or "a short story or novella." Returning to this original definition, I propose a new way of viewing the work known contemporarily as the novel as a collection, or sequence, of united short stories rather than a single indivisible work, with the component short stories or novellas comprising the sequence renamed as "novels." A brief examination of several classic works traditionally considered novels serves to illustrate how this change in definition will affect reading.

  20. Infantile Short Bowel Syndrome: short and long term evaluation

    NARCIS (Netherlands)

    J.F. Olieman (Joanne)

    2009-01-01

    textabstractInfantile short bowel syndrome is a condition which is characterized by malabsorption of nutrients, as a result of congenital intestinal shortening or massive small bowel resection. Survival rates have improved over the years, but morbidity remains high and clinical management of these

  1. Short Communication Effects of short chain fatty acid (SCFA ...

    African Journals Online (AJOL)

    A study was conducted to determine the effect of supplementing the diet of breeder hens with a short-chain fatty acid (SCFA) premix, containing 509 g fatty acid salts/kg of which 285 g were calcium butyrate, on their eggshell characteristics and the hatching percentage of the eggs. One thousand six hundred 66-week old ...

  2. Short Communication Short-term variability in alongshore winds and ...

    African Journals Online (AJOL)

    Swakopmund is a popular coastal resort in Namibia, especially during the summer holiday season when daily sea temperatures can fluctuate several degrees in a short period. Hourly measurements of the near bottom water temperature were collected off the Swakopmund Jetty to investigate the thermal variability in ...

  3. Short-circuit impedance measurement

    DEFF Research Database (Denmark)

    Pedersen, Knud Ole Helgesen; Nielsen, Arne Hejde; Poulsen, Niels Kjølstad

    2003-01-01

    Methods for estimating the short-circuit impedance in the power grid are investigated for various voltage levels and situations. The short-circuit impedance is measured, preferably from naturally occurring load changes in the grid, and it is shown that such a measurement system faces different...... kinds of problems at different locations in the grid. This means that the best measurement methodology changes depending on the location in the grid. Three typical examples with different measurement problems at 400 kV, 132 kV and 400 V voltage level are discussed....

  4. A Short Term Analogue Memory

    DEFF Research Database (Denmark)

    Shah, Peter Jivan

    1992-01-01

    A short term analogue memory is described. It is based on a well-known sample-hold topology in which leakage currents have been minimized partly by circuit design and partly by layout techniques. Measurements on a test chip implemented in a standard 2.4 micron analogue CMOS process show a droop...

  5. Primes in short arithmetic progressions

    Science.gov (United States)

    Puchta, Jan-Christoph

    We give a large sieve type inequality for functions supported on primes. As application we prove a conjecture by Elliott, and give bounds for short character sums over primes. The proves uses a combination of the large sieve and the Selberg sieve.

  6. Astronomical Spectroscopy A Short History

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 3; Issue 5. Astronomical Spectroscopy A Short History. J C Bhattacharyya. General Article Volume 3 Issue 5 May 1998 pp 24-29. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/003/05/0024-0029 ...

  7. SHORT COMMUNICATION CARCINOGENIC POTENCY OF ...

    African Journals Online (AJOL)

    SHORT COMMUNICATION. CARCINOGENIC POTENCY OF POLYCYCLIC AROMATIC HYDROCARBONS IN. SOIL. Okoro Dukea* and Ikolo O. Albertb. aDepartment of Chemistry, Delta State University, Abraka, Delta State, Nigeria. bNational Agency for Food and Drug Administration and Control, Central Laboratory, ...

  8. Downlink Transmission of Short Packets

    DEFF Research Database (Denmark)

    Trillingsgaard, Kasper Fløe; Popovski, Petar

    2017-01-01

    the principles of frame design and show the impact of the new design in scenarios that feature short data packets, which are central to various 5G and Internet of Things applications. We~treat framing for downlink transmission in an AWGN broadcast channel with $K$ users, where the sizes of the messages...

  9. Astronomical Spectroscopy A Short History

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 3; Issue 5. Astronomical Spectroscopy A Short History. J C Bhattacharyya. General Article Volume 3 Issue 5 May 1998 pp 24-29. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/003/05/0024-0029 ...

  10. Structure of a Stable G-Hairpin

    Czech Academy of Sciences Publication Activity Database

    Gajarský, M.; Zivkovic, M.L.; Stadlbauer, Petr; Pagano, B.; Fiala, R.; Amato, J.; Tomáška, L´.; Šponer, Jiří; Plavec, J.; Trantírek, L.

    2017-01-01

    Roč. 139, č. 10 (2017), s. 3591-3594 ISSN 0002-7863 R&D Projects: GA ČR GA13-28310S; GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : g-quadruplex structures * human telomeric dna * single-stranded- dna * g-triplex Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 13.858, year: 2016

  11. Review of short wavelength lasers

    International Nuclear Information System (INIS)

    Hagelstein, P.L.

    1985-01-01

    There has recently been a substantial amount of research devoted to the development of short wavelength amplifiers and lasers. A number of experimental results have been published wherein the observation of significant gain has been claimed on transitions in the EUV and soft x-ray regimes. The present review is intended to discuss the main approaches to the creation of population inversions and laser media in the short wavelength regime, and hopefully aid workers in the field by helping to provide access to a growing literature. The approaches to pumping EUV and soft x-ray lasers are discussed according to inversion mechanism. The approaches may be divided into roughly seven categories, including collisional excitation pumping, recombination pumping, direct photoionization and photoexcitation pumping, metastable state storage plus optical pumping, charge exchange pumping, and finally, the extension of free electron laser techniques into the EUV and soft x-ray regimes. 250 references

  12. Review of short wavelength lasers

    Energy Technology Data Exchange (ETDEWEB)

    Hagelstein, P.L.

    1985-03-18

    There has recently been a substantial amount of research devoted to the development of short wavelength amplifiers and lasers. A number of experimental results have been published wherein the observation of significant gain has been claimed on transitions in the EUV and soft x-ray regimes. The present review is intended to discuss the main approaches to the creation of population inversions and laser media in the short wavelength regime, and hopefully aid workers in the field by helping to provide access to a growing literature. The approaches to pumping EUV and soft x-ray lasers are discussed according to inversion mechanism. The approaches may be divided into roughly seven categories, including collisional excitation pumping, recombination pumping, direct photoionization and photoexcitation pumping, metastable state storage plus optical pumping, charge exchange pumping, and finally, the extension of free electron laser techniques into the EUV and soft x-ray regimes. 250 references.

