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Sample records for efficiently retarget lentiviral

  1. 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....

  2. 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

  3. 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

  4. Personalization in social retargeting - A field experiment

    NARCIS (Netherlands)

    Frick, T.W. (Thomas W.); T. Li (Ting)

    2016-01-01

    textabstractThis study compares the effectiveness of product- and category-specific advertising personalization in Social Retargeting. Social Retargeting combines the features of social advertising, targeting consumers based on social connections, and retargeting, using consumers' browsing behavior

  5. 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.

  6. Structured Scan Patterns Retargeting for Dynamic Instruments Access

    NARCIS (Netherlands)

    Ibrahim, Ahmed Mohammed Youssef; Kerkhoff, Hans G.

    2017-01-01

    The IEEE 1687 (iJTAG) standard introduces an efficient access network based on reconfigurable scan, for accessing the increasing number of embedded instruments. Pattern retargeting is defined as the process of translating an instrument pattern to several network-level scan vectors. In this paper, an

  7. A Framework for Retargetable Code Generation using Simulated Annealing

    NARCIS (Netherlands)

    Visser, B.S.

    2000-01-01

    embedded systems. Retargetable code generation is a co-designing method to map a high-level software description onto a variety of hardware architectures without the need to rewrite a compiler. Highly efficient code generation is required to meet, for example, timing, area and low-power constraints.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Saliency detection in the compressed domain for adaptive image retargeting.

    Science.gov (United States)

    Fang, Yuming; Chen, Zhenzhong; Lin, Weisi; Lin, Chia-Wen

    2012-09-01

    Saliency detection plays important roles in many image processing applications, such as regions of interest extraction and image resizing. Existing saliency detection models are built in the uncompressed domain. Since most images over Internet are typically stored in the compressed domain such as joint photographic experts group (JPEG), we propose a novel saliency detection model in the compressed domain in this paper. The intensity, color, and texture features of the image are extracted from discrete cosine transform (DCT) coefficients in the JPEG bit-stream. Saliency value of each DCT block is obtained based on the Hausdorff distance calculation and feature map fusion. Based on the proposed saliency detection model, we further design an adaptive image retargeting algorithm in the compressed domain. The proposed image retargeting algorithm utilizes multioperator operation comprised of the block-based seam carving and the image scaling to resize images. A new definition of texture homogeneity is given to determine the amount of removal block-based seams. Thanks to the directly derived accurate saliency information from the compressed domain, the proposed image retargeting algorithm effectively preserves the visually important regions for images, efficiently removes the less crucial regions, and therefore significantly outperforms the relevant state-of-the-art algorithms, as demonstrated with the in-depth analysis in the extensive experiments.

  15. Retargeting of digital photos and documents

    Science.gov (United States)

    Bucha, V.; Safonov, I.; Rychagov, M.; Hong, J. K.; Kim, S. H.

    2009-01-01

    Present paper generally relates to content-aware image resizing and image inscribing into particular predetermined areas. The problem consists in transformation of the image to a new size with or without modification of aspect ratio in a manner that preserves the recognizability and proportions of the important features of the image. Most close solutions presented in prior art cover along with standard image linear scaling, including down-sampling and up-sampling, image cropping, image retargeting, seam carving and some special image manipulations which similar to some kind of image retouching. Present approach provides a method for digital image retargeting by means of erasing or addition of less significant image pixels. The defined above retargeting approach can be easily used for image shrinking easily. However, for image enlargement there are some limitations as a stretching artifact. History map with relaxation is introduced to avoid such drawback and overcome some known limits of retargeting. In proposed approach means for important objects preservation are taken into account. It allows significant improvement of resulting quality of retargeting. Retargeting applications for different devices such as display, copier, facsimile and photo-printer are described as well.

  16. A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

    Science.gov (United States)

    Leoni, Valerio; Gatta, Valentina; Casiraghi, Costanza; Nicosia, Alfredo; Petrovic, Biljana; Campadelli-Fiume, Gabriella

    2017-05-15

    The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ 1 34.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells. IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity

  17. Retargeting Strategies for Oncolytic Herpes Simplex Viruses.

    Science.gov (United States)

    Campadelli-Fiume, Gabriella; Petrovic, Biljana; Leoni, Valerio; Gianni, Tatiana; Avitabile, Elisa; Casiraghi, Costanza; Gatta, Valentina

    2016-02-26

    Most of the oncolytic herpes simplex viruses (HSVs) exhibit a high safety profile achieved through attenuation. They carry defects in virulence proteins that antagonize host cell response to the virus, including innate response, apoptosis, authophagy, and depend on tumor cell proliferation. They grow robustly in cancer cells, provided that these are deficient in host cell responses, which is often the case. To overcome the attenuation limits, a strategy is to render the virus highly cancer-specific, e.g., by retargeting their tropism to cancer-specific receptors, and detargeting from natural receptors. The target we selected is HER-2, overexpressed in breast, ovarian and other cancers. Entry of wt-HSV requires the essential glycoproteins gD, gH/gL and gB. Here, we reviewed that oncolytic HSV retargeting was achieved through modifications in gD: the addition of a single-chain antibody (scFv) to HER-2 coupled with appropriate deletions to remove part of the natural receptors' binding sites. Recently, we showed that also gH/gL can be a retargeting tool. The insertion of an scFv to HER-2 at the gH N-terminus, coupled with deletions in gD, led to a recombinant capable to use HER-2 as the sole receptor. The retargeted oncolytic HSVs can be administered systemically by means of carrier cells-forcedly-infected mesenchymal stem cells. Altogether, the retargeted oncolytic HSVs are highly cancer-specific and their replication is not dependent on intrinsic defects of the tumor cells. They might be further modified to express immunomodulatory molecules.

  18. Recommendation advertising method based on behavior retargeting

    Science.gov (United States)

    Zhao, Yao; YIN, Xin-Chun; CHEN, Zhi-Min

    2011-10-01

    Online advertising has become an important business in e-commerce. Ad recommended algorithms are the most critical part in recommendation systems. We propose a recommendation advertising method based on behavior retargeting which can avoid leakage click of advertising due to objective reasons and can observe the changes of the user's interest in time. Experiments show that our new method can have a significant effect and can be further to apply to online system.

  19. Dynamic Retargeting : -The Holy Grail of Marketing?

    OpenAIRE

    Johansson, Christoffer; Wengberg, Patrik

    2017-01-01

    To reach consumers with marketing in today's digital climate is in need of highly accurate and relevant ads. Consumer are in constant information bombardment and increasingly tougher competition is making it more complicated to reach your target consumers that wants to see and get what they want whenever they want it. Dynamic retargeting is highly intelligent, utilizing the latest technology for performance marketing, which enables ads that are highly accurate and relevant through personaliza...

  20. Antibody neutralization of retargeted measles viruses

    Science.gov (United States)

    Lech, Patrycja J.; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J.; Nara, Peter L.; Russell, Stephen J.

    2014-01-01

    The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

  1. 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

  2. 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.

  3. Groupwise Retargeted Least-Squares Regression.

    Science.gov (United States)

    Wang, Lingfeng; Pan, Chunhong

    2018-04-01

    In this brief, we propose a new groupwise retargeted least squares regression (GReLSR) model for multicategory classification. The main motivation behind GReLSR is to utilize an additional regularization to restrict the translation values of ReLSR, so that they should be similar within same class. By analyzing the regression targets of ReLSR, we propose a new formulation of ReLSR, where the translation values are expressed explicitly. On the basis of the new formulation, discriminative least-squares regression can be regarded as a special case of ReLSR with zero translation values. Moreover, a groupwise constraint is added to ReLSR to form the new GReLSR model. Extensive experiments on various machine leaning data sets illustrate that our method outperforms the current state-of-the-art approaches.

  4. Tunable CD44-specific cellular retargeting with hyaluronic acid nanoshells

    DEFF Research Database (Denmark)

    Ebbesen, Morten F.; Olesen, Morten T. J.; Gjelstrup, Mikkel C.

    2014-01-01

    of different Mw was covalently grafted onto model polystyrene nanoparticles and advanced surface analysis by X-ray photoelectron spectroscopy performed to quantify and evaluate the effect of the coating procedure. Specific CD44-mediated retargeting was investigated by flow cytometry and confocal microscopy...... that indicates an extended and nonconstricted HA polymer surface. Reduced non-specific particle interaction in CD44- cells was shown for all HA nanoshells but CD44-dependent cellular retargeting and internalization in CD44+ cells was highly dependent on the coating HA Mw properties. Conclusion The combination...... of advanced surface characterization and evaluation of particle interactions in isogenic cells with and without CD44 receptor demonstrates direct evidence for the dual capacity of HA for stealth and CD44-mediated retargeting tunable by the HA molecular weight....

  5. 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.

  6. PIL : A platform independent language for retargetable DSLs

    NARCIS (Netherlands)

    Hemel, Z.; Visser, E.

    2009-01-01

    This paper is a pre-print of: Zef Hemel, Eelco Visser. PIL: A Platform Independent Language for Retargetable DSLs. In Mark G. J. van den Brand, Jeff Gray, editors, Software Language Engineering, Second International Conference, SLE 2009, Denver, USA, October, 2009. Lecture Notes in Computer Science,

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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.

  9. 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.

  10. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    Directory of Open Access Journals (Sweden)

    Merry ZC Ruan

    2016-01-01

    Full Text Available Osteoarthritis (OA is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs. Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab. We show that a10mab-conjugated HDV (a10mabHDV-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4 into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

  11. 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.

  12. 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.

  13. 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.

  14. Interactive visibility retargeting in VR using conformal visualization.

    Science.gov (United States)

    Petkov, Kaloian; Papadopoulos, Charilaos; Zhang, Min; Kaufman, Arie E; Gu, Xianfeng David

    2012-07-01

    In Virtual Reality, immersive systems such as the CAVE provide an important tool for the collaborative exploration of large 3D data. Unlike head-mounted displays, these systems are often only partially immersive due to space, access, or cost constraints. The resulting loss of visual information becomes a major obstacle for critical tasks that need to utilize the users' entire field of vision. We have developed a conformal visualization technique that establishes a conformal mapping between the full 360° field of view and the display geometry of a given visualization system. The mapping is provably angle-preserving and has the desirable property of preserving shapes locally, which is important for identifying shape-based features in the visual data. We apply the conformal visualization to both forward and backward rendering pipelines in a variety of retargeting scenarios, including CAVEs and angled arrangements of flat panel displays. In contrast to image-based retargeting approaches, our technique constructs accurate stereoscopic images that are free of resampling artifacts. Our user study shows that on the visual polyp detection task in Immersive Virtual Colonoscopy, conformal visualization leads to improved sensitivity at comparable examination times against the traditional rendering approach. We also develop a novel user interface based on the interactive recreation of the conformal mapping and the real-time regeneration of the view direction correspondence.

  15. Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8.

    Directory of Open Access Journals (Sweden)

    Irene Michalk

    Full Text Available There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs. Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.

  16. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    Science.gov (United States)

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

  17. Expediting analog design retargeting by design knowledge re-use and circuit synthesis: a practical example on a Delta-Sigma modulator

    Science.gov (United States)

    Webb, Matthew; Tang, Hua

    2016-08-01

    In the past decade or two, due to constant and rapid technology changes, analog design re-use or design retargeting to newer technologies has been brought to the table in order to expedite the design process and improve time-to-market. If properly conducted, analog design retargeting could significantly cut down design cycle compared to designs starting from the scratch. In this article, we present an empirical and general method for efficient analog design retargeting by design knowledge re-use and circuit synthesis (CS). The method first identifies circuit blocks that compose the source system and extracts the performance parameter specifications of each circuit block. Then, for each circuit block, it scales the values of design variables (DV) from the source design to derive an initial design in the target technology. Depending on the performance of this initial target design, a design space is defined for synthesis. Subsequently, each circuit block is automatically synthesised using state-of-art analog synthesis tools based on a combination of global and local optimisation techniques to achieve comparable performance specifications to those extracted from the source system. Finally, the overall system is composed of those synthesised circuit blocks in the target technology. We illustrate the method using a practical example of a complex Delta-Sigma modulator (DSM) circuit.

  18. Autonomous Collaborative Agents for Onboard Multi-Sensor Re-Targeting, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — In our Phase I effort we developed a prototype software-agent based framework to provide for autonomous re-targeting of sensors hosted on satellites in polar orbits,...

  19. Autonomous Collaborative Agents for Onboard Multi-Sensor Re-Targeting Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Technologies that support the configuration of sensors and satellites, and allow the reconfiguration or retargeting of sensors in response to user demand or...

  20. Autonomous Collaborative Agents for Onboard Multi-Sensor Re-Targeting Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In our Phase I effort we developed a prototype software-agent based framework to provide for autonomous re-targeting of sensors hosted on satellites in polar orbits,...

  1. 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.

  2. Improved content aware scene retargeting for retinitis pigmentosa patients

    Directory of Open Access Journals (Sweden)

    Al-Atabany Walid I

    2010-09-01

    Full Text Available Abstract Background In this paper we present a novel scene retargeting technique to reduce the visual scene while maintaining the size of the key features. The algorithm is scalable to implementation onto portable devices, and thus, has potential for augmented reality systems to provide visual support for those with tunnel vision. We therefore test the efficacy of our algorithm on shrinking the visual scene into the remaining field of view for those patients. Methods Simple spatial compression of visual scenes makes objects appear further away. We have therefore developed an algorithm which removes low importance information, maintaining the size of the significant features. Previous approaches in this field have included seam carving, which removes low importance seams from the scene, and shrinkability which dynamically shrinks the scene according to a generated importance map. The former method causes significant artifacts and the latter is inefficient. In this work we have developed a new algorithm, combining the best aspects of both these two previous methods. In particular, our approach is to generate a shrinkability importance map using as seam based approach. We then use it to dynamically shrink the scene in similar fashion to the shrinkability method. Importantly, we have implemented it so that it can be used in real time without prior knowledge of future frames. Results We have evaluated and compared our algorithm to the seam carving and image shrinkability approaches from a content preservation perspective and a compression quality perspective. Also our technique has been evaluated and tested on a trial included 20 participants with simulated tunnel vision. Results show the robustness of our method at reducing scenes up to 50% with minimal distortion. We also demonstrate efficacy in its use for those with simulated tunnel vision of 22 degrees of field of view or less. Conclusions Our approach allows us to perform content aware video

  3. 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.