  13. Complexity a very short introduction

    CERN Document Server

    Holland, John H

    2014-01-01

    The importance of complexity is well-captured by Hawking's comment: "Complexity is the science of the 21st century". From the movement of flocks of birds to the Internet, environmental sustainability, and market regulation, the study and understanding of complex non-linear systems has become highly influential over the last 30 years. In this Very Short Introduction, one of the leading figures in the field, John Holland, introduces the key elements and conceptual framework of complexity. From complex physical systems such as fluid flow and the difficulties of predicting weather, to complex adaptive systems such as the highly diverse and interdependent ecosystems of rainforests, he combines simple, well-known examples - Adam Smith's pin factory, Darwin's comet orchid, and Simon's 'watchmaker' - with an account of the approaches, involving agents and urn models, taken by complexity theory. ABOUT THE SERIES: The Very Short Introductions series from Oxford University Press contains hundreds of titles in almost eve...

  14. Optimal short-sighted rules

    Directory of Open Access Journals (Sweden)

    Sacha eBourgeois-Gironde

    2012-09-01

    Full Text Available The aim of this paper is to assess the relevance of methodological transfers from behavioral ecology to experimental economics with respect to the elicitation of intertemporal preferences. More precisely our discussion will stem from the analysis of Stephens and Anderson’s (2001 seminal article. In their study with blue jays they document that foraging behavior typically implements short sighted choice rules which are beneficial in the long-run. Such long term profitability of short-sighted behavior cannot be evidenced when using a self-control paradigm (one which contrasts in a binary way sooner smaller and later larger payoffs but becomes apparent when ecological patch-paradigms (replicating economic situations in which the main trade-off consists in staying on a food patch or leaving for another patch are implemented. We transfer this methodology in view of contrasting foraging strategies and self-control in human intertemporal choices.

  15. Short-range communication system

    Science.gov (United States)

    Alhorn, Dean C. (Inventor); Howard, David E. (Inventor); Smith, Dennis A. (Inventor)

    2012-01-01

    A short-range communication system includes an antenna, a transmitter, and a receiver. The antenna is an electrical conductor formed as a planar coil with rings thereof being uniformly spaced. The transmitter is spaced apart from the plane of the coil by a gap. An amplitude-modulated and asynchronous signal indicative of a data stream of known peak amplitude is transmitted into the gap. The receiver detects the coil's resonance and decodes same to recover the data stream.

  16. Short Course in the Microbiome

    Directory of Open Access Journals (Sweden)

    Kimberly Falana

    2015-07-01

    provides us with ideas as to how we can manipulate our microbiome to prevent certain diseases. However, given the variety of applications, there are scientific challenges, though there are very promising areas in reference to the clinical benefits of understanding more about our micro‐ biome, whether in our gut or on our skin: the outlook is bright. A summary of the short course is presented as a meeting dispatch.

  17. Statistical inference a short course

    CERN Document Server

    Panik, Michael J

    2012-01-01

    A concise, easily accessible introduction to descriptive and inferential techniques Statistical Inference: A Short Course offers a concise presentation of the essentials of basic statistics for readers seeking to acquire a working knowledge of statistical concepts, measures, and procedures. The author conducts tests on the assumption of randomness and normality, provides nonparametric methods when parametric approaches might not work. The book also explores how to determine a confidence interval for a population median while also providing coverage of ratio estimation, randomness, and causal

  18. Immunogenetics of Small Ruminant Lentiviral Infections

    Directory of Open Access Journals (Sweden)

    Nancy Stonos

    2014-08-01

    Full Text Available The small ruminant lentiviruses (SRLV include the caprine arthritis encephalitis virus (CAEV and the Maedi-Visna virus (MVV. Both of these viruses limit production and can be a major source of economic loss to producers. Little is known about how the immune system recognizes and responds to SRLVs, but due to similarities with the human immunodeficiency virus (HIV, HIV research can shed light on the possible immune mechanisms that control or lead to disease progression. This review will focus on the host immune response to HIV-1 and SRLV, and will discuss the possibility of breeding for enhanced SRLV disease resistance.

  19. Lentiviral vectors for treatment of haemophilia

    OpenAIRE

    Ward, N. J.

    2010-01-01

    Haemophilia A and B are X‐linked recessive disorders caused by defects in coagulation factors (F) VIII and IX, respectively. Severe cases of haemophilia are characterised by episodes of spontaneous bleeding, predominantly into the joints and muscles, and can result in permanent disability and even mortality if left untreated. The haemophilias are compelling candidates for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagul...

  20. Design of lentivirally expressed siRNAs

    NARCIS (Netherlands)

    Liu, Ying Poi; Berkhout, Ben

    2013-01-01

    RNA interference (RNAi) has been widely used as a tool for gene knockdown in fundamental research and for the development of new RNA-based therapeutics. The RNAi pathway is typically induced by expression of ∼22 base pair (bp) small interfering RNAs (siRNAs), which can be transfected into cells. For

  1. Gene Expression Profile Changes After Short-activating RNA-mediated Induction of Endogenous Pluripotency Factors in Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Jon Voutila

    2012-01-01

    Full Text Available It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4 and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research.

  2. Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.

    Science.gov (United States)

    Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan

    2016-09-01

    In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Folding very short peptides using molecular dynamics.

    Directory of Open Access Journals (Sweden)

    Bosco K Ho

    2006-04-01

    Full Text Available Peptides often have conformational preferences. We simulated 133 peptide 8-mer fragments from six different proteins, sampled by replica-exchange molecular dynamics using Amber7 with a GB/SA (generalized-Born/solvent-accessible electrostatic approximation to water implicit solvent. We found that 85 of the peptides have no preferred structure, while 48 of them converge to a preferred structure. In 85% of the converged cases (41 peptides, the structures found by the simulations bear some resemblance to their native structures, based on a coarse-grained backbone description. In particular, all seven of the beta hairpins in the native structures contain a fragment in the turn that is highly structured. In the eight cases where the bioinformatics-based I-sites library picks out native-like structures, the present simulations are largely in agreement. Such physics-based modeling may be useful for identifying early nuclei in folding kinetics and for assisting in protein-structure prediction methods that utilize the assembly of peptide fragments.

  4. Short Stature Diagnosis and Referral

    Directory of Open Access Journals (Sweden)

    Mohamad Maghnie

    2018-01-01

    Full Text Available The “360° GH in Europe” meeting, which examined various aspects of GH diseases, was held in Lisbon, Portugal, in June 2016. The Merck KGaA (Germany funded meeting comprised three sessions entitled “Short Stature Diagnosis and Referral,” “Optimizing Patient Management,” and “Managing Transition.” Each session had three speaker presentations, followed by a discussion period, and is reported as a manuscript, authored by the speakers. The first session examined current processes of diagnosis and referral by endocrine specialists for pediatric patients with short stature. Requirements for referral vary widely, by country and by patient characteristics such as age. A balance must be made to ensure eligible patients get referred while healthcare systems are not over-burdened by excessive referrals. Late referral and diagnosis of non-GH deficiency conditions can result in increased morbidity and mortality. The consequent delays in making a diagnosis may compromise the effectiveness of GH treatment. Algorithms for growth monitoring and evaluation of skeletal disproportions can improve identification of non-GH deficiency conditions. Performance and validation of guidelines for diagnosis of GH deficiency have not been sufficiently tested. Provocative tests for investigation of GH deficiency remain equivocal, with insufficient information on variations due to patient characteristics, and cutoff values for definition differ not only by country but also by the assay used. When referring and diagnosing causes of short stature in pediatric patients, clinicians need to rely on many factors, but the most essential is clinical experience.

  5. Bombesin in short bowel syndrome.

    Science.gov (United States)

    Uluutku, Ahmet Haldun; Akin, Mehmet Levhi; Kurt, Yavuz; Yucel, Ergun; Cermik, Hakan; Avsar, Kadir; Celenk, Tuncay

    2004-01-01

    Short bowel syndrome comprises the sequel of nutrient, fluid, and weight loss that occurs subsequent to greatly reduced functional surface area of the small intestine. The aim of this study is to investigate the trophic and functional effects of bombesin on remaining gut in rats with experimentally induced short bowel syndrome. Thirty-two rats were allocated randomly and experimental short bowel syndrome was induced by 80% bowel resection in all rats. A regular enteral diet and isocaloric elemental enteral nutrition for 12 days were given in the control group and the elemental nutrition group, respectively. In the bombesin group 10 microg/kg subcutaneous bombesin (t.i.d.) for 10 days with regular enteral diet for 12 days was given. In the elemental nutrition and bombesin group the diet consisted of 10 microg/kg subcutaneous bombesin (t.i.d.) for 10 days with isocaloric elemental enteral nutrition for 12 days was given. All rats underwent physical, histological, and biochemical evaluation. Reduction in weight loss, bowel diameter, fecal fat content, and glycemia, increase in cellularity, and d-xylose absorption were observed in all treatment groups. These changes were more evident in the bombesin treatment groups. Increases in serum protein and albumin levels were seen with bombesin treatment with or without elemental diet, whereas reductions in villous height and crypt depth were observed only with bombesin treatment without elemental diet. Serum calcium, iron, and vitamin B(12) levels were not affected with any treatment. It is concluded that bombesin may be a useful trophic agent contributing to increased absorptive capacity and improved biochemical values even in the absence of elemental nutrition.

  6. Sound a very short introduction

    CERN Document Server

    Goldsmith, Mike

    2015-01-01

    Sound is integral to how we experience the world, in the form of noise as well as music. But what is sound? What is the physical basis of pitch and harmony? And how are sound waves exploited in musical instruments? Sound: A Very Short Introduction looks at the science of sound and the behaviour of sound waves with their different frequencies. It also explores sound in different contexts, covering the audible and inaudible, sound underground and underwater, acoustic and electronic sound, and hearing in humans and animals. It concludes with the problem of sound out of place—noise and its reduction.

  7. Radioactivity a very short introduction

    CERN Document Server

    Tuniz, Claudio

    2012-01-01

    Radioactivity: A Very Short Introduction explains radioactivity and discusses its fundamental role in nature. Radioactivity remains misunderstood and feared perhaps because nuclear radiation cannot be detected by human senses, and can undoubtedly do great harm if appropriate precautions are not taken. Radioactivity in the stars and in the Earth and its wide range of applications in biomedicine, science, industry, agriculture are described, as well as the mechanisms of nuclear fission and fusion, and the harnessing of nuclear power. The issues surrounding safety and security and the increasing concerns about nuclear terrorism are also considered.

  8. Ultra-short pulse generator

    Science.gov (United States)

    McEwan, T.E.

    1993-12-28

    An inexpensive pulse generating circuit is disclosed that generates ultra-short, 200 picosecond, and high voltage 100 kW, pulses suitable for wideband radar and other wideband applications. The circuit implements a nonlinear transmission line with series inductors and variable capacitors coupled to ground made from reverse biased diodes to sharpen and increase the amplitude of a high-voltage power MOSFET driver input pulse until it causes non-destructive transit time breakdown in a final avalanche shock wave diode, which increases and sharpens the pulse even more. 5 figures.

  9. Ultra-short pulse generator

    Science.gov (United States)

    McEwan, Thomas E.

    1993-01-01

    An inexpensive pulse generating circuit is disclosed that generates ultra-short, 200 picosecond, and high voltage 100 kW, pulses suitable for wideband radar and other wideband applications. The circuit implements a nonlinear transmission line with series inductors and variable capacitors coupled to ground made from reverse biased diodes to sharpen and increase the amplitude of a high-voltage power MOSFET driver input pulse until it causes non-destructive transit time breakdown in a final avalanche shockwave diode, which increases and sharpens the pulse even more.

  10. Moons a very short introduction

    CERN Document Server

    Rothery, David A

    2015-01-01

    Moons: A Very Short Introduction introduces the reader to the varied and fascinating moons of our Solar System. Beginning with the early discoveries of Galileo and others, it describes their variety of mostly mythological names, and the early use of Jupiter’s moons to establish position at sea and to estimate the speed of light. It discusses the structure, formation, and profound influence of our Moon, those of the other planets, and ends with the recent discovery of moons orbiting asteroids, whilst looking forward to the possibility of discovering microbial life beyond Earth and of finding moons of exoplanets in planetary systems far beyond our own.