  4. 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.

  5. Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells

    Czech Academy of Sciences Publication Activity Database

    Stevenson, M.; Boos, E.; Herbert, C. W.; Hale, A. B.; Green, N.; Lyons, M.; Chandler, L.; Ulbrich, Karel; van Rooijen, N.; Mautner, V.; Fisher, K.; Seymour, L.

    2006-01-01

    Roč. 13, č. 4 (2006), s. 356-368 ISSN 0969-7128 EU Projects: European Commission(XE) 512087 - GIANT Institutional research plan: CEZ:AV0Z40500505 Keywords : CELO virus * N-(2-hydroxypropyl)methacrylamide * retargeting Subject RIV: CE - Biochemistry Impact factor: 4.782, year: 2006

  6. 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.

  7. Retargeting T cells for HER2-positive tumor killing by a bispecific Fv-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Lei Wang

    Full Text Available To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding, a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2 developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

  8. Design Considerations for Miniaturized Control Moment Gyroscopes for Rapid Retargeting and Precision Pointing of Small Satellites

    Science.gov (United States)

    Patankar, Kunal; Fitz-Coy, Norman; Roithmayr, Carlos M.

    2014-01-01

    This paper presents the design as well as characterization of a practical control moment gyroscope (CMG) based attitude control system (ACS) for small satellites in the 15-20 kilogram mass range performing rapid retargeting and precision pointing maneuvers. The paper focuses on the approach taken in the design of miniaturized CMGs while considering the constraints imposed by the use of commercial off-the-shelf (COTS) components as well as the size of the satellite. It is shown that a hybrid mode is more suitable for COTS based moment exchange actuators; a mode that uses the torque amplification of CMGs for rapid retargeting and direct torque capabilities of the flywheel motors for precision pointing. A simulation is provided to demonstrate on-orbit slew and pointing performance.

  9. Online tracking and retargeting with applications to optical biopsy in gastrointestinal endoscopic examinations.

    Science.gov (United States)

    Ye, Menglong; Giannarou, Stamatia; Meining, Alexander; Yang, Guang-Zhong

    2016-05-01

    With recent advances in biophotonics, techniques such as narrow band imaging, confocal laser endomicroscopy, fluorescence spectroscopy, and optical coherence tomography, can be combined with normal white-light endoscopes to provide in vivo microscopic tissue characterisation, potentially avoiding the need for offline histological analysis. Despite the advantages of these techniques to provide online optical biopsy in situ, it is challenging for gastroenterologists to retarget the optical biopsy sites during endoscopic examinations. This is because optical biopsy does not leave any mark on the tissue. Furthermore, typical endoscopic cameras only have a limited field-of-view and the biopsy sites often enter or exit the camera view as the endoscope moves. In this paper, a framework for online tracking and retargeting is proposed based on the concept of tracking-by-detection. An online detection cascade is proposed where a random binary descriptor using Haar-like features is included as a random forest classifier. For robust retargeting, we have also proposed a RANSAC-based location verification component that incorporates shape context. The proposed detection cascade can be readily integrated with other temporal trackers. Detailed performance evaluation on in vivo gastrointestinal video sequences demonstrates the performance advantage of the proposed method over the current state-of-the-art. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. [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.

  11. 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 ...

  12. 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.

  13. 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

  14. 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.

  15. 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.

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

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    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.

  17. [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.

  18. 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

  19. 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

  20. Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid.

    Science.gov (United States)

    Allaume, Xavier; El-Andaloussi, Nazim; Leuchs, Barbara; Bonifati, Serena; Kulkarni, Amit; Marttila, Tiina; Kaufmann, Johanna K; Nettelbeck, Dirk M; Kleinschmidt, Jürgen; Rommelaere, Jean; Marchini, Antonio

    2012-04-01

    The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)β(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

  1. 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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

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    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.

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

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    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.

  7. Retargeting of existing FORTRAN program and development of parallel compilers

    Science.gov (United States)

    Agrawal, Dharma P.

    1988-01-01

    The software models used in implementing the parallelizing compiler for the B-HIVE multiprocessor system are described. The various models and strategies used in the compiler development are: flexible granularity model, which allows a compromise between two extreme granularity models; communication model, which is capable of precisely describing the interprocessor communication timings and patterns; loop type detection strategy, which identifies different types of loops; critical path with coloring scheme, which is a versatile scheduling strategy for any multicomputer with some associated communication costs; and loop allocation strategy, which realizes optimum overlapped operations between computation and communication of the system. Using these models, several sample routines of the AIR3D package are examined and tested. It may be noted that automatically generated codes are highly parallelized to provide the maximized degree of parallelism, obtaining the speedup up to a 28 to 32-processor system. A comparison of parallel codes for both the existing and proposed communication model, is performed and the corresponding expected speedup factors are obtained. The experimentation shows that the B-HIVE compiler produces more efficient codes than existing techniques. Work is progressing well in completing the final phase of the compiler. Numerous enhancements are needed to improve the capabilities of the parallelizing compiler.

  8. 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.

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

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    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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies

    Czech Academy of Sciences Publication Activity Database

    Fisher, K. D.; Stallwood, Y.; Green, N. K.; Ulbrich, Karel; Mautner, V.; Seymour, L. W.

    2001-01-01

    Roč. 8, - (2001), s. 341-348 ISSN 0969-7128 R&D Projects: GA AV ČR KSK4055109 Institutional research plan: CEZ:AV0Z4050913 Keywords : adenovirus * gene therapy * targetting Subject RIV: CC - Organic Chemistry Impact factor: 5.893, year: 2001

  16. 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.

  17. In vivo antimetastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer

    Science.gov (United States)

    Jing, Yuqi; Bejarano, Marcela Toro; Zaias, Julia; Merchan, Jaime R.

    2014-01-01

    Purpose The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases, and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. Methods In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles and immunostaining of MV particles in lungs from tumor bearing mice. Results In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment related toxicity was observed in treated mice. Conclusions The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer. PMID:25519042

  18. 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.

  19. 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.

  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. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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 ...

  6. 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.

  7. Dual Ligand Insertion in gB and gD of Oncolytic Herpes Simplex Viruses for Retargeting to a Producer Vero Cell Line and to Cancer Cells.

    Science.gov (United States)

    Petrovic, Biljana; Leoni, Valerio; Gatta, Valentina; Zaghini, Anna; Vannini, Andrea; Campadelli-Fiume, Gabriella

    2018-03-15

    Oncolytic viruses gain cancer specificity in several ways. Like the majority of viruses, they grow better in cancer cells that are defective in mounting the host response to viruses. Often, they are attenuated by deletion or mutation of virulence genes that counteract the host response or are naturally occurring oncolytic mutants. In contrast, retargeted viruses are not attenuated or deleted; their cancer specificity rests on a modified, specific tropism for cancer receptors. For herpes simplex virus (HSV)-based oncolytics, the detargeting-retargeting strategies employed so far were based on genetic modifications of gD. Recently, we showed that even gH or gB can serve as retargeting tools. To enable the growth of retargeted HSVs in cells that can be used for clinical-grade virus production, a double-retargeting strategy has been developed. Here we show that several sites in the N terminus of gB are suitable to harbor the 20-amino-acid (aa)-long GCN4 peptide, which readdresses HSV tropism to Vero cells expressing the artificial GCN4 receptor and thus enables virus cultivation in the producer noncancer Vero-GCN4R cell line. The gB modifications can be combined with a minimal detargeting modification in gD, consisting in the deletion of two residues, aa 30 and 38, and replacement of aa 38 with the scFv to human epidermal growth factor receptor 2 (HER2), for retargeting to the cancer receptor. The panel of recombinants was analyzed comparatively in terms of virus growth, cell-to-cell spread, cytotoxicity, and in vivo antitumor efficacy to define the best double-retargeting strategy. IMPORTANCE There is increasing interest in oncolytic viruses, following FDA and the European Medicines Agency (EMA) approval of HSV Oncovex GM-CSF , and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly checkpoint inhibitors. A strategy to gain cancer specificity and avoid virus attenuation is to retarget the

  8. 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

    we use molecular techniques to evaluate transmission, fitness, and adaptation of puma lentivirus A (PLVA) between bobcats and mountain lions in two geographic regions. Our findings illustrate that mountain lion exposure to PLVA is relatively common, but does not routinely result in infections communicable in the new host. This is attributed to efficient species barriers that largely prevent lentiviral adaptation. However, the evolutionary capacity for lentiviruses to adapt to novel environments may ultimately overcome host restriction mechanisms over time and under certain ecological circumstances. This phenomenon provides a unique opportunity to examine cross-species transmission events leading to new lentiviral emergence.

  9. 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

    outcome will occur are complex, and the risk of new virus emergence is therefore difficult to predict. We used molecular techniques to evaluate the transmission, fitness, and adaptation of puma lentivirus A (PLVA) between bobcats and mountain lions in two geographic regions. Our findings illustrate that mountain lion exposure to PLVA is relatively common but does not routinely result in communicable infections in the new host. This is attributed to efficient species barriers that largely prevent lentiviral adaptation. However, the evolutionary capacity for lentiviruses to adapt to novel environments may ultimately overcome host restriction mechanisms over time and under certain ecological circumstances. This phenomenon provides a unique opportunity to examine cross-species transmission events leading to new lentiviral emergence. Copyright © 2017 American Society for Microbiology.

  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. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Low-Energy Ultrasound Treatment Improves Regional Tumor Vessel Infarction by Retargeted Tissue Factor.

    Science.gov (United States)

    Brand, Caroline; Dencks, Stefanie; Schmitz, Georg; Mühlmeister, Mareike; Stypmann, Jörg; Ross, Rebecca; Hintelmann, Heike; Schliemann, Christoph; Müller-Tidow, Carsten; Mesters, Rolf M; Berdel, Wolfgang E; Schwöppe, Christian

    2015-07-01

    To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment. For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days). Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity. Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy. © 2015 by the American Institute of Ultrasound in Medicine.

  19. 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

  20. 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.

  1. 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...

  2. 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 ...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. In vivo anti-metastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer.

    Science.gov (United States)

    Jing, Yuqi; Bejarano, Marcela Toro; Zaias, Julia; Merchan, Jaime R

    2015-01-01

    The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles, and immunostaining of MV particles in lungs from tumor bearing mice. In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated, and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA, and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment-related toxicity was observed in treated mice. The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer.

  9. 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.

  10. An optimized content-aware image retargeting method: toward expanding the perceived visual field of the high-density retinal prosthesis recipients

    Science.gov (United States)

    Li, Heng; Zeng, Yajie; Lu, Zhuofan; Cao, Xiaofei; Su, Xiaofan; Sui, Xiaohong; Wang, Jing; Chai, Xinyu

    2018-04-01

    Objective. Retinal prosthesis devices have shown great value in restoring some sight for individuals with profoundly impaired vision, but the visual acuity and visual field provided by prostheses greatly limit recipients’ visual experience. In this paper, we employ computer vision approaches to seek to expand the perceptible visual field in patients implanted potentially with a high-density retinal prosthesis while maintaining visual acuity as much as possible. Approach. We propose an optimized content-aware image retargeting method, by introducing salient object detection based on color and intensity-difference contrast, aiming to remap important information of a scene into a small visual field and preserve their original scale as much as possible. It may improve prosthetic recipients’ perceived visual field and aid in performing some visual tasks (e.g. object detection and object recognition). To verify our method, psychophysical experiments, detecting object number and recognizing objects, are conducted under simulated prosthetic vision. As control, we use three other image retargeting techniques, including Cropping, Scaling, and seam-assisted shrinkability. Main results. Results show that our method outperforms in preserving more key features and has significantly higher recognition accuracy in comparison with other three image retargeting methods under the condition of small visual field and low-resolution. Significance. The proposed method is beneficial to expand the perceived visual field of prosthesis recipients and improve their object detection and recognition performance. It suggests that our method may provide an effective option for image processing module in future high-density retinal implants.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. 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.

  16. 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.

  17. Nucleus-to-nucleus gene transfer and protein retargeting into a remnant cytoplasm of cryptophytes and diatoms.

    Science.gov (United States)

    Gould, Sven B; Sommer, Maik S; Kroth, Peter G; Gile, Gillian H; Keeling, Patrick J; Maier, Uwe-G

    2006-12-01

    The complex plastid of the cryptophyte Guillardia theta and of the diatom Phaeodactylum tricornutum can both be traced back to an engulfed eukaryotic red alga. The eukaryotic origin of these plastids is most obvious in cryptophytes, where the organelle still possesses a remnant nucleus, the nucleomorph. The nucleomorph itself is embedded in the periplastid compartment (PPC), the remnant of the former red algal cytosol. In the cryptophyte and diatom, the complex plastid is surrounded by 4 membranes, the outer one being continuous with the host rough endoplasmatic reticulum. In a recent report, we have shown that a nuclear encoded PPC protein of G. theta expressed in P. tricornutum leads to a localization, recently described as being a "bloblike structure," which can be obtained by mutation of plastid protein-targeting sequences of the diatom itself. Here we present further nucleus-encoded PPC proteins from G. theta, such as the eukaryotic translation elongation factor-1alpha, evidence for their nucleus-to-nucleus gene transfer, and retargeting of the proteins. We also investigated the first nuclear encoded PPC-targeted protein of P. tricornutum (Hsp70) and analyzed it for in vivo localization together with the identified G. theta PPC proteins. This revealed that all localize to the bloblike structures, which we suggest is the highly reduced PPC of P. tricornutum. Furthermore, the described cryptophyte PPC proteins possibly allow the elucidation of the processes by which proteins are involved in different levels of host control over its eukaryotic organelle.