  11. Planets a very short introduction

    CERN Document Server

    Rothery, David A

    2010-01-01

    Planets: A Very Short Introduction demonstrates the excitement, uncertainties, and challenges faced by planetary scientists, and provides an overview of our Solar System and its origins, nature, and evolution. Terrestrial planets, giant planets, dwarf planets and various other objects such as satellites (moons), asteroids, trans-Neptunian objects, and exoplanets are discussed. Our knowledge about planets has advanced over the centuries, and has expanded at a rapidly growing rate in recent years. Controversial issues are outlined, such as What qualifies as a planet? What conditions are required for a planetary body to be potentially inhabited by life? Why does Pluto no longer have planet status? And Is there life on other planets?

  12. Superconductivity a very short introduction

    CERN Document Server

    Blundell, Stephen

    2009-01-01

    Superconductivity is one of the most exciting areas of research in physics today. Outlining the history of its discovery, and the race to understand its many mysterious and counter-intuitive phenomena, this Very Short Introduction explains in accessible terms the theories that have been developed, and how they have influenced other areas of science, including the Higgs boson of particle physics and ideas about the early Universe. It is an engaging and informative accountof a fascinating scientific detective story, and an intelligible insight into some deep and beautiful ideas of physics

  13. Stars a very short introduction

    CERN Document Server

    King, Andrew

    2012-01-01

    Stars: A Very Short Introduction looks at how stars live, producing all the chemical elements beyond helium, and how they die, leaving remnants such as black holes. Every atom of our bodies has been part of a star. Our very own star, the Sun, is crucial to the development and sustainability of life on Earth. Understanding stars is key to understanding the galaxies they inhabit, the existence of planets, and the history of our entire Universe. This VSI explores the science of stars, the mechanisms that allow them to form, the processes that allow them to shine, and the results of their death.

  14. Galaxies a very short introduction

    CERN Document Server

    Gribbin, John

    2008-01-01

    Galaxies: A Very Short Introduction explores the building blocks of the Universe. Standing like islands in space, each is made up of many hundreds of millions of stars in which the chemical elements are made, around which planets form, and where on at least one of those planets intelligent life has emerged. Our own galaxy, the Milky Way, is just one of several hundred million other galaxies. Yet it was only in the 1920s that we realised that there is more to the Universe. Since then, many exciting discoveries have been made about our own galaxy and about those beyond.

  15. Robin Jenkins: the short stories

    OpenAIRE

    Riach, Alan

    2017-01-01

    Robin Jenkins’s short stories are collected in two books: A Far Cry from Bowmore (1973), set in the far east, in locations such as Malaysia, Afghanistan and Burma, and Lunderston Tales (1996), set in a small seaside town on the west coast of Scotland. This chapter begins by discussing ideas of distance and proximity in Jenkins’s writing, showing how stylistically he brings together apparently ‘objective’ prose describing events ‘externally’ with more insidious and sly representations of ‘subj...

  16. Magnetism a very short introduction

    CERN Document Server

    Blundell, Stephen J

    2012-01-01

    Magnetism: A Very Short Introduction explains the mysteries and importance of magnetism. For centuries magnetism has been used for various exploits: as a great healer, a navigation aid through compasses, and through motors, generators, and turbines it has given us power. Our understanding of electricity and magnetism, from the work of Galvani, Ampère, Faraday, and Tesla is explored, and how Maxwell and Faraday's work led to the unification of electricity and magnetism is explained. With a discussion of the relationship between magnetism and relativity, quantum magnetism, and its impact on computers and information storage, how magnetism has changed our fundamental understanding of the Universe is shown.

  17. Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Agerstam, Helena

    2009-01-01

    OBJECTIVE: The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. MATERIALS AND METHODS: We investigated...... and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. RESULTS: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells...... and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under...

  18. Short rotation Wood Crops Program

    Energy Technology Data Exchange (ETDEWEB)

    Wright, L.L.; Ehrenshaft, A.R.

    1990-08-01

    This report synthesizes the technical progress of research projects in the Short Rotation Woody Crops Program for the year ending September 30, 1989. The primary goal of this research program, sponsored by the US Department of Energy's Biofuels and Municipal Waste Technology Division, is the development of a viable technology for producing renewable feedstocks for conversion to biofuels. One of the more significant accomplishments was the documentation that short-rotation woody crops total delivered costs could be $40/Mg or less under optimistic but attainable conditions. By taking advantage of federal subsidies such as those offered under the Conservation Reserve Program, wood energy feedstock costs could be lower. Genetic improvement studies are broadening species performance within geographic regions and under less-than-optimum site conditions. Advances in physiological research are identifying key characteristics of species productivity and response to nutrient applications. Recent developments utilizing biotechnology have achieved success in cell and tissue culture, somaclonal variation, and gene-insertion studies. Productivity gains have been realized with advanced cultural studies of spacing, coppice, and mixed-species trials. 8 figs., 20 tabs.

  19. Isotopes a very short introduction

    CERN Document Server

    Ellam, Rob

    2016-01-01

    An isotope is a variant form of a chemical element, containing a different number of neutrons in its nucleus. Most elements exist as several isotopes. Many are stable while others are radioactive, and some may only exist fleetingly before decaying into other elements. In this Very Short Introduction, Rob Ellam explains how isotopes have proved enormously important across all the sciences and in archaeology. Radioactive isotopes may be familiar from their use in nuclear weapons, nuclear power, and in medicine, as well as in carbon dating. They have been central to establishing the age of the Earth and the origins of the solar system. Combining previous and new research, Ellam provides an overview of the nature of stable and radioactive isotopes, and considers their wide range of modern applications. ABOUT THE SERIES: The Very Short Introductions series from Oxford University Press contains hundreds of titles in almost every subject area. These pocket-sized books are the perfect way to get ahead in a new subjec...

  20. Economic impact analysis of short line railroads.

    Science.gov (United States)

    2014-10-01

    This research project assesses the economic role and impact of short line railroads in the state of Louisiana. While relatively small in : scope, with 11 operators and approximately 500 miles of track, short line railroads play a significant role in ...

  1. Pregnancy Complications: Cervical Insufficiency and Short Cervix

    Science.gov (United States)

    ... insufficiency and short cervix Cervical insufficiency and short cervix E-mail to a friend Please fill in ... the past or repeat miscarriages. What is the cervix? The cervix is the opening to the uterus ( ...

  2. WPC's Short Range Forecast Coded Bulletin

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Short Range Forecast Coded Bulletin. The Short Range Forecast Coded Bulletin describes the expected locations of high and low pressure centers, surface frontal...