  18. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts

    International Nuclear Information System (INIS)

    Cartellieri, M; Feldmann, A; Koristka, S; Arndt, C; Loff, S; Ehninger, A; Bonin, M von; Bejestani, E P; Ehninger, G; Bachmann, M P

    2016-01-01

    The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide ‘proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo

  19. 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.

  20. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. 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

  3. Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef.

    Science.gov (United States)

    Chase, Amanda J; Wombacher, Rebecka; Fackler, Oliver T

    2018-03-27

    The HIV-1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network (TGN), while the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18 amino acid SH4 domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for TGN retargeting by Nef, as well as the importance of the amino acid at position 8 for determining Nef-sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone (LPC) Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that (i) analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and (ii) intrinsic properties define SH4 domains as cargo of this Nef-sensitive LPC-GTPase transport cycle. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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...

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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.

  18. 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...

  19. 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...

  20. 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...

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Lentiviral modification of enriched populations of bovine male gonocytes.

    Science.gov (United States)

    Kim, K-J; Cho, C M; Kim, B-G; Lee, Y-A; Kim, B-J; Kim, Y-H; Kim, C G; Schmidt, J A; Ryu, B-Y

    2014-01-01

    Undifferentiated germ cells have the capacity to develop into sperm capable of fertilizing oocytes and contributing genetic material to subsequent generations. The most primitive prepubertal undifferentiated germ cells include gonocytes and undifferentiated spermatogonia, including spermatogonial stem cells (SSC). Gonocytes, present in the testis at birth, differentiate into SSC, which maintain spermatogenesis for the remainder of the male's life. Because of their capacity to contribute to lifelong spermatogenesis, undifferentiated germ cells are attractive targets for genetic modification to produce transgenic animals, including cattle. To maximize the efficiency of genetic modification of bovine gonocytes and SSC, effective enrichment techniques need to be developed. Selection of bovine gonocytes using differential plating was improved 8-fold (P green fluorescent protein. Transduction efficiency was 17%. Collectively, these data demonstrate effective methods for the selection and genetic modification of bovine undifferentiated germ cells.

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

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    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.

  10. [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.

  11. 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.

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

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    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.

  13. 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.

  14. 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.

  15. 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.

  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. 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.

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

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    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.

  20. 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

  1. 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...

  2. 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.

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

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    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.

  4. 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.

  5. Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

    Science.gov (United States)

    Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott

    2015-01-01

    HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983

  6. 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.

  7. 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.

  8. 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

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  11. 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.

  12. 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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  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. 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.

  5. 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Recommendations for a Retargetable Compiler.

    Science.gov (United States)

    1980-03-01

    than that, whereas some need much more data concerning the target machine. Many such operations are available in the literature . Some of the bet- ter...global analysis in the literature , some quite recent. The one that will be used in this discussion is the one developed by Geschke Ges72I in his thesis...modularization. goth of these definitions are available throuqhout the descriotlon in which they appear, A DO construct is provided to envoke orede- fined

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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.

  14. 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...

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

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    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.

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

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    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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Lentiviral gene transfer into the dorsal root ganglion of adult rats

    Directory of Open Access Journals (Sweden)

    Park Frank

    2011-08-01

    Full Text Available Abstract Background Lentivector-mediated gene delivery into the dorsal root ganglion (DRG is a promising method for exploring pain pathophysiology and for genetic treatment of chronic neuropathic pain. In this study, a series of modified lentivector particles with different cellular promoters, envelope glycoproteins, and viral accessory proteins were generated to evaluate the requirements for efficient transduction into neuronal cells in vitro and adult rat DRG in vivo. Results In vitro, lentivectors expressing enhanced green fluorescent protein (EGFP under control of the human elongation factor 1α (EF1α promoter and pseudotyped with the conventional vesicular stomatitis virus G protein (VSV-G envelope exhibited the best performance in the transfer of EGFP into an immortalized DRG sensory neuron cell line at low multiplicities of infection (MOIs, and into primary cultured DRG neurons at higher MOIs. In vivo, injection of either first or second-generation EF1α-EGFP lentivectors directly into adult rat DRGs led to transduction rates of 19 ± 9% and 20 ± 8% EGFP-positive DRG neurons, respectively, detected at 4 weeks post injection. Transduced cells included a full range of neuronal phenotypes, including myelinated neurons as well as both non-peptidergic and peptidergic nociceptive unmyelinated neurons. Conclusion VSV-G pseudotyped lentivectors containing the human elongation factor 1α (EF1α-EGFP expression cassette demonstrated relatively efficient transduction to sensory neurons following direct injection into the DRG. These results clearly show the potential of lentivectors as a viable system for delivering target genes into DRGs to explore basic mechanisms of neuropathic pain, with the potential for future clinical use in treating chronic pain.

  4. 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.

  5. 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.

  6. 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...

  7. Retargeting Clostridium difficile Toxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism

    Directory of Open Access Journals (Sweden)

    Greice Krautz-Peterson

    2012-01-01

    Full Text Available Botulinum neurotoxins (BoNTs deliver a protease to neurons which can cause a flaccid paralysis called botulism. Development of botulism antidotes will require neuronal delivery of agents that inhibit or destroy the BoNT protease. Here, we investigated the potential of engineering Clostridium difficile toxin B (TcdB as a neuronal delivery vehicle by testing two recombinant TcdB chimeras. For AGT-TcdB chimera, an alkyltransferase (AGT was appended to the N-terminal glucosyltransferase (GT of TcdB. Recombinant AGT-TcdB had alkyltransferase activity, and the chimera was nearly as toxic to Vero cells as wild-type TcdB, suggesting efficient cytosolic delivery of the AGT/GT fusion. For AGT-TcdB-BoNT/A-Hc, the receptor-binding domain (RBD of TcdB was replaced by the equivalent RBD from BoNT/A (BoNT/A-Hc. AGT-TcdB-BoNT/A-Hc was >25-fold more toxic to neuronal cells and >25-fold less toxic to Vero cells than AGT-TcdB. Thus, TcdB can be engineered for cytosolic delivery of biomolecules and improved targeting of neuronal cells.

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

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    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.

  9. 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.

  10. 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.

  11. 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.

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

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    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

  13. 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.

  14. 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

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    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.

  15. Highly efficient generation of transgenic sheep by lentivirus accompanying the alteration of methylation status.

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    Chenxi Liu

    Full Text Available BACKGROUND: Low efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed. METHODOLOGY/PRINCIPLE FINDINGS: EGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep. CONCLUSIONS/SIGNIFICANCE: Injection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by

  16. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

    NARCIS (Netherlands)

    Ahmed, Bushra Y; Chakravarthy, Sridhara; Eggers, Ruben; Hermens, Wim T J M C; Zhang, Jing Ying; Niclou, Simone P; Levelt, Christiaan; Sablitzky, Fred; Anderson, Patrick N; Lieberman, A Robert; Verhaagen, J.

    2004-01-01

    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

  17. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

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    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

  18. A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

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    Mathieu Desclaux

    Full Text Available BACKGROUND: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi. METHODS AND FINDINGS: In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. CONCLUSIONS: Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for

  19. 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

  20. 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.

  1. Immortalization and Characterization of Porcine Macrophages That Had Been Transduced with Lentiviral Vectors Encoding the SV40 Large T Antigen and Porcine Telomerase Reverse Transcriptase

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    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.

  2. 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.

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    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.

  3. 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.

  4. 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

  5. Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

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    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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN vectors and multi-colour analyses

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    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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

  13. 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

  14. 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.

  15. Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains.

    Science.gov (United States)

    Wu, Shi-Hao; Liao, Zhi-Xing; D Rizak, Joshua; Zheng, Na; Zhang, Lin-Heng; Tang, Hen; He, Xiao-Bin; Wu, Yang; He, Xia-Ping; Yang, Mei-Feng; Li, Zheng-Hui; Qin, Dong-Dong; Hu, Xin-Tian

    2017-03-18

    Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non-human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca 2+ /calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs). These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.

  16. Energy efficiency

    International Nuclear Information System (INIS)

    2010-01-01

    After a speech of the CEA's (Commissariat a l'Energie Atomique) general administrator about energy efficiency as a first rank challenge for the planet and for France, this publications proposes several contributions: a discussion of the efficiency of nuclear energy, an economic analysis of R and D's value in the field of fourth generation fast reactors, discussions about biofuels and the relationship between energy efficiency and economic competitiveness, and a discussion about solar photovoltaic efficiency

  17. Development of all-in-one multicistronic Tet-On lentiviral vectors for inducible co-expression of two transgenes.

    Science.gov (United States)

    Huang, Yide; Zhen, Ruonan; Jiang, Meiqin; Yang, Jie; Yang, Yun; Huang, Zhen; Lin, Yao

    2015-01-01

    Inducible co-expression of multiple genes is often needed in research. Here we describe a single-vector-based Tet-On inducible system for co-expression of two transgenes. The two transgenes (DsRed1 and eGFP as model genes) and reverse tetracycline-controlled transactivator were separated by internal ribosomal entry sites and 2A sequences, and their transcription was controlled by the same tetracycline responsive element. Two novel vectors with different internal ribosomal entry sites and 2A positions on the vectors were constructed. The DsRed1 and eGFP in cells transduced with both vectors are undetectable in the absence of doxycycline and can be efficiently induced in the presence of doxycycline in vitro and in vivo. These two vectors can be useful tools when regulated co-expression of two ecotopic genes is needed. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. Batch efficiency

    International Nuclear Information System (INIS)

    Schwickerath, Ulrich; Silva, Ricardo; Uria, Christian

    2010-01-01

    A frequent source of concern for resource providers is the efficient use of computing resources in their centers. This has a direct impact on requests for new resources. There are two different but strongly correlated aspects to be considered: while users are mostly interested in a good turn-around time for their jobs, resource providers are mostly interested in a high and efficient usage of their available resources. Both things, the box usage and the efficiency of individual user jobs, need to be closely monitored so that the sources of the inefficiencies can be identified. At CERN, the Lemon monitoring system is used for both purposes. Examples of such sources are poorly written user code, inefficient access to mass storage systems, and dedication of resources to specific user groups. As a first step for improvements CERN has launched a project to develop a scheduler add-on that allows careful overloading of worker nodes that run idle jobs.

  4. Efficient ICT for efficient smart grids

    NARCIS (Netherlands)

    Smit, Gerardus Johannes Maria

    2012-01-01

    In this extended abstract the need for efficient and reliable ICT is discussed. Efficiency of ICT not only deals with energy-efficient ICT hardware, but also deals with efficient algorithms, efficient design methods, efficient networking infrastructures, etc. Efficient and reliable ICT is a

  5. Offsetting efficiency

    International Nuclear Information System (INIS)

    Katz, M.

    1995-01-01

    Whichever way the local distribution company (LDC) tries to convert residential customers to gas or expand their use of it, the process itself has become essential for the natural gas industry. The amount of gas used by each residential customer has been decreasing for 25 years -- since the energy crisis of the early 1970s. It's a direct result of better-insulated homes and more-efficient gas appliances, and that trend is continuing. So, LDCs have a choice of either finding new users and uses for gas, or recognizing that their throughput per customer is going to continue declining. The paper discusses strategies that several gas utilities are using to increase the number of gas appliances in the customer's homes. These and other strategies keep the gas industry optimistic about the future of the residential market: A.G.A. has projected that by 2010 demand will expand, from 1994's 5.1 quadrillion Btu (quads) to 5.7 quads, even with continued improvements in appliance efficiency. That estimate, however, will depend on the industry-s utilities and whether they keep converting, proselytizing, persuading and influencing customers to use more natural gas

  6. Degradation of cellular mir-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo.

    Directory of Open Access Journals (Sweden)

    Lisa Marcinowski

    2012-02-01

    Full Text Available Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.

  7. Efficient STFT

    International Nuclear Information System (INIS)

    Aamir, K.M.; Maud, M.A.

    2004-01-01

    Small perturbations in signals (or any time series), at some particular instant, affect the whole frequency spectrum due to the global function e/sup j omega t/ in Fourier Transform formulation. However, the Fourier spectrum does not convey the time instant at which the perturbation occurred. Consequently the information on the particular time instance of occurrence of that perturbation is lost when spectrum is observed. Apparently Fourier analysis seems to be inadequate in such situations. This inadequacy is overcome by the use of Short Time Fourier Transform (STFT), which keeps track of time as well as frequency information. In STFT analysis, a fixed length window, say of length N, is moved sample by sample as the data arrives. The Discrete Fourier Transform (DFT) of this fixed window of length N is calculated using Fast Fourier Transform (FFT) algorithm. If the total number of points is M > N, the computational complexity of this scheme works out to be at least ((M-N) N log/sub 2/N). On the other hand, STFT is shown to be of computational complexity 6NM and 8NM in the literature. In this paper, two algorithms are presented which compute the same STFT more efficiently. The computational complexity of the proposed algorithms works out to be MN of one algorithm and even lesser in the other algorithm. This reduction in complexity becomes significant for large data sets. This algorithm also remains valid if a stationary part of signal is skipped. (author)

  8. Generation of transgene-free lung disease-specific human iPS cells using a single excisable lentiviral stem cell cassette

    OpenAIRE

    Somers, A.; Jean, J.C.; Sommer, C.A.; Omari, A.; Ford, C.C.; Mills, J.A.; Ying, L.; Sommer Gianotti, A.; Jean, J.M.; Smith, B.W.; Lafyatis, R.; Demierre, M.F.; Weiss, D.J.; French, D.L.; Gadue, P.

    2010-01-01

    The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step towards the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. While several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed...

  9. High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing

    Directory of Open Access Journals (Sweden)

    Duran Sürün

    2018-03-01

    Full Text Available The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB, whose inactivation causes chronic granulomatous disease (XCGD—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS. We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs. Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

  10. Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells.

    Science.gov (United States)

    von Levetzow, Gregor; Spanholtz, Jan; Beckmann, Julia; Fischer, Johannes; Kögler, Gesine; Wernet, Peter; Punzel, Michael; Giebel, Bernd

    2006-04-01

    The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

  13. 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.

  14. An Inducible Lentiviral Guide RNA Platform Enables the Identification of Tumor-Essential Genes and Tumor-Promoting Mutations In Vivo

    Directory of Open Access Journals (Sweden)

    Brandon J. Aubrey

    2015-03-01

    Full Text Available The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.

  15. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection.

    Directory of Open Access Journals (Sweden)

    Smita Srivastava

    2008-05-01

    Full Text Available Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4-based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

  16. Technical efficiency, efficiency change, technical progress and ...

    African Journals Online (AJOL)

    Technical efficiency, efficiency change, technical progress and productivity growth in the national health systems of continental African countries. ... a view to analysing changes in efficiency and changes in technology. The analysis was based on a five-year panel data (1999-2003) from all the 53 countries of continental ...

  17. Highly efficient inhibition of human immunodeficiency virus type 1 reverse transcriptase by aptamers functionalized gold nanoparticles

    Science.gov (United States)

    Shiang, Yen-Chun; Ou, Chung-Mao; Chen, Shih-Ju; Ou, Ting-Yu; Lin, Han-Jia; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-03-01

    We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45-Au NPs shows inhibitory efficiency in the retroviral replication cycle with a decreasing infectivity (40.2%).We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45

  18. RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope.

    Science.gov (United States)

    Marin, Virna; Stornaiuolo, Anna; Piovan, Claudia; Corna, Stefano; Bossi, Sergio; Pema, Monika; Giuliani, Erica; Scavullo, Cinzia; Zucchelli, Eleonora; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2016-01-01

    To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

  19. “Marker of Self” CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors

    Directory of Open Access Journals (Sweden)

    Nisha G Sosale

    2016-01-01

    Full Text Available Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress “Marker of Self” CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show “hCD47-Lenti” display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rg−/− (NSG mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known “Self” signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors and also in targeting various SIRPA-expressing tumors such as glioblastomas.

  20. Reconsidering energy efficiency

    International Nuclear Information System (INIS)

    Goldoni, Giovanni

    2007-01-01

    Energy and environmental policies are reconsidering energy efficiency. In a perfect market, rational and well informed consumers reach economic efficiency which, at the given prices of energy and capital, corresponds to physical efficiency. In the real world, market failures and cognitive frictions distort the consumers from perfectly rational and informed choices. Green incentive schemes aim at balancing market failures and directing consumers toward more efficient goods and services. The problem is to fine tune the incentive schemes [it

  1. Energy efficiency; Efficacite energetique

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-06-15

    This road-map proposes by the Group Total aims to inform the public on the energy efficiency. It presents the energy efficiency and intensity around the world with a particular focus on Europe, the energy efficiency in industry and Total commitment. (A.L.B.)

  2. Efficiency of Brownian Motors

    OpenAIRE

    Parrondo, J. M. R.; Blanco, J. M.; Cao, F. J.; Brito, R.

    1998-01-01

    The efficiency of different types of Brownian motors is calculated analytically and numerically. We find that motors based on flashing ratchets present a,low efficiency and an unavoidable entropy production. On the other hand, a certain class of motors based on adiabatically changing potentials, named reversible ratchets, exhibit a higher efficiency and the entropy production can be arbitrarily reduced.

  3. Revisiting energy efficiency fundamentals

    Energy Technology Data Exchange (ETDEWEB)

    Perez-Lombard, L.; Velazquez, D. [Grupo de Termotecnia, Escuela Superior de Ingenieros, Universidad de Sevilla, Camino de los Descubrimientos s/n, 41092 Seville (Spain); Ortiz, J. [Building Research Establishment (BRE), Garston, Watford, WD25 9XX (United Kingdom)

    2013-05-15

    Energy efficiency is a central target for energy policy and a keystone to mitigate climate change and to achieve a sustainable development. Although great efforts have been carried out during the last four decades to investigate the issue, focusing into measuring energy efficiency, understanding its trends and impacts on energy consumption and to design effective energy efficiency policies, many energy efficiency-related concepts, some methodological problems for the construction of energy efficiency indicators (EEI) and even some of the energy efficiency potential gains are often ignored or misunderstood, causing no little confusion and controversy not only for laymen but even for specialists. This paper aims to revisit, analyse and discuss some efficiency fundamental topics that could improve understanding and critical judgement of efficiency stakeholders and that could help in avoiding unfounded judgements and misleading statements. Firstly, we address the problem of measuring energy efficiency both in qualitative and quantitative terms. Secondly, main methodological problems standing in the way of the construction of EEI are discussed, and a sequence of actions is proposed to tackle them in an ordered fashion. Finally, two key topics are discussed in detail: the links between energy efficiency and energy savings, and the border between energy efficiency improvement and renewable sources promotion.

  4. 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.

  5. 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...

  6. 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

  7. More efficient together

    DEFF Research Database (Denmark)

    Zhang, Tian

    2015-01-01

    approach to make photosynthesis more efficient is to build hybrid systems that combine inorganic and microbial components to produce specific chemicals. Such hybrid bioinorganic systems lead to improved efficiency and specificity and do not require processed vegetable biomass. They thus prevent harmful......The solar-to-biomass conversion efficiency of natural photosynthesis is between 2.9 and 4.3% for most crops (1, 2). Improving the efficiency of photosynthesis could help increase the appeal of biologically derived fuels and chemicals in comparison with traditional petrochemical processes. One...

  8. Nitrogen use efficiency (NUE)

    NARCIS (Netherlands)

    Oenema, O.

    2015-01-01

    There is a need for communications about resource use efficiency and for measures to increase the use efficiency of nutrients in relation to food production. This holds especially for nitrogen. Nitrogen (N) is essential for life and a main nutrient element. It is needed in relatively large

  9. Energy Efficient TCP

    NARCIS (Netherlands)

    Donckers, L.; Smit, Gerardus Johannes Maria; Havinga, Paul J.M.; Smit, L.T.

    This paper describes the design of an energy-efficient transport protocol for mobile wireless communication. First we describe the metrics used to measure the energy efficiency of transport protocols. We identify several problem areas that prevent TCP/IP from reaching high levels of energy

  10. Energy Efficiency Collaboratives

    Energy Technology Data Exchange (ETDEWEB)

    Li, Michael [US Department of Energy, Washington, DC (United States); Bryson, Joe [US Environmental Protection Agency, Washington, DC (United States)

    2015-09-01

    Collaboratives for energy efficiency have a long and successful history and are currently used, in some form, in more than half of the states. Historically, many state utility commissions have used some form of collaborative group process to resolve complex issues that emerge during a rate proceeding. Rather than debate the issues through the formality of a commission proceeding, disagreeing parties are sent to discuss issues in a less-formal setting and bring back resolutions to the commission. Energy efficiency collaboratives take this concept and apply it specifically to energy efficiency programs—often in anticipation of future issues as opposed to reacting to a present disagreement. Energy efficiency collaboratives can operate long term and can address the full suite of issues associated with designing, implementing, and improving energy efficiency programs. Collaboratives can be useful to gather stakeholder input on changing program budgets and program changes in response to performance or market shifts, as well as to provide continuity while regulators come and go, identify additional energy efficiency opportunities and innovations, assess the role of energy efficiency in new regulatory contexts, and draw on lessons learned and best practices from a diverse group. Details about specific collaboratives in the United States are in the appendix to this guide. Collectively, they demonstrate the value of collaborative stakeholder processes in producing successful energy efficiency programs.

  11. Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells.

    Science.gov (United States)

    Bazley, Faith A; Liu, Cyndi F; Yuan, Xuan; Hao, Haiping; All, Angelo H; De Los Angeles, Alejandro; Zambidis, Elias T; Gearhart, John D; Kerr, Candace L

    2015-11-15

    Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

  12. The Efficient Windows Collaborative

    Energy Technology Data Exchange (ETDEWEB)

    Petermann, Nils

    2006-03-31

    The Efficient Windows Collaborative (EWC) is a coalition of manufacturers, component suppliers, government agencies, research institutions, and others who partner to expand the market for energy efficient window products. Funded through a cooperative agreement with the U.S. Department of Energy, the EWC provides education, communication and outreach in order to transform the residential window market to 70% energy efficient products by 2005. Implementation of the EWC is managed by the Alliance to Save Energy, with support from the University of Minnesota and Lawrence Berkeley National Laboratory.

  13. Learning efficient correlated equilibria

    KAUST Repository

    Borowski, Holly P.

    2014-12-15

    The majority of distributed learning literature focuses on convergence to Nash equilibria. Correlated equilibria, on the other hand, can often characterize more efficient collective behavior than even the best Nash equilibrium. However, there are no existing distributed learning algorithms that converge to specific correlated equilibria. In this paper, we provide one such algorithm which guarantees that the agents\\' collective joint strategy will constitute an efficient correlated equilibrium with high probability. The key to attaining efficient correlated behavior through distributed learning involves incorporating a common random signal into the learning environment.

  14. Transport Energy Efficiency

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2010-07-01

    Transport is the sector with the highest final energy consumption and, without any significant policy changes, is forecast to remain so. In 2008, the IEA published 25 energy efficiency recommendations, among which four are for the transport sector. The recommendations focus on road transport and include policies on improving tyre energy efficiency, fuel economy standards for both light-duty vehicles and heavy-duty vehicles, and eco-driving. Implementation of the recommendations has been weaker in the transport sector than others. This paper updates the progress that has been made in implementing the transport energy efficiency recommendations in IEA countries since March 2009. Many countries have in the last year moved from 'planning to implement' to 'implementation underway', but none have fully implemented all transport energy efficiency recommendations. The IEA calls therefore for full and immediate implementation of the recommendations.

  15. Energy Efficient Cryogenics

    Science.gov (United States)

    Meneghelli, Barry J.; Notardonato, William; Fesmire, James E.

    2016-01-01

    The Cryogenics Test Laboratory, NASA Kennedy Space Center, works to provide practical solutions to low-temperature problems while focusing on long-term technology targets for the energy-efficient use of cryogenics on Earth and in space.

  16. Educated for Efficiency

    DEFF Research Database (Denmark)

    Amore, Mario Daniele; Bennedsen, Morten; Larsen, Birthe

    We study the effect of CEO education on a firm’s energy efficiency. Using a unique dataset of Danish firms, we document that firms led by more educated CEOs exhibit greater energy efficiency. We establish causality by employing exogenous CEO hospitalization episodes: the hospitalization of highly......-educated CEOs induces a drop in a firm’s energy efficiency, whereas the hospitalization of low-education CEOs does not have any significant effect. Disentangling the effect of educational length from that of the field of study, we find that the greater energy efficiency is mostly driven by the cumulated years...... of CEO education rather than by having obtained business or technical-related degrees....

  17. Energy efficiency and behaviour

    DEFF Research Database (Denmark)

    Carstensen, Trine Agervig; Kunnasvirta, Annika; Kiviluoto, Katariina

    The purpose of Work Package 5 Deliverable 5.1., “Case study reports on energy efficiency and behaviour” is to present examples of behavioral interventions to promote energy efficiency in cities. The case studies were collected in January – June 2014, and they represent behavioural interventions...... from different sectors of energy efficiency from the following PLEEC partner countries: Denmark, Sweden, Finland, the UK, the Netherlands, Estonia, Bulgaria and Spain. Each case is presented shortly with key details of budget, target group, and methods as well as a short assessment of main success...... factors. The main addressees of D5.1. are city officials, NGO representatives, private sector actors and any other relevant actors who plan and realize behavioural energy efficiency interventions in European cities. The WP5 team will also further apply results from D5.1. with a more general model on how...

  18. Efficient incremental relaying

    KAUST Repository

    Fareed, Muhammad Mehboob

    2013-07-01

    We propose a novel relaying scheme which improves the spectral efficiency of cooperative diversity systems by utilizing limited feedback from destination. Our scheme capitalizes on the fact that relaying is only required when direct transmission suffers deep fading. We calculate the packet error rate for the proposed efficient incremental relaying scheme with both amplify and forward and decode and forward relaying. Numerical results are also presented to verify their analytical counterparts. © 2013 IEEE.

  19. Centrifugal Contactor Efficiency Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Mincher, Bruce Jay [Idaho National Lab. (INL), Idaho Falls, ID (United States); Tillotson, Richard Dean [Idaho National Lab. (INL), Idaho Falls, ID (United States); Grimes, Travis Shane [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2017-01-01

    The contactor efficiency of a 2-cm acrylic centrifugal contactor, fabricated by ANL using 3D printer technology was measured by comparing a contactor test run to 5-min batch contacts. The aqueous phase was ~ 3 ppm depleted uranium in 3 M HNO3, and the organic phase was 1 M DAAP/dodecane. Sampling during the contactor run showed that equilibrium was achieved within < 3 minutes. The contactor efficiency at equilibrium was 95% to 100 %, depending on flowrate.

  20. Efficient Windows Collaborative

    Energy Technology Data Exchange (ETDEWEB)

    Nils Petermann

    2010-02-28

    The project goals covered both the residential and commercial windows markets and involved a range of audiences such as window manufacturers, builders, homeowners, design professionals, utilities, and public agencies. Essential goals included: (1) Creation of 'Master Toolkits' of information that integrate diverse tools, rating systems, and incentive programs, customized for key audiences such as window manufacturers, design professionals, and utility programs. (2) Delivery of education and outreach programs to multiple audiences through conference presentations, publication of articles for builders and other industry professionals, and targeted dissemination of efficient window curricula to professionals and students. (3) Design and implementation of mechanisms to encourage and track sales of more efficient products through the existing Window Products Database as an incentive for manufacturers to improve products and participate in programs such as NFRC and ENERGY STAR. (4) Development of utility incentive programs to promote more efficient residential and commercial windows. Partnership with regional and local entities on the development of programs and customized information to move the market toward the highest performing products. An overarching project goal was to ensure that different audiences adopt and use the developed information, design and promotion tools and thus increase the market penetration of energy efficient fenestration products. In particular, a crucial success criterion was to move gas and electric utilities to increase the promotion of energy efficient windows through demand side management programs as an important step toward increasing the market share of energy efficient windows.