  3. Coulomb repulsion in short polypeptides.

    Science.gov (United States)

    Norouzy, Amir; Assaf, Khaleel I; Zhang, Shuai; Jacob, Maik H; Nau, Werner M

    2015-01-08

    Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge-charge repulsion, side chain functional group solvation, N-terminal and side chain charge-charge repulsion, and side chain-side chain as well as side chain-backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each

  4. 46 CFR 15.725 - Sailing short.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Sailing short. 15.725 Section 15.725 Shipping COAST... Limitations and Qualifying Factors § 15.725 Sailing short. Whenever a vessel is deprived of the service of a... vessel is sufficiently manned for the voyage. A report of sailing short must be filed in writing with the...

  5. Copernicus a very short introduction

    CERN Document Server

    Gingerich, Owen

    2016-01-01

    Nicolaus Copernicus (1473–1543) was the astronomer whose shocking vision of a sun-centered universe turned out to be the essential blueprint for a physical understanding of celestial motions. Copernicus: A Very Short Introduction offers a fascinating portrayal of the man who launched the modern vision of the universe. It sets Copernicus in the context of a rapidly changing world, where the recent invention of printing with movable type not only made sources more readily available to him, but also fueled Martin Luther’s transformation of the religious landscape. Copernicus’s heliocentric revolution is revealed as an aesthetic achievement not dictated by observational “proofs,” but another new way of looking at the ancient cosmos.

  6. Shapeable short circuit resistant capacitor

    Science.gov (United States)

    Taylor, Ralph S.; Myers, John D.; Baney, William J.

    2015-10-06

    A ceramic short circuit resistant capacitor that is bendable and/or shapeable to provide a multiple layer capacitor that is extremely compact and amenable to desirable geometries. The capacitor that exhibits a benign failure mode in which a multitude of discrete failure events result in a gradual loss of capacitance. Each event is a localized event in which localized heating causes an adjacent portion of one or both of the electrodes to vaporize, physically cleaning away electrode material from the failure site. A first metal electrode, a second metal electrode, and a ceramic dielectric layer between the electrodes are thin enough to be formed in a serpentine-arrangement with gaps between the first electrode and the second electrode that allow venting of vaporized electrode material in the event of a benign failure.

  7. Infinity a very short introduction

    CERN Document Server

    Stewart, Ian

    2017-01-01

    Infinity is an intriguing topic, with connections to religion, philosophy, metaphysics, logic, and physics as well as mathematics. Its history goes back to ancient times, with especially important contributions from Euclid, Aristotle, Eudoxus, and Archimedes. The infinitely large (infinite) is intimately related to the infinitely small (infinitesimal). Cosmologists consider sweeping questions about whether space and time are infinite. Philosophers and mathematicians ranging from Zeno to Russell have posed numerous paradoxes about infinity and infinitesimals. Many vital areas of mathematics rest upon some version of infinity. The most obvious, and the first context in which major new techniques depended on formulating infinite processes, is calculus. But there are many others, for example Fourier analysis and fractals. In this Very Short Introduction, Ian Stewart discusses infinity in mathematics while also drawing in the various other aspects of infinity and explaining some of the major problems and insights ...

  8. Astrophysics a very short introduction

    CERN Document Server

    Binney, James

    2016-01-01

    Astrophysics is the physics of the stars, and more widely the physics of the Universe. It enables us to understand the structure and evolution of planetary systems, stars, galaxies, interstellar gas, and the cosmos as a whole. In this Very Short Introduction, the leading astrophysicist James Binney shows how the field of astrophysics has expanded rapidly in the past century, with vast quantities of data gathered by telescopes exploiting all parts of the electromagnetic spectrum, combined with the rapid advance of computing power, which has allowed increasingly effective mathematical modelling. He illustrates how the application of fundamental principles of physics - the consideration of energy and mass, and momentum - and the two pillars of relativity and quantum mechanics, has provided insights into phenomena ranging from rapidly spinning millisecond pulsars to the collision of giant spiral galaxies. This is a clear, rigorous introduction to astrophysics for those keen to cut their teeth on a conceptual trea...

  9. Intestino Corto Short bowel syndrome

    Directory of Open Access Journals (Sweden)

    María Matilde Socarrás Suárez

    2004-06-01

    Full Text Available El intestino corto está asociado a pérdida o disfunción del intestino delgado por resección del mismo, que causa diarreas, tránsito intestinal acelerado, malabsorción intestinal, y eventualmente la pérdida de peso y el desgaste muscular. El objetivo de este trabajo fue actualizar el conocimiento acerca de este síndrome. Se realiza una revisión del tema de intestino corto donde se refiere a su definición, causas fundamentales frecuentes e infrecuentes en el niño y en el adulto, cómo se adapta el intestino a la resección de diferentes extensiones, las funciones del íleon terminal. Se hacen una valoración clínica inicial, con el interrogatorio médico, revisión minuciosa de la historia clínica para cuantificar la capacidad de absorción. Se habla de los síntomas y signos de deficiencia nutricional. Se explican las estrategias del tratamiento, que tienen 3 etapas de evolución clínica. Se concluye que se indica la dietoterapia adecuada según el estado nutricional del paciente y la resección intestinal realizada, evitando las complicaciones para lograr una calidad máxima de vidaShort bowel is associated with loss or dysfunction of the small bowel due to its resection, which causes diarrheas, accelerated intestinal transit, intestinal malabsorption and, eventually, weight loss ansd muscular waste. The objective of this paper was to update knowledge about this syndrome. A review of the short intestine topic is made, making reference to its definition, common and uncommon main cuases in the child and adult, how the bowel adapts itslef to resection of different extensions, and the functions of the terminal ileum. An initial clinical assessment is made with the medical questionnaire and a detailed review of the medical history to quantify the absorption capacity. The symptoms and signs of nutritional deficiency are dealt with. The strategies of the treatment consisting of 3 stages of clinical evolution are explained. It is concluded

  10. Gravity a very short introduction

    CERN Document Server

    Clifton, Timothy

    2017-01-01

    Gravity is one of the four fundamental interactions that exist in nature. It also has the distinction of being the oldest, weakest, and most difficult force to quantize. Understanding gravity is not only essential for understanding the motion of objects on Earth, but also the motion of all celestial objects, and even the expansion of the Universe itself. It was the study of gravity that led Einstein to his profound realizations about the nature of space and time. Gravity is not only universal, it is also essential for understanding the behavior of the Universe, and all astrophysical bodies within it. In this Very Short Introduction Timothy Clifton looks at the development of our understanding of gravity since the early observations of Kepler and Newtonian theory. He discusses Einstein's theory of gravity, which now supplants Newton's, showing how it allows us to understand why the frequency of light changes as it passes through a gravitational field, why GPS satellites need their clocks corrected as they orbi...