  1. Supernovae Discovery Efficiency

    Science.gov (United States)

    John, Colin

    2018-01-01

    Abstract:We present supernovae (SN) search efficiency measurements for recent Hubble Space Telescope (HST) surveys. Efficiency is a key component to any search, and is important parameter as a correction factor for SN rates. To achieve an accurate value for efficiency, many supernovae need to be discoverable in surveys. This cannot be achieved from real SN only, due to their scarcity, so fake SN are planted. These fake supernovae—with a goal of realism in mind—yield an understanding of efficiency based on position related to other celestial objects, and brightness. To improve realism, we built a more accurate model of supernovae using a point-spread function. The next improvement to realism is planting these objects close to galaxies and of various parameters of brightness, magnitude, local galactic brightness and redshift. Once these are planted, a very accurate SN is visible and discoverable by the searcher. It is very important to find factors that affect this discovery efficiency. Exploring the factors that effect detection yields a more accurate correction factor. Further inquires into efficiency give us a better understanding of image processing, searching techniques and survey strategies, and result in an overall higher likelihood to find these events in future surveys with Hubble, James Webb, and WFIRST telescopes. After efficiency is discovered and refined with many unique surveys, it factors into measurements of SN rates versus redshift. By comparing SN rates vs redshift against the star formation rate we can test models to determine how long star systems take from the point of inception to explosion (delay time distribution). This delay time distribution is compared to SN progenitors models to get an accurate idea of what these stars were like before their deaths.

  2. Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

    Directory of Open Access Journals (Sweden)

    Friederike Knipping

    2017-03-01

    Full Text Available In T cells with transgenic high-avidity T cell receptors (TCRs, endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs and five guide RNAs (gRNAs from the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas9 system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

  3. Multi-directional program efficiency

    DEFF Research Database (Denmark)

    Asmild, Mette; Balezentis, Tomas; Hougaard, Jens Leth

    2016-01-01

    approach is used to estimate efficiency. This enables a consideration of input-specific efficiencies. The study shows clear differences between the efficiency scores on the different inputs as well as between the farm types of crop, livestock and mixed farms respectively. We furthermore find that crop...... farms have the highest program efficiency, but the lowest managerial efficiency and that the mixed farms have the lowest program efficiency (yet not the highest managerial efficiency)....

  4. Span efficiency in hawkmoths.

    Science.gov (United States)

    Henningsson, Per; Bomphrey, Richard J

    2013-07-06

    Flight in animals is the result of aerodynamic forces generated as flight muscles drive the wings through air. Aerial performance is therefore limited by the efficiency with which momentum is imparted to the air, a property that can be measured using modern techniques. We measured the induced flow fields around six hawkmoth species flying tethered in a wind tunnel to assess span efficiency, ei, and from these measurements, determined the morphological and kinematic characters that predict efficient flight. The species were selected to represent a range in wingspan from 40 to 110 mm (2.75 times) and in mass from 0.2 to 1.5 g (7.5 times) but they were similar in their overall shape and their ecology. From high spatio-temporal resolution quantitative wake images, we extracted time-resolved downwash distributions behind the hawkmoths, calculating instantaneous values of ei throughout the wingbeat cycle as well as multi-wingbeat averages. Span efficiency correlated positively with normalized lift and negatively with advance ratio. Average span efficiencies for the moths ranged from 0.31 to 0.60 showing that the standard generic value of 0.83 used in previous studies of animal flight is not a suitable approximation of aerodynamic performance in insects.

  5. National energy efficiency programme

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    This paper focusses on energy conservation and specifically on energy efficiency which includes efficiency in the production, delivery and utilisation of energy as part of the total energy system of the economy. A National Energy Efficiency Programme is being launched in the Eighth Plan that will take into account both macro level and policy and planning considerations as well as micro level responses for different category of users in the industry, agriculture, transport and domestic sectors. The need for such a National Energy Efficiency Programme after making an assessment of existing energy conservation activities in the country is discussed. The broad framework and contents of the National Energy Efficiency Programme have been outlined and the Eighth Plan targets for energy conservation and their break-up have been given. These targets, as per the Eighth Plan document are 5000 MW in electricity installed capacity and 6 million tonnes of petroleum products by the terminal year of the Eighth Plan. The issues that need to be examined for each sector for achieving the above targets for energy conservation in the Eighth Plan are discussed briefly. They are: (a) policy and planning, (b) implementation arrangements which include the institutional setup and selective legislation, (c) technological requirements, and (d) resource requirements which include human resources and financial resources. (author)

  6. Energy Efficiency Center - Overview

    International Nuclear Information System (INIS)

    Obryk, E.

    2000-01-01

    Full text: The Energy Efficiency Center (EEC) activities have been concentrated on Energy Efficiency Network (SEGE), education and training of energy auditors. EEC has started studies related to renewable fuels (bio fuel, wastes) and other topics related to environment protection. EEC has continued close collaboration with Institute for Energy Technology, Kjeller, Norway. It has been organized and conducted Seminar and Workshop on ''How to Reduce Energy and Water Cost in Higher Education Buildings'' for general and technical managers of the higher education institutions. This Seminar was proceeded by the working meeting on energy efficiency strategy in higher education at the Ministry of National Education. EEC has worked out proposal for activities of Cracow Regional Agency for Energy Efficiency and Environment and has made offer to provide services for this Agency in the field of training, education and consulting. The vast knowledge and experiences in the field of energy audits have been used by the members of EEC in lecturing at energy auditors courses authorized by the National Energy Efficiency Agency (KAPE). Altogether 20 lectures have been delivered. (author)

  7. Efficiency of scanning automatons

    International Nuclear Information System (INIS)

    Shkundenkov, V.N.

    1977-01-01

    Investigated are the methods for improving the efficiency of the picture processing system based on an automatic scanner. Discussed are two types of such a system. In the first case the system contains both automatic and semi-automatic scanners. In the second case the system includes only the automatic scanners with the man-to-computer dialog facilities. For analyzing the role of the automatic scanner and the role of the operator in the processing system use is made of the processing system balance equation. It is proved that the picture processing system should be designed in two steps. The first step should, by all means, insure high efficiency in processing but the high capacity is not obligatory. The second step is aimed at higher capacity along with high efficiency. So, such a two-step designing makes it possible to solve the problem of higher capacity and lesser cost of picture processing

  8. EFFICIENCY OF LOGISTIC SYSTEMS

    Directory of Open Access Journals (Sweden)

    S. M Salum

    2006-01-01

    Full Text Available Main missions of production and distributive logistics are defined in the paper. The paper gives the concept of logistic distribution channel. The possibility of its selection is analyzed, and that is a significant reserve to improve efficiency of logistic processes.The paper considers efficiency of logistic and transport-technological systems on the basis of the relationship of aggregate gross revenue and aggregate costs of an enterprise.The paper cites a concept of economic criterion of efficiency describing the whole logistic chain, that is from the moment of primary raw materials output to consumption of products at all stages of production and transportation of primary raw materials, semi-finished and finished products.

  9. Measuring efficiency in logistics

    Directory of Open Access Journals (Sweden)

    Milan Milovan Andrejić

    2013-06-01

    Full Text Available Dynamic market and environmental changes greatly affect operating of logistics systems. Logistics systems have to realize their activities and processes in an efficient way. The main objective of this paper is to analyze different aspects of efficiency measurement in logistics and to propose appropriate models of measurement. Measuring efficiency in logistics is a complex process that requires consideration of all subsystems, processes and activities as well as the impact of various financial, operational, environmental, quality and other factors. The proposed models have a basis in the Data Envelopment Analysis method. They could help managers in decision making and corrective actions processes. The tests and results of the model show the importance of input and output variables selection.

  10. Efficient Learning Design

    DEFF Research Database (Denmark)

    Godsk, Mikkel

    engaging educators in the design process and developing teaching and learning, it is a shift in educational practice that potentially requires a stakeholder analysis and ultimately a business model for the deployment. What is most important is to balance the institutional, educator, and student......This paper presents the current approach to implementing educational technology with learning design at the Faculty of Science and Technology, Aarhus University, by introducing the concept of ‘efficient learning design’. The underlying hypothesis is that implementing learning design is more than...... perspectives and to consider all these in conjunction in order to obtain a sustainable, efficient learning design. The approach to deploying learning design in terms of the concept of efficient learning design, the catalyst for educational development, i.e. the learning design model and how it is being used...

  11. Danish Energy Efficiency Policy

    DEFF Research Database (Denmark)

    Togeby, Mikael; Larsen, Anders; Dyhr-Mikkelsen, Kirsten

    2009-01-01

    Ten groups of policy instruments for promoting energy efficiency are actively used in Denmark. Among these are the EU instruments such as the CO2 emissions trading scheme and labelling of appliances, labelling of all buildings, combined with national instruments such as high taxes especially...... of the entire Danish energy efficiency policy portfolio must be carried out before end 2008 and put forward for discussion among governing parties no later than February 2009. A consortium comprising Ea Energy Analyses, Niras, the Department of Society and Globalisation (Roskilde University) and 4-Fact...... was assigned with this task. The evaluation aimed to answer the crucial questions: Is the overall design of the portfolio of instruments appropriate? Does the impact of the instruments justify the costs, so that we reach the national goals in a cost efficient way? Will the current instrument portfolio be able...

  12. Efficiency in Microfinance Cooperatives

    Directory of Open Access Journals (Sweden)

    HARTARSKA, Valentina

    2012-12-01

    Full Text Available In recognition of cooperatives’ contribution to the socio-economic well-being of their participants, the United Nations has declared 2012 as the International Year of Cooperatives. Microfinance cooperatives make a large part of the microfinance industry. We study efficiency of microfinance cooperatives and provide estimates of the optimal size of such organizations. We employ the classical efficiency analysis consisting of estimating a system of equations and identify the optimal size of microfinance cooperatives in terms of their number of clients (outreach efficiency, as well as dollar value of lending and deposits (sustainability. We find that microfinance cooperatives have increasing returns to scale which means that the vast majority can lower cost if they become larger. We calculate that the optimal size is around $100 million in lending and half of that in deposits. We find less robust estimates in terms of reaching many clients with a range from 40,000 to 180,000 borrowers.

  13. Efficient convolutional sparse coding

    Energy Technology Data Exchange (ETDEWEB)

    Wohlberg, Brendt

    2017-06-20

    Computationally efficient algorithms may be applied for fast dictionary learning solving the convolutional sparse coding problem in the Fourier domain. More specifically, efficient convolutional sparse coding may be derived within an alternating direction method of multipliers (ADMM) framework that utilizes fast Fourier transforms (FFT) to solve the main linear system in the frequency domain. Such algorithms may enable a significant reduction in computational cost over conventional approaches by implementing a linear solver for the most critical and computationally expensive component of the conventional iterative algorithm. The theoretical computational cost of the algorithm may be reduced from O(M.sup.3N) to O(MN log N), where N is the dimensionality of the data and M is the number of elements in the dictionary. This significant improvement in efficiency may greatly increase the range of problems that can practically be addressed via convolutional sparse representations.

  14. Energy efficiency; Energieffektivisering

    Energy Technology Data Exchange (ETDEWEB)

    2009-06-15

    The Low Energy Panel will halve the consumption in buildings. The Panel has proposed a halving of consumption in the construction within 2040 and 20 percent reduction in the consumption in the industry within 2020. The Panel consider it as possible to gradually reduce consumption in buildings from the current level of 80 TWh with 10 TWh in 2020, 25 TWh in 2030 and 40 TWh in 2040. According the committee one such halving can be reached by significant efforts relating to energy efficiency, by greater rehabilitations, energy efficiency in consisting building stock and stricter requirements for new construction. For the industry field the Panel recommend a political goal to be set at least 20 percent reduction in specific energy consumption in the industry and primary industry beyond general technological development by the end of 2020. This is equivalent to approximately 17 TWh based on current level of activity. The Panel believes that a 5 percent reduction should be achieved by the end of 2012 by carrying out simple measures. The Low Energy Panel has since March 2009 considered possibilities to strengthen the authorities' work with energy efficiency in Norway. The wide complex panel adds up proposals for a comprehensive approach for increased energy efficiency in particular in the building- and industry field. The Panel has looked into the potential for energy efficiency, barriers for energy efficiency, assessment of strengths and weaknesses in the existing policy instruments and members of the Panel's recommendations. In addition the report contains a review of theoretical principles for effects of instruments together with an extensive background. One of the committee members have chosen to take special notes on the main recommendations in the report. (AG)

  15. Efficient use of energy

    CERN Document Server

    Dryden, IGC

    2013-01-01

    The Efficient Use of Energy, Second Edition is a compendium of papers discussing the efficiency with which energy is used in industry. The collection covers relevant topics in energy handling and describes the more important features of plant and equipment. The book is organized into six parts. Part I presents the various methods of heat production. The second part discusses the use of heat in industry and includes topics in furnace design, industrial heating, boiler plants, and water treatment. Part III deals with the production of mechanical and electrical energy. It tackles the principles o

  16. efficience technico-economique

    African Journals Online (AJOL)

    USER

    Les indices d'inefficience coût varient de 1 à 2,86 pour l'oignon et de 1 à 1,77 pour la pomme de terre. ..... Ci ûi. Ci*. Cj*. Xi. Xj. Coût Moyen. Coût Minimale. Figure 3 : Principe de l'efficience coût selon l'approche FFSC. Cost efficiency principle according to SFCF approach. ... différentes quantités des divers engrais utilisés.