  11. Light a very short introduction

    CERN Document Server

    Walmsley, Ian

    2015-01-01

    Light enables us to see the world around us. Our sense of sight provides us with direct information about space and time, the physical arrangement of the world, and how it changes. This almost universal shared sensation of vision has led to a fascination with the nature and properties of light across the ages. But the light we see is just a small part of the whole spectrum of electromagnetic radiation, ranging from radio waves to gamma rays. In this Very Short Introduction Ian Walmsley discusses early attempts to explain light, and the development of apparently opposing particulate and wave theories by scientists such as Isaac Newton and Christiaan Huygens. He shows how light was recognized as an electromagnetic wave in the 19th century, and the development of the quantum mechanics view of wave-particle duality in the 20th century. He also describes the many applications of light, domestic and scientific, such as microwaves, DVDs, and lasers. We now use the whole range of electromagnetic radiation to peer bot...

  12. Onboard Short Term Plan Viewer

    Science.gov (United States)

    Hall, Tim; LeBlanc, Troy; Ulman, Brian; McDonald, Aaron; Gramm, Paul; Chang, Li-Min; Keerthi, Suman; Kivlovitz, Dov; Hadlock, Jason

    2011-01-01

    Onboard Short Term Plan Viewer (OSTPV) is a computer program for electronic display of mission plans and timelines, both aboard the International Space Station (ISS) and in ISS ground control stations located in several countries. OSTPV was specifically designed both (1) for use within the limited ISS computing environment and (2) to be compatible with computers used in ground control stations. OSTPV supplants a prior system in which, aboard the ISS, timelines were printed on paper and incorporated into files that also contained other paper documents. Hence, the introduction of OSTPV has both reduced the consumption of resources and saved time in updating plans and timelines. OSTPV accepts, as input, the mission timeline output of a legacy, print-oriented, UNIX-based program called "Consolidated Planning System" and converts the timeline information for display in an interactive, dynamic, Windows Web-based graphical user interface that is used by both the ISS crew and ground control teams in real time. OSTPV enables the ISS crew to electronically indicate execution of timeline steps, launch electronic procedures, and efficiently report to ground control teams on the statuses of ISS activities, all by use of laptop computers aboard the ISS.

  13. Short QT syndrome presenting as syncope: how short is too short?

    Science.gov (United States)

    Portugal, Guilherme; Martins Oliveira, Mário; Silva Cunha, Pedro; Ferreira, Filipa; Lousinha, Ana; Fiarresga, António; Nogueira da Silva, Manuel; Cruz Ferreira, Rui

    2014-10-01

    We report the case of a 52-year-old man who presented to our emergency department (ED) after three episodes of syncope in the seven hours before admission. During his stay in the ED he had recurrent ventricular tachycardia (VT) requiring external electrical cardioversion. A 12-lead electrocardiogram (ECG) showed a short QT (SQT) interval (270 ms, QTc 327 ms), with frequent R-on-T extrasystoles triggering sustained polymorphic VT. After exclusion of other precipitating causes, the patient was diagnosed as having SQT syndrome (SQTS) according to the Gollob criteria. To our knowledge, this is the first known documentation of an SQT-caused arrhythmic episode on a 12-lead ECG, as well as the first reported case of SQTS in Portugal. The patient received an implantable cardioverter-defibrillator and was discharged. At a follow-up assessment 14 months later he was symptom-free, interrogation of the device showed no arrhythmic events, and the ECG showed a QT interval of 320 ms (QTc 347 ms). Copyright © 2013 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

  14. Short-Selling, Leverage and Systemic Risk

    OpenAIRE

    Pais, Amelia; Stork, Philip A.

    2013-01-01

    During the Global Financial Crisis, regulators imposed short-selling bans to protect financial institutions. The rationale behind the bans was that “bear raids”, driven by short-sellers, would increase the individual and systemic risk of financial institutions, especially for institutions with high leverage. This study uses Extreme Value Theory to estimate the effect of short-selling on financial institutions’ individual and systemic risks in France, Italy and Spain; it also analyses the rela...

  15. Short-sale Constraints and Credit Runs

    DEFF Research Database (Denmark)

    Venter, Gyuri

    This paper studies how short-sale constraints affect the informational efficiency of market prices and the link between prices and economic activity. I show that under short-sale constraints security prices contain less information. However, short-sale constraints increase the informativeness...... of prices to some agents who learn about the quality of an investment opportunity from market prices and have additional private information. Then I apply this observation when modeling a run on an investment bank by its short-term creditors, who are endowed with dispersed information and also learn from...

  16. Chaos: A Very Short Introduction

    International Nuclear Information System (INIS)

    Klages, R

    2007-01-01

    is also getting a bit too intricate for the complete layman, and experts may not agree on all details of the more conceptual discussions. Altogether I thoroughly enjoyed reading this book. It was a happy companion while travelling and a nice bedtime literature. It is furthermore an excellent reminder of the 'big picture' underlying nonlinear science as it applies to the real world. I will gladly recommend this book as background literature for students in my introductory course on dynamical systems. However, the book will be of interest to anyone who is looking for a very short account on fundamental problems and principles in modern nonlinear science. (book review)

  17. Chaos: A Very Short Introduction

    Energy Technology Data Exchange (ETDEWEB)

    Klages, R [School of Mathematical Sciences, Mile End Road, London, E1 4NS (United Kingdom)

    2007-07-20

    field. I feel that occasionally the book is also getting a bit too intricate for the complete layman, and experts may not agree on all details of the more conceptual discussions. Altogether I thoroughly enjoyed reading this book. It was a happy companion while travelling and a nice bedtime literature. It is furthermore an excellent reminder of the 'big picture' underlying nonlinear science as it applies to the real world. I will gladly recommend this book as background literature for students in my introductory course on dynamical systems. However, the book will be of interest to anyone who is looking for a very short account on fundamental problems and principles in modern nonlinear science. (book review)

  18. Short Communication Health Workers' Knowledge of and ...

    African Journals Online (AJOL)

    2011-08-30

    Aug 30, 2011 ... Short Communication. Health Workers' Knowledge of and Experience with Female ... associated with both short-term and long-term complications (Behrendt and Moritz, 2005). It is common in ..... Stress Disorder and Memory Problems after Female. Genital Mutilation. Am J Psychiatry. 162: 1000–1002.