  17. Financing Energy Efficient Homes

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2007-07-01

    Existing buildings require over 40% of the world's total final energy consumption, and account for 24% of world CO2 emissions (IEA, 2006). Much of this consumption could be avoided through improved efficiency of building energy systems (IEA, 2006) using current, commercially-viable technology. In most cases, these technologies make economic sense on a life-cycle cost analysis (IEA, 2006b). Moreover, to the extent that they reduce dependence on risk-prone fossil energy sources, energy efficient technologies also address concerns of energy security.

  18. Institutions, Equilibria and Efficiency

    DEFF Research Database (Denmark)

    Competition and efficiency is at the core of economic theory. This volume collects papers of leading scholars, which extend the conventional general equilibrium model in important ways. Efficiency and price regulation are studied when markets are incomplete and existence of equilibria...... membership are evaluated. Core equivalence is shown for bargaining economies. The theory of risk aversion is extended and the relation between risk taking and wealth is experimentally investigated. Other topics include: determinacy in OLG with cash-in-advance constraints, income distribution and democracy...... in OLG, learning in OLG and in games, optimal pricing of derivative securities, the impact of heterogeneity...

  19. Dimensions of energy efficiency

    International Nuclear Information System (INIS)

    Ramani, K.V.

    1992-01-01

    In this address the author describes three dimensions of energy efficiency in order of increasing costs: conservation, resource and technology substitution, and changes in economic structure. He emphasizes the importance of economic rather than environmental rationales for energy efficiency improvements in developing countries. These countries do not place high priority on the problems of global climate change. Opportunities for new technologies may exist in resource transfer, new fuels and, possibly, small reactors. More research on economic and social impacts of technologies with greater sensitivity to user preferences is needed

  20. Fuzzy efficiency without convexity

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Balezentis, Tomas

    2014-01-01

    In this paper we develop a fuzzy version of the crisp Free Disposal Hull (FDH) method for measuring technical efficiency for samples of similar production units. The FDH-method is basically Data Envelopment Analysis (DEA) without the underlying assumption of convexity of the technology set. Our...... approach builds directly upon the definition of Farrell's indexes of technical efficiency used in crisp FDH. Therefore we do not require the use of fuzzy programming techniques but only utilize ranking probabilities of intervals as well as a related definition of dominance between pairs of intervals. We...

  1. ENERGY EFFICIENT LAUNDRY PROCESS

    Energy Technology Data Exchange (ETDEWEB)

    Tim Richter

    2005-04-01

    With the rising cost of energy and increased concerns for pollution and greenhouse gas emissions from power generation, increased focus is being put on energy efficiency. This study looks at several approaches to reducing energy consumption in clothes care appliances by considering the appliances and laundry chemistry as a system, rather than individually.

  2. The efficiency of freedom

    DEFF Research Database (Denmark)

    Madsen, Christian Østergaard; Kræmmergaard, Pernille

    2015-01-01

    The Danish e-government strategy aims to increase the efficiency of public sector administration by making e-government channels mandatory for citizens by 2015. Although Danish citizens have adopted e-government channels to interact with public authorities, many also keep using traditional channels...

  3. Robust efficient video fingerprinting

    Science.gov (United States)

    Puri, Manika; Lubin, Jeffrey

    2009-02-01

    We have developed a video fingerprinting system with robustness and efficiency as the primary and secondary design criteria. In extensive testing, the system has shown robustness to cropping, letter-boxing, sub-titling, blur, drastic compression, frame rate changes, size changes and color changes, as well as to the geometric distortions often associated with camcorder capture in cinema settings. Efficiency is afforded by a novel two-stage detection process in which a fast matching process first computes a number of likely candidates, which are then passed to a second slower process that computes the overall best match with minimal false alarm probability. One key component of the algorithm is a maximally stable volume computation - a three-dimensional generalization of maximally stable extremal regions - that provides a content-centric coordinate system for subsequent hash function computation, independent of any affine transformation or extensive cropping. Other key features include an efficient bin-based polling strategy for initial candidate selection, and a final SIFT feature-based computation for final verification. We describe the algorithm and its performance, and then discuss additional modifications that can provide further improvement to efficiency and accuracy.

  4. ERP=Efficiency

    Science.gov (United States)

    Violino, Bob

    2008-01-01

    This article discusses the enterprise resource planning (ERP) system. Deploying an ERP system is one of the most extensive--and expensive--IT projects a college or university can undertake. The potential benefits of ERP are significant: a more smoothly running operation with efficiencies in virtually every area of administration, from automated…

  5. Web anonymization efficiency study

    Science.gov (United States)

    Sochor, Tomas

    2017-11-01

    The analysis of TOR, JonDo and CyberGhost efficiency (measured the as latency increase and transmission speed decrease) is presented in the paper. Results showed that all tools have relatively favorable latency increase (no more than 60% RTT increase). The transmission speed increase was much more significant (more than 60%), and even more for JonDo (above 90%).

  6. An efficiency correction model

    NARCIS (Netherlands)

    Francke, M.K.; de Vos, A.F.

    2009-01-01

    We analyze a dataset containing costs and outputs of 67 American local exchange carriers in a period of 11 years. This data has been used to judge the efficiency of BT and KPN using static stochastic frontier models. We show that these models are dynamically misspecified. As an alternative we

  7. Microeconomics : Equilibrium and Efficiency

    NARCIS (Netherlands)

    Ten Raa, T.

    2013-01-01

    Microeconomics: Equilibrium and Efficiency teaches how to apply microeconomic theory in an innovative, intuitive and concise way. Using real-world, empirical examples, this book not only covers the building blocks of the subject, but helps gain a broad understanding of microeconomic theory and

  8. Combining Expert Advice Efficiently

    NARCIS (Netherlands)

    W.M. Koolen-Wijkstra (Wouter); S. de Rooij (Steven)

    2008-01-01

    htmlabstractWe show how models for prediction with expert advice can be defined concisely and clearly using hidden Markov models (HMMs); standard HMM algorithms can then be used to efficiently calculate, among other things, how the expert predictions should be weighted according to the model. We

  9. Corporate efficiency in Europe

    Czech Academy of Sciences Publication Activity Database

    Hanousek, Jan; Kočenda, E.; Shamshur, Anastasiya

    2015-01-01

    Roč. 32, June (2015), s. 24-40 ISSN 0929-1199 R&D Projects: GA ČR(CZ) GA15-15927S Institutional support: PRVOUK-P23 Keywords : efficiency * ownership structure * firms Subject RIV: AH - Economics Impact factor: 1.286, year: 2015

  10. Higher Efficiency HVAC Motors

    Energy Technology Data Exchange (ETDEWEB)

    Flynn, Charles Joseph [QM Power, Inc., Kansas City, MO (United States)

    2018-02-13

    The objective of this project was to design and build a cost competitive, more efficient heating, ventilation, and air conditioning (HVAC) motor than what is currently available on the market. Though different potential motor architectures among QMP’s primary technology platforms were investigated and evaluated, including through the building of numerous prototypes, the project ultimately focused on scaling up QM Power, Inc.’s (QMP) Q-Sync permanent magnet synchronous motors from available sub-fractional horsepower (HP) sizes for commercial refrigeration fan applications to larger fractional horsepower sizes appropriate for HVAC applications, and to add multi-speed functionality. The more specific goal became the research, design, development, and testing of a prototype 1/2 HP Q-Sync motor that has at least two operating speeds and 87% peak efficiency compared to incumbent electronically commutated motors (EC or ECM, also known as brushless direct current (DC) motors), the heretofore highest efficiency HVACR fan motor solution, at approximately 82% peak efficiency. The resulting motor prototype built achieved these goals, hitting 90% efficiency and .95 power factor at full load and speed, and 80% efficiency and .7 power factor at half speed. Q-Sync, developed in part through a DOE SBIR grant (Award # DE-SC0006311), is a novel, patented motor technology that improves on electronically commutated permanent magnet motors through an advanced electronic circuit technology. It allows a motor to “sync” with the alternating current (AC) power flow. It does so by eliminating the constant, wasteful power conversions from AC to DC and back to AC through the synthetic creation of a new AC wave on the primary circuit board (PCB) by a process called pulse width modulation (PWM; aka electronic commutation) that is incessantly required to sustain motor operation in an EC permanent magnet motor. The Q-Sync circuit improves the power factor of the motor by removing all

  11. Chlorific efficiency of coal hydrogenation

    Energy Technology Data Exchange (ETDEWEB)

    Schappert, H.

    1942-10-20

    In studies on the calorific efficiency of coal hydrogenation, the efficiency for H/sub 2/ production was calculated to be 26%, the efficiency for hydrogenation was calculated to be 49%, and the efficiency of hydrogenation including H/sub 2/ production was 27.2%. The efficiency of hydrogenation plus hydrogen production was almost equal to the efficiency of hydrogen production alone, even though this was not expected because of the total energy calculated in the efficiency of hydrogenation proper. It was entirely possible, but did not affect computations, that the efficiency of one or the other components of hydrogenation process differed somewhat from 49%. The average efficiency for all cases was 49%. However, when hydrogen was not bought, but was produced--(efficiency of hydrogen production was 26%, not 100%-- then the total energy changed and the efficiency of hydrogen production and combination was not 26%, but 13%. This lower value explained the drop of hydrogenation efficiency to 27.2%.

  12. Energy Efficiency Project Development

    Energy Technology Data Exchange (ETDEWEB)

    IUEP

    2004-03-01

    The International Utility Efficiency Partnerships, Inc. (IUEP) has been a leader among the industry groups that have supported voluntary initiatives to promote international energy efficiency projects and address global climate change. The IUEP maintains its leadership by both supporting international greenhouse gas (GHG) reduction projects under the auspices of the U.S. Department of Energy (DOE) and by partnering with U.S. and international organizations to develop and implement strategies and specific energy efficiency projects. The goals of the IUEP program are to (1) provide a way for U.S. industry to maintain a leadership role in international energy efficiency infrastructure projects; (2) identify international energy project development opportunities to continue its leadership in supporting voluntary market-based mechanisms to reduce GHG emissions; and (3) demonstrate private sector commitment to voluntary approaches to global climate issues. The IUEP is dedicated to identifying, promoting, managing, and assisting in the registration of international energy efficiency projects that result in demonstrated voluntary reductions of GHG emissions. This Final Technical Report summarizes the IUEP's work in identifying, promoting, managing, and assisting in development of these projects and IUEP's effort in creating international cooperative partnerships to support project development activities that develop and deploy technologies that (1) increase efficiency in the production, delivery and use of energy; (2) increase the use of cleaner, low-carbon fuels in processing products; and (3) capture/sequester carbon gases from energy systems. Through international cooperative efforts, the IUEP intends to strengthen partnerships for energy technology innovation and demonstration projects capable of providing cleaner energy in a cost-effective manner. As detailed in this report, the IUEP met program objectives and goals during the reporting period January 1

  13. Energy efficiency system development

    Science.gov (United States)

    Leman, A. M.; Rahman, K. A.; Chong, Haw Jie; Salleh, Mohd Najib Mohd; Yusof, M. Z. M.

    2017-09-01

    By subjecting to the massive usage of electrical energy in Malaysia, energy efficiency is now one of the key areas of focus in climate change mitigation. This paper focuses on the development of an energy efficiency system of household electrical appliances for residential areas. Distribution of Questionnaires and pay a visit to few selected residential areas are conducted during the fulfilment of the project as well as some advice on how to save energy are shared with the participants. Based on the collected data, the system developed by the UTHM Energy Team is then evaluated from the aspect of the consumers' behaviour in using electrical appliances and the potential reduction targeted by the team. By the end of the project, 60% of the participants had successfully reduced the electrical power consumption set by the UTHM Energy Team. The reasons for whether the success and the failure is further analysed in this project.

  14. Negotiating Efficient PPP Contracts

    DEFF Research Database (Denmark)

    Tvarnø, Christina D.

    This paper concerns Public Private Partnership (PPP) contracts in concern to the coming new 2014/24IEU public procurement directive. The new EU public procurement directive gives the public authority the opportunity to negotiate PPPs much more when they are implemented in national law....... An opportunity the member states should consider using when procuring a PPP. This paper looks at the negotiation and contracting of a PPP in an economic theoretical and EU public procurement perspective and discusses how to establish an efficient PPP contract under a strong public law doctrine. Governments...... procurement law. Furthermore, the paper seeks to establish a connection between public law, private law and the efficient PPP contract by drawing upon economic theory and empirical contract data from UK, US and Danish partnering contracts from the construction industry and the aim of contracting joint utility...

  15. SparkJet Efficiency

    Science.gov (United States)

    Golbabaei-Asl, Mona; Knight, Doyle; Anderson, Kellie; Wilkinson, Stephen

    2013-01-01

    A novel method for determining the thermal efficiency of the SparkJet is proposed. A SparkJet is attached to the end of a pendulum. The motion of the pendulum subsequent to a single spark discharge is measured using a laser displacement sensor. The measured displacement vs time is compared with the predictions of a theoretical perfect gas model to estimate the fraction of the spark discharge energy which results in heating the gas (i.e., increasing the translational-rotational temperature). The results from multiple runs for different capacitances of c = 3, 5, 10, 20, and 40 micro-F show that the thermal efficiency decreases with higher capacitive discharges.

  16. Superstructure high efficiency photovoltaics

    Science.gov (United States)

    Wagner, M.; So, L. C.; Leburton, J. P.

    1987-01-01

    A novel class of photovoltaic cascade structures is introduced which features multijunction upper subcells. These superstructure high efficiency photovoltaics (SHEP's) exhibit enhanced upper subcell spectral response because of the additional junctions which serve to reduce bulk recombination losses by decreasing the mean collection distance for photogenerated minority carriers. Two possible electrical configurations were studied and compared: a three-terminal scheme that allows both subcells to be operated at their individual maximum power points and a two-terminal configuration with an intercell ohmic contact for series interconnection. The three-terminal devices were found to be superior both in terms of beginning-of-life expectancy and radiation tolerance. Realistic simulations of three-terminal AlGaAs/GaAs SHEP's show that one sun AMO efficiencies in excess of 26 percent are possible.

  17. Efficiency and Logistics

    CERN Document Server

    Hompel, Michael; Klumpp, Matthias

    2013-01-01

    The „EffizienzCluster LogistikRuhr“ was a winner in the Leading Edge Science Cluster competition run by the German federal Ministry of Education and Research. The mission and aim of the „EffizienzCluster LogistikRuhr“ is to facilitate tomorrow’s individuality – in the sense of individual goods supply, mobility, and production – using 75 percent of today’s resources. Efficiency – both in economical and ecological terms – is enabled by state-of-the-art and innovative logistical solutions including transportation, production and intralogistics. These proceedings “Efficiency and Logistics” give first answers from 27 research projects as an insight into the current state of research of Europe’s leading research and development cluster in logistics and as a contribution to the discussion on how logistics as a science can help to cope with foreseeable resource shortage and sustainability as global challenges.

  18. Solar bowl component efficiencies

    International Nuclear Information System (INIS)

    O'Hair, E.A.; Green, B.L.

    1992-01-01

    Battelle Pacific Northwest Laboratory has published two volumes on the economic evaluation of various proposed configurations and plant sizes for the four solar thermal technologies. These are the latest in a series of publications sponsored by the Department of Energy (DOE) on plant and operational costs and are more complete in that they include calculations of electrical output. These latest Battelle volumes use the 1976 solar data from Barstow, Calif., and by calculating or estimating the energy conversion efficiency of each element in the process from sun to electricity predict the output and cost of electricity from different plant sizes for each of the four technologies. In this paper a comparison is presented of the component efficiencies developed by Battelle and those of the solar bowl at Crosbyton, Tex

  19. Efficiently Controllable Graphs.

    Science.gov (United States)

    Gokler, Can; Lloyd, Seth; Shor, Peter; Thompson, Kevin

    2017-06-30

    We investigate graphs that can be disconnected into small components by removing a vanishingly small fraction of their vertices. We show that, when a controllable quantum network is described by such a graph and the gaps in eigenfrequencies and in transition frequencies are bounded exponentially in the number of vertices, the network is efficiently controllable, in the sense that universal quantum computation can be performed using a control sequence polynomial in the size of the network while controlling a vanishingly small fraction of subsystems. We show that networks corresponding to finite-dimensional lattices are efficiently controllable and explore generalizations to percolation clusters and random graphs. We show that the classical computational complexity of estimating the ground state of Hamiltonians described by controllable graphs is polynomial in the number of subsystems or qubits.

  20. Energy efficiency: A proposal

    Directory of Open Access Journals (Sweden)

    Pendić Zoran R.

    2015-01-01

    Full Text Available Increase of energy efficiency in Serbia is one of the priority tasks. Sector of electric lighting offers great opportunities for increasing energy efficiency due to the rapid development of LED technology. Nowadays LED lighting is still expensive and have relatively little application in the domain of electric lighting. However, it is anticipated that LED products would soon greatly transform the electric lighting market. In recent years, LED technology has significantly improved, and constantly evolving. It is expected that advanced and future LED bulbs / lamps to be getting cheaper and with a better technical characteristics and that they will soon be dominant on the lighting market. Serbia must prepare for the LED future, creating appropriate legislation and promotion of appropriate incentive measures.

  1. CONTROLLING AND BUSINESS EFFICIENCY

    OpenAIRE

    Vuko, Tina; Ojvan, Ivica

    2013-01-01

    Managing business successfully in dynamic environment requires effective controlling system. Controlling is the process of defining objectives, planning and management control so that every decision maker can act in accordance with agreed objectives. Controlling function as a separate department contributes business efficiency trough ensuring transparency of business result and business processes. Controlling takes place when manager and controller cooperate. The aim of this paper is to inves...

  2. Carbon Efficient Building Solutions

    Directory of Open Access Journals (Sweden)

    Pellervo Matilainen

    2010-03-01

    Full Text Available Traditionally, the Finnish legislation have focused on energy use and especially on energy used for heating space in buildings. However, in many cases this does not lead to the optimal concept in respect to minimizing green house gases. This paper studies how CO2 emission levels are affected by different measures to reduce energy use in buildings. This paper presents two real apartment buildings with different options of energy efficiency and power sources. The calculations clearly show that in the future electricity and domestic hot water use will have high importance in respect to energy efficiency, and therefore also CO2 equivalent (eq emissions. The importance increases when the energy efficiency of the building increases. There are big differences between average Finnish production and individual power plants; CO2 eq emissions might nearly double depending on the energy source and the power plant type. Both a building with an efficient district heating as a power source, and a building with ground heat in addition to nuclear power electricity as a complimentary electricity source performed very similarly to each other in respect to CO2 eq emissions. However, it is dangerous to conclude that it is not important which energy source is chosen. If hypothetically, the use of district heating would dramatically drop, the primary energy factor and CO2 eq emissions from electricity would rise, which in turn would lead to the increase of the ground heat systems emissions. A problem in the yearly calculations is that the fact that it is very important, sometimes even crucial, when energy is needed, is always excluded.

  3. Determinants of Government Efficiency

    OpenAIRE

    David Hauner; Annette J Kyobe

    2008-01-01

    We compile the first large cross-country panel dataset of public sector performance and efficiency, encompassing 114 countries on all income levels from 1980 to 2006, with about 1,800 country-year observations for the education sector and about 900 observations for health. We regress these indicators on potential economic, institutional, demographic, and geographic determinants. Our most resounding conclusion is that higher government expenditure relative to GDP tends to be associated with lo...

  4. Measuring Tax Efficiency

    DEFF Research Database (Denmark)

    Raimondos-Møller, Pascalis; Woodland, Alan D.

    2004-01-01

    , thereby, the degree of efficiency of a taxsystem. We call this number the Tax Optimality Index. We show howthe basic method can be altered in order to derive a revenue equivalentuniform tax, which measures the size of the public sector. A numericalexample is used to illustrate the method developed.......JEL Code: H21, H41.Keywords: Tax optimality index, excess burden, distance function.Authors Affiliations: Raimondos-Møller: Copenhagen Business School, CEPR,CESifo, and EPRU. Woodland: University of Sydney....

  5. SOURgraphs for efficient completion

    OpenAIRE

    Christopher Lynch; Polina Strogova

    1998-01-01

    We introduce a data structure called SOUR graphs and present an efficient Knuth-Bendix completion procedure based on it. SOUR graphs allow for a maximal structure sharing of terms in rewriting systems. The term representation is a dag representation, except that edges are labelled with equational constraints and variable renamings. The rewrite rules correspond to rewrite edges, the unification problems to unification edges. The Critical Pair and Simplification inferences are recogniz...

  6. Efficient Non Linear Loudspeakers

    DEFF Research Database (Denmark)

    Petersen, Bo R.; Agerkvist, Finn T.

    2006-01-01

    Loudspeakers have traditionally been designed to be as linear as possible. However, as techniques for compensating non linearities are emerging, it becomes possible to use other design criteria. This paper present and examines a new idea for improving the efficiency of loudspeakers at high levels...... by changing the voice coil layout. This deliberate non-linear design has the benefit that a smaller amplifier can be used, which has the benefit of reducing system cost as well as reducing power consumption....

  7. Efficient HVAC. New products

    International Nuclear Information System (INIS)

    2016-01-01

    Jung is responding to the challenge of energy efficiency, ease of operation and economic profitability in all of its solutions for the tertiary sector, whether for newly constructed buildings or refurbishments, for full management of the electrical system or the partial control of lighting, HVAC, mood settings, access control, etc., for the bedrooms or specific areas of the building. In the specific case of hotels, Jung offers each a custom-made solution in line with its possibilities and objectives. (Author)

  8. Energy efficiency fallacies revisited

    International Nuclear Information System (INIS)

    Brookes, Leonard

    2000-01-01

    A number of governments including that of the UK subscribe to the belief that a national program devoted to raising energy efficiency throughout the economy provides a costless - indeed profitable - route to meeting international environmental obligations. This is a seductive policy. It constitutes the proverbial free lunch - not only avoiding politically unpopular measures like outlawing, taxing or rationing offending fuels or expanding non-carboniferous sources of energy like nuclear power but doing so with economic benefit. The author of this contribution came to doubt the validity of this solution when it was offered as a way of mitigating the effect of the OPEC price hikes of the 1970s, maintaining that economically justified improvement in energy efficiency led to higher levels of energy consumption at the economy-wide level than in the absence of any efficiency response. More fundamentally, he argues that there is no case for preferentially singling out energy, from among all the resources available to us, for efficiency maximisation. The least damaging policy is to determine targets, enact the restrictive measures needed to curb consumption, and then leave it to consumers - intermediate and final - to reallocate all the resources available to them to best effect subject to the new enacted constraints and any others they might be experiencing. There is no reason to suppose that it is right for all the economic adjustment following a new resource constraint to take the form of improvements in the productivity of that resource alone. As many others have argued, any action to impose resource constraint entails an inevitable economic cost in the shape of a reduction in production and consumption possibilities: there would be no free lunch. In the last few years debate about the validity of these contentions has blossomed, especially under the influence of writers on the western side of the Atlantic. In this contribution the author outlines the original arguments

  9. High-efficiency CARM

    Energy Technology Data Exchange (ETDEWEB)

    Bratman, V.L.; Kol`chugin, B.D.; Samsonov, S.V.; Volkov, A.B. [Institute of Applied Physics, Nizhny Novgorod (Russian Federation)

    1995-12-31

    The Cyclotron Autoresonance Maser (CARM) is a well-known variety of FEMs. Unlike the ubitron in which electrons move in a periodical undulator field, in the CARM the particles move along helical trajectories in a uniform magnetic field. Since it is much simpler to generate strong homogeneous magnetic fields than periodical ones for a relatively low electron energy ({Brit_pounds}{le}1-3 MeV) the period of particles` trajectories in the CARM can be sufficiently smaller than in the undulator in which, moreover, the field decreases rapidly in the transverse direction. In spite of this evident advantage, the number of papers on CARM is an order less than on ubitron, which is apparently caused by the low (not more than 10 %) CARM efficiency in experiments. At the same time, ubitrons operating in two rather complicated regimes-trapping and adiabatic deceleration of particles and combined undulator and reversed guiding fields - yielded efficiencies of 34 % and 27 %, respectively. The aim of this work is to demonstrate that high efficiency can be reached even for a simplest version of the CARM. In order to reduce sensitivity to an axial velocity spread of particles, a short interaction length where electrons underwent only 4-5 cyclotron oscillations was used in this work. Like experiments, a narrow anode outlet of a field-emission electron gun cut out the {open_quotes}most rectilinear{close_quotes} near-axis part of the electron beam. Additionally, magnetic field of a small correcting coil compensated spurious electron oscillations pumped by the anode aperture. A kicker in the form of a sloping to the axis frame with current provided a control value of rotary velocity at a small additional velocity spread. A simple cavity consisting of a cylindrical waveguide section restricted by a cut-off waveguide on the cathode side and by a Bragg reflector on the collector side was used as the CARM-oscillator microwave system.

  10. Cost efficient command management

    Science.gov (United States)

    Brandt, Theresa; Murphy, C. W.; Kuntz, Jon; Barlett, Tom

    1996-01-01

    The design and implementation of a command management system (CMS) for a NASA control center, is described. The technology innovations implemented in the CMS provide the infrastructure required for operations cost reduction and future development cost reduction through increased operational efficiency and reuse in future missions. The command management design facilitates error-free operations which enables the automation of the routine control center functions and allows for the distribution of scheduling responsibility to the instrument teams. The reusable system was developed using object oriented methodologies.

  11. Energy Efficient Digital Networks

    Energy Technology Data Exchange (ETDEWEB)

    Lanzisera, Steven [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Brown, Richard [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2013-01-01

    Digital networks are the foundation of the information services, and play an expanding and indispensable role in our lives, via the Internet, email, mobile phones, etc. However, these networks consume energy, both through the direct energy use of the network interfaces and equipment that comprise the network, and in the effect they have on the operating patterns of devices connected to the network. The purpose of this research was to investigate a variety of technology and policy issues related to the energy use caused by digital networks, and to further develop several energy-efficiency technologies targeted at networks.

  12. Merging {DBMs} Efficiently

    DEFF Research Database (Denmark)

    David, Alexandre

    2005-01-01

    In this paper we present different algorithms to reduce the number of DBMs in federations by merging them. Federations are unions of DBMs and are used to represent non-convex zones. Inclusion checking between DBMs is a limited technique to reduce the size of federations and how to choose some DBMs...... to merge them into a larger one is a combi-natorial problem. We present a number of simple but efficient techniques to avoid searching the combinations while still being able to merge any number of DBMs...

  13. Improving efficiency in stereology

    DEFF Research Database (Denmark)

    Keller, Kresten Krarup; Andersen, Ina Trolle; Andersen, Johnnie Bremholm

    2013-01-01

    of the study was to investigate the time efficiency of the proportionator and the autodisector on virtual slides compared with traditional methods in a practical application, namely the estimation of osteoclast numbers in paws from mice with experimental arthritis and control mice. Tissue slides were scanned...... in a digital slide scanner and the autodisector was applied on the obtained virtual tissue slides. Every slide was partitioned into fields of view, and cells were counted in all of them. Based on the original exhaustive data set comprising 100% of fields of view and covering the total section area...

  14. Late Washing efficiency

    International Nuclear Information System (INIS)

    Morrissey, M.F.