  19. Worst Asymmetrical Short-Circuit Current

    DEFF Research Database (Denmark)

    Arana Aristi, Iván; Holmstrøm, O; Grastrup, L

    2010-01-01

    In a typical power plant, the production scenario and the short-circuit time were found for the worst asymmetrical short-circuit current. Then, a sensitivity analysis on the missing generator values was realized in order to minimize the uncertainty of the results. Afterward the worst asymmetrical...

  20. 31 CFR 505.01 - Short title.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Short title. 505.01 Section 505.01 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... CERTAIN MERCHANDISE BETWEEN FOREIGN COUNTRIES § 505.01 Short title. The regulations in this part may be...

  1. 33 CFR 401.1 - Short title.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Short title. 401.1 Section 401.1 Navigation and Navigable Waters SAINT LAWRENCE SEAWAY DEVELOPMENT CORPORATION, DEPARTMENT OF TRANSPORTATION SEAWAY REGULATIONS AND RULES Regulations § 401.1 Short title. These regulations may be cited as the...

  2. 18 CFR 415.1 - Short title.

    Science.gov (United States)

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Short title. 415.1 Section 415.1 Conservation of Power and Water Resources DELAWARE RIVER BASIN COMMISSION ADMINISTRATIVE MANUAL BASIN REGULATIONS-FLOOD PLAIN REGULATIONS Generally § 415.1 Short title. This part shall be known...

  3. Genetics Home Reference: short QT syndrome

    Science.gov (United States)

    ... and can lead to the abnormal heart rhythms characteristic of short QT syndrome . Some affected individuals do not have an identified ... C, Giustetto C, Gaita F, Borggrefe M. Clinical characteristics and treatment of short QT syndrome. Expert Rev Cardiovasc Ther. 2005 Jul;3(4): ...

  4. Are short GRBs powered by magnetars?

    NARCIS (Netherlands)

    O'Brien, P.T.; Rowlinson, A.

    2012-01-01

    The standard model for a short duration Gamma-Ray Burst (GRB) involves the merger of a neutron star binary system, resulting in a black hole which accretes for a brief period of time. However, some of the short-duration GRBs observed by the Swift satellite show features in their light curves which

  5. Short range radio research in Twente

    NARCIS (Netherlands)

    Meijerink, Arjan

    2010-01-01

    The research and education by the Telecommunication Engineering Group at the University of Twente is dedicated to physical layer topics in communications. Three research tracks have prominence: Short Range Radio, Microwave Photonics, and Electromagnetic Compatibility. Arjan is active in the Short

  6. Proposition algebra and short-circuit logic

    NARCIS (Netherlands)

    Bergstra, J.A.; Ponse, A.

    2012-01-01

    Short-circuit evaluation denotes the semantics of propositional connectives in which the second argument is only evaluated if the first argument does not suffice to determine the value of the expression. In programming, short-circuit evaluation is widely used. We review proposition algebra [2010],

  7. Magnetic short range order in Gd

    International Nuclear Information System (INIS)

    Child, H.R.

    1976-01-01

    Quasielastic neutron scattering has been used to investigate magnetic short range order in Gd for 80 0 K 0 K. Short range order exists throughout this range from well below T/sub C/ = 291 0 K to well above it and can be reasonably well described by an anisotropic Orstein-Zernike form for chi

  8. Short oestrous cycles post partum in cattle

    International Nuclear Information System (INIS)

    Edqvist, L.E.; Frederiksson, G.; Kindahl, H.; Larsson, K.; Madej, A.

    1984-01-01

    Oestrous cycles of shorter duration (8-12 days) are usually found when either dairy or beef cows resume cyclicity post partum. The incidence of short cycle is so high that it should be considered a normal physiological phenomenon. Strong evidence in beef cattle suggests that the short cycle follows an ovulation (probably the same in dairy cows). In dairy cows it is likely that a premature release of prostaglandin is responsible for the short luteal phase (probably the same in beef cows). The occurrence of short oestrous cycles is incompatible with the establishment of pregnancy. In water buffaloes the occurrence of short oestrous cycles following weaning has to be considered and breeding of animals subsequenty adjusted. (author)

  9. Short term memory in echo state networks

    OpenAIRE

    Jaeger, H.

    2001-01-01

    The report investigates the short-term memory capacity of echo state recurrent neural networks. A quantitative measure MC of short-term memory capacity is introduced. The main result is that MC 5 N for networks with linear Output units and i.i.d. input, where N is network size. Conditions under which these maximal memory capacities are realized are described. Several theoretical and practical examples demonstrate how the short-term memory capacities of echo state networks can be exploited for...

  10. Models of Short-Term Synaptic Plasticity.

    Science.gov (United States)

    Barroso-Flores, Janet; Herrera-Valdez, Marco A; Galarraga, Elvira; Bargas, José

    2017-01-01

    We focus on dynamical descriptions of short-term synaptic plasticity. Instead of focusing on the molecular machinery that has been reviewed recently by several authors, we concentrate on the dynamics and functional significance of synaptic plasticity, and review some mathematical models that reproduce different properties of the dynamics of short term synaptic plasticity that have been observed experimentally. The complexity and shortcomings of these models point to the need of simple, yet physiologically meaningful models. We propose a simplified model to be tested in synapses displaying different types of short-term plasticity.

  11. 24th geotechnical laboratory testing short course

    Science.gov (United States)

    2008-02-01

    This is a 3-day workshop/short course to teach practicing professionals techniques and procedures for conducting high quality geotechnical laboratory tests. Transportation facility design and construction begins with an investigation of the type, ext...

  12. Minimalism in the modern short story

    Directory of Open Access Journals (Sweden)

    A Razi

    2009-09-01

    Full Text Available Short story has recently become the focus of attention in the late decades in Iran. The expanding value of writing short story is actually a reasonable outcome of the dominance of minimalism- a movement which is based upon simplicity and shortness. Minimalist writers, leaving out redundant features of narration, mainly focus on essentialities through applying a variety of techniques such as cuttings from the interesting moments of real life, evading introduction, applying inter-referents, choice of words, short stanzas and sentences and so on. Looking upon critic’s opinion about such a tendency over the past and present, this article will come up with a brief explanation of the properties of such stories. Finally a sample story “candles will never go dead” will be analyzed and discussed in the lights of such techniques.

  13. Optimal parametric modelling of measured short waves

    Digital Repository Service at National Institute of Oceanography (India)

    Mandal, S.

    The spectral analysis of measured short waves can efficiently be carried out by the fast Fourier transform technique. Even though many present techniques can be used for the simulation of time series waves, these may not provide accurate...

  14. Short-distance structure of instantons

    International Nuclear Information System (INIS)

    Carlitz, R.D.; Creamer, D.B.

    The short distance structure of a quark propagator in an instanton background field is analyzed. This analysis describes the influence of instantons upon physical processes such as electron-position annihilation and the deep inelastic scattering of leptons from hadrons

  15. New shorting mechanism for pelletron accelerator

    International Nuclear Information System (INIS)

    Ansari, Q.N.; Bhagwat, P.V.; D'Costa, C.; Sarkar, S.K.

    2001-01-01

    A new mechanism to electrically short Pelletron Accelerator modules has been developed to replace the present manually operated system that contains some limitations. Its salient features over the existing system are that the operation will be fully automatic, any module can be independently shorted from control console and its status read back will be available on the control console. Since the new system is not extended to the main Accelerator Tank, chance of SF 6 leakage is eliminated. (author)

  16. Fund managers’ contracts and short-termism

    OpenAIRE

    Casamatta, Catherine; Pouget, Sébastien

    2009-01-01

    This paper considers the problem faced by long-term investors who have to delegate the management of their money to professional fund managers. Investors can earn profits if fund managers collect long-term information. We investigate to what extent the delegation of fund management prevents long-term information acquisition, inducing short-termism. We also study the design of long-term fund managers' compensation contracts. Absent moral hazard, short-termism arises only because of the cost...

  17. Modern treatment of short bowel syndrome

    DEFF Research Database (Denmark)

    Jeppesen, Palle B

    2013-01-01

    Recently, the US Food and Drug Administration and the European Medicines Agency approved the glucagon-like peptide 2 analogue, teduglutide, for the treatment of short bowel syndrome (SBS), and this review describes the physiological basis for its clinical use.......Recently, the US Food and Drug Administration and the European Medicines Agency approved the glucagon-like peptide 2 analogue, teduglutide, for the treatment of short bowel syndrome (SBS), and this review describes the physiological basis for its clinical use....

  18. Short-term memory across eye blinks.

    Science.gov (United States)

    Irwin, David E

    2014-01-01

    The effect of eye blinks on short-term memory was examined in two experiments. On each trial, participants viewed an initial display of coloured, oriented lines, then after a retention interval they viewed a test display that was either identical or different by one feature. Participants kept their eyes open throughout the retention interval on some blocks of trials, whereas on others they made a single eye blink. Accuracy was measured as a function of the number of items in the display to determine the capacity of short-term memory on blink and no-blink trials. In separate blocks of trials participants were instructed to remember colour only, orientation only, or both colour and orientation. Eye blinks reduced short-term memory capacity by approximately 0.6-0.8 items for both feature and conjunction stimuli. A third, control, experiment showed that a button press during the retention interval had no effect on short-term memory capacity, indicating that the effect of an eye blink was not due to general motoric dual-task interference. Eye blinks might instead reduce short-term memory capacity by interfering with attention-based rehearsal processes.

  19. Short Implants: New Horizon in Implant Dentistry

    Science.gov (United States)

    Gulati, Manisha; Garg, Meenu; Pathak, Chetan

    2016-01-01

    The choice of implant length is an essential factor in deciding the survival rates of these implants and the overall success of the prosthesis. Placing an implant in the posterior part of the maxilla and mandible has always been very critical due to poor bone quality and quantity. Long implants can be placed in association with complex surgical procedures such as sinus lift and bone augmentation. These techniques are associated with higher cost, increased treatment time and greater morbidity. Hence, there is need for a less invasive treatment option in areas of poor bone quantity and quality. Data related to survival rates of short implants, their design and prosthetic considerations has been compiled and structured in this manuscript with emphasis on the indications, advantages of short implants and critical biomechanical factors to be taken into consideration when choosing to place them. Studies have shown that comparable success rates can be achieved with short implants as those with long implants by decreasing the lateral forces to the prosthesis, eliminating cantilevers, increasing implant surface area and improving implant to abutment connection. Short implants can be considered as an effective treatment alternative in resorbed ridges. Short implants can be considered as a viable treatment option in atrophic ridge cases in order to avoid complex surgical procedures required to place long implants. With improvement in the implant surface geometry and surface texture, there is an increase in the bone implant contact area which provides a good primary stability during osseo-integration. PMID:27790598

  20. Short Implants: New Horizon in Implant Dentistry.

    Science.gov (United States)

    Jain, Neha; Gulati, Manisha; Garg, Meenu; Pathak, Chetan

    2016-09-01

    The choice of implant length is an essential factor in deciding the survival rates of these implants and the overall success of the prosthesis. Placing an implant in the posterior part of the maxilla and mandible has always been very critical due to poor bone quality and quantity. Long implants can be placed in association with complex surgical procedures such as sinus lift and bone augmentation. These techniques are associated with higher cost, increased treatment time and greater morbidity. Hence, there is need for a less invasive treatment option in areas of poor bone quantity and quality. Data related to survival rates of short implants, their design and prosthetic considerations has been compiled and structured in this manuscript with emphasis on the indications, advantages of short implants and critical biomechanical factors to be taken into consideration when choosing to place them. Studies have shown that comparable success rates can be achieved with short implants as those with long implants by decreasing the lateral forces to the prosthesis, eliminating cantilevers, increasing implant surface area and improving implant to abutment connection. Short implants can be considered as an effective treatment alternative in resorbed ridges. Short implants can be considered as a viable treatment option in atrophic ridge cases in order to avoid complex surgical procedures required to place long implants. With improvement in the implant surface geometry and surface texture, there is an increase in the bone implant contact area which provides a good primary stability during osseo-integration.