    1992-01-01

    Interim Waste Technology has demonstrated the Late Washing concept on the Experimental Laboratory Filter (ELF) at TNX. In two tests, washing reduced the [NO 2 - ] from 0.08 M to approximately 0.01 M on slurries with 2 year equivalent radiation exposures and 9.5 wt. % solids. For both washes, the [NO 2 - ] decreased at rates near theoretical for a constant volume stirred vessel, indicating approximately l00% washing efficiency. Permeate flux was greater than 0.05 gpm/ft 2 for both washes at a transmembrane pressure of 50 psi and flow velocity of 9 ft/sec

  15. Fuze Gear Train Efficiency

    Science.gov (United States)

    1981-11-01

    P6.06. S a1.0 POINTIFr .94;11 ALPHA)) -02 .O~ 5 La1. POINTEr a94 AIPHA)) a Pfl.3E S *1.0 P’) INTEF a 94149 AIPHAO a Pnl*h& S v 1.0 POINTirr a .949!. ALPHA...conditions, the point efficiency is computed according to equation 3 (ref E-1), i.e., Cŕ PO INTEF KRA 3IO M-in where, withb Ka lt ny The cycle

  16. Energy efficient data centers

    Energy Technology Data Exchange (ETDEWEB)

    Tschudi, William; Xu, Tengfang; Sartor, Dale; Koomey, Jon; Nordman, Bruce; Sezgen, Osman

    2004-03-30

    Data Center facilities, prevalent in many industries and institutions are essential to California's economy. Energy intensive data centers are crucial to California's industries, and many other institutions (such as universities) in the state, and they play an important role in the constantly evolving communications industry. To better understand the impact of the energy requirements and energy efficiency improvement potential in these facilities, the California Energy Commission's PIER Industrial Program initiated this project with two primary focus areas: First, to characterize current data center electricity use; and secondly, to develop a research ''roadmap'' defining and prioritizing possible future public interest research and deployment efforts that would improve energy efficiency. Although there are many opinions concerning the energy intensity of data centers and the aggregate effect on California's electrical power systems, there is very little publicly available information. Through this project, actual energy consumption at its end use was measured in a number of data centers. This benchmark data was documented in case study reports, along with site-specific energy efficiency recommendations. Additionally, other data center energy benchmarks were obtained through synergistic projects, prior PG&E studies, and industry contacts. In total, energy benchmarks for sixteen data centers were obtained. For this project, a broad definition of ''data center'' was adopted which included internet hosting, corporate, institutional, governmental, educational and other miscellaneous data centers. Typically these facilities require specialized infrastructure to provide high quality power and cooling for IT equipment. All of these data center types were considered in the development of an estimate of the total power consumption in California. Finally, a research ''roadmap'' was developed

  17. The efficient learner.

    Science.gov (United States)

    Almåsbakk, B; Whiting, H T; Helgerud, J

    2001-02-01

    The research to be reported examines the concept of efficiency, defined as the relation of metabolic energy expended to mechanical work done. The development of movement coordination was investigated in the context of the hypothesis that an increase in coordination would be accompanied by a parallel reduction in overall energy expenditure, relative to the increase in energy expenditure demanded by improvements in work output. The task involved learning to make cyclical, slalom-like, ski movements on a ski apparatus. Development of coordination was indexed by changes in the timing of forcing while on-line measurements of oxygen consumption were used as indices of energy expenditure. Six female volunteers served as subjects in nine training sessions. The change in the coordination pattern, between the subject and the apparatus, as indexed by the development of the phase lag, was paralleled by an improvement in efficiency. It was concluded that learning the skill used in this study involves a search by the system for perception-action couplings (phase relations in the present study) that require minimal energy expenditure.

  18. Negotiating Efficient PPP Contracts

    DEFF Research Database (Denmark)

    Tvarnø, Christina D.

    This paper concerns Public Private Partnership (PPP) contracts in concern to the coming new 2014/24IEU public procurement directive. The new EU public procurement directive gives the public authority the opportunity to negotiate PPPs much more when they are implemented in national law. An opportu......This paper concerns Public Private Partnership (PPP) contracts in concern to the coming new 2014/24IEU public procurement directive. The new EU public procurement directive gives the public authority the opportunity to negotiate PPPs much more when they are implemented in national law....... An opportunity the member states should consider using when procuring a PPP. This paper looks at the negotiation and contracting of a PPP in an economic theoretical and EU public procurement perspective and discusses how to establish an efficient PPP contract under a strong public law doctrine. Governments...... procurement law. Furthermore, the paper seeks to establish a connection between public law, private law and the efficient PPP contract by drawing upon economic theory and empirical contract data from UK, US and Danish partnering contracts from the construction industry and the aim of contracting joint utility...

  19. Plerixafor enables the safe, rapid, efficient mobilization of haematopoietic stem cells in sickle cell disease patients after exchange transfusion.

    Science.gov (United States)

    Lagresle-Peyrou, Chantal; Lefrère, François; Magrin, Elisa; Ribeil, Jean-Antoine; Romano, Oriana; Weber, Leslie; Magnani, Alessandra; Sadek, Hanem; Plantier, Clémence; Gabrion, Aurélie; Ternaux, Brigitte; Félix, Tristan; Couzin, Chloé; Stanislas, Aurélie; Tréluyer, Jean-Marc; Lamhaut, Lionel; Joseph, Laure; Delville, Marianne; Miccio, Annarita; André-Schmutz, Isabelle; Cavazzana, Marina

    2018-02-22

    Sickle cell disease is characterized by chronic anaemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Haematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected haematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and the red blood cells' properties hamper the harvesting and immunoselection of patient stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of haematopoietic stem and progenitor cells (HSPC) in the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a Phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients under RBC exchanges to decrease Haemoglobin S level below 30%. The secondary objective was to measure the efficiency of HSPC mobilization and isolation. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of haematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize haematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Copyright © 2018, Ferrata Storti Foundation.

  20. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Shimojo, Daisuke; Onodera, Kazunari; Doi-Torii, Yukiko; Ishihara, Yasuharu; Hattori, Chinatsu; Miwa, Yukino; Tanaka, Satoshi; Okada, Rina; Ohyama, Manabu; Shoji, Masanobu; Nakanishi, Atsushi; Doyu, Manabu; Okano, Hideyuki; Okada, Yohei

    2015-12-01

    Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves. In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6 (+) and SOX1 (+) neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9(+) and ISL-1(+) motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9 (e438) ::Venus). The specificity of this reporter was confirmed through immunocytochemistry and

  1. ENERGY EFFICIENT DESALINATOR

    Directory of Open Access Journals (Sweden)

    T. A. Ismailov

    2017-01-01

    Full Text Available Objectives. The aim of the research is to develop a thin-film semiconductor thermoelectric heat pump of cylindrical shape for the desalination of sea water.Methods. To improve the efficiency of the desalination device, a  special thin-film semiconductor thermoelectric heat pump of  cylindrical shape is developed. The construction of the thin-film  semiconductor thermoelectric heat pump allows the flow rates of  incoming sea water and outflowing fresh water and brine to be  equalised by changing the geometric dimensions of the desalinator.  The cross-sectional area of the pipeline for incoming sea water is equal to the total area of outflowing fresh water and brine.Results. The use of thin-film semiconductor p- and n-type branches  in a thermo-module reduces their electrical resistance virtually to  zero and completely eliminates Joule's parasitic heat release. The  Peltier thermoelectric effect on heating and cooling is completely  preserved, bringing the efficiency of the heat pump to almost 100%, improving the energy-saving characteristics of the  desalinator as a whole. To further increase the efficiency of the  proposed desalinator, thermoelectric modules with radiation can be  used as thermoelectric devices.Conclusion. As a consequence of the creation of conditions of high rarefaction under which water will be converted to steam, which, at  20° C, is cold (as is the condensed distilled water, energy costs can  be reduced. In this case, the energy for heating and cooling is not  wasted; moreover, sterilisation is also achieved using the ultraviolet  radiation used in the thermoelectric devices, which, on the one hand, generate electromagnetic ultraviolet radiation, and, on the other, cooling. Such devices operate in optimal mode without heat  release. The desalination device can be used to produce fresh water and concentrated solutions from any aqueous solutions, including wastewater from industrial

  2. Refines Efficiency Improvement

    Energy Technology Data Exchange (ETDEWEB)

    WRI

    2002-05-15

    Refinery processes that convert heavy oils to lighter distillate fuels require heating for distillation, hydrogen addition or carbon rejection (coking). Efficiency is limited by the formation of insoluble carbon-rich coke deposits. Heat exchangers and other refinery units must be shut down for mechanical coke removal, resulting in a significant loss of output and revenue. When a residuum is heated above the temperature at which pyrolysis occurs (340 C, 650 F), there is typically an induction period before coke formation begins (Magaril and Aksenova 1968, Wiehe 1993). To avoid fouling, refiners often stop heating a residuum before coke formation begins, using arbitrary criteria. In many cases, this heating is stopped sooner than need be, resulting in less than maximum product yield. Western Research Institute (WRI) has developed innovative Coking Index concepts (patent pending) which can be used for process control by refiners to heat residua to the threshold, but not beyond the point at which coke formation begins when petroleum residua materials are heated at pyrolysis temperatures (Schabron et al. 2001). The development of this universal predictor solves a long standing problem in petroleum refining. These Coking Indexes have great potential value in improving the efficiency of distillation processes. The Coking Indexes were found to apply to residua in a universal manner, and the theoretical basis for the indexes has been established (Schabron et al. 2001a, 2001b, 2001c). For the first time, a few simple measurements indicates how close undesired coke formation is on the coke formation induction time line. The Coking Indexes can lead to new process controls that can improve refinery distillation efficiency by several percentage points. Petroleum residua consist of an ordered continuum of solvated polar materials usually referred to as asphaltenes dispersed in a lower polarity solvent phase held together by intermediate polarity materials usually referred to as

  3. Information Systems Efficiency Model

    Directory of Open Access Journals (Sweden)

    Milos Koch

    2017-07-01

    Full Text Available This contribution discusses the basic concept of creating a new model for the efficiency and effectiveness assessment of company information systems. The present trends in this field are taken into account, and the attributes are retained of measuring the optimal solutions for a company’s ICT (the implementation, functionality, service, innovations, safety, relationships, costs, etc.. The proposal of a new model of assessment comes from our experience with formerly implemented and employed methods, methods which we have modified in time and adapted to companies’ needs but also to the necessaries of our research that has been done through the ZEFIS portal. The most noteworthy of them is the HOS method that we have discussed in a number of forums. Its main feature is the fact that it respects the complexity of an information system in correlation with the balanced state of its individual parts.

  4. CONTROLLING AND BUSINESS EFFICIENCY

    Directory of Open Access Journals (Sweden)

    Tina Vuko

    2013-02-01

    Full Text Available Managing business successfully in dynamic environment requires effective controlling system. Controlling is the process of defining objectives, planning and management control so that every decision maker can act in accordance with agreed objectives. Controlling function as a separate department contributes business efficiency trough ensuring transparency of business result and business processes. Controlling takes place when manager and controller cooperate. The aim of this paper is to investigate the effectiveness of controlling function (i.e. controlling department in Croatian companies and to address the specific features of the function that contribute significantly to overall business performance. The research is conducted on the sample of companies listed on the Regulated market of the Zagreb Stock Exchange. Survey is used as a method to collect the data regarding the controlling function, while financial data necessary for the research are extracted from the published financial statements. Results of the research indicate that controlling department has positive effects on the business performance.

  5. SOURgraphs for efficient completion

    Directory of Open Access Journals (Sweden)

    Christopher Lynch

    1998-12-01

    Full Text Available We introduce a data structure called SOUR graphs and present an efficient Knuth-Bendix completion procedure based on it. SOUR graphs allow for a maximal structure sharing of terms in rewriting systems. The term representation is a dag representation, except that edges are labelled with equational constraints and variable renamings. The rewrite rules correspond to rewrite edges, the unification problems to unification edges. The Critical Pair and Simplification inferences are recognized as patterns in the graph and are performed as local graph transformations. Our algorithm avoids duplicating term structure while performing inferences, which causes exponential behavior in the standard procedure. This approach gives a basis to design other completion algorithms, such as goal-oriented completion, concurrent completion and group completion procedures.

  6. HIGH EFFICIENCY SYNGAS GENERATION

    Energy Technology Data Exchange (ETDEWEB)

    Robert J. Copeland; Yevgenia Gershanovich; Brian Windecker

    2005-02-01

    This project investigated an efficient and low cost method of auto-thermally reforming natural gas to hydrogen and carbon monoxide. Reforming is the highest cost step in producing products such as methanol and Fisher Tropsch liquids (i.e., gas to liquids); and reducing the cost of reforming is the key to reducing the cost of these products. Steam reforming is expensive because of the high cost of the high nickel alloy reforming tubes (i.e., indirectly fired reforming tubes). Conventional auto-thermal or Partial Oxidation (POX) reforming minimizes the size and cost of the reformers and provides a near optimum mixture of CO and hydrogen. However POX requires pure oxygen, which consumes power and significantly increases the cost to reforming. Our high efficiency process extracts oxygen from low-pressure air with novel oxygen sorbent and transfers the oxygen to a nickel-catalyzed reformer. The syngas is generated at process pressure (typically 20 to 40 bar) without nitrogen dilution and has a 1CO to 2H{sub 2} ratio that is near optimum for the subsequent production of Fisher-Tropsch liquid to liquids and other chemicals (i.e., Gas to Liquids, GTL). Our high process efficiency comes from the way we transfer the oxygen into the reformer. All of the components of the process, except for the oxygen sorbent, are commonly used in commercial practice. A process based on a longlived, regenerable, oxygen transfer sorbent could substantially reduce the cost of natural gas reforming to syngas. Lower cost syngas (CO + 2H{sub 2}) that is the feedstock for GTL would reduce the cost of GTL and for other commercial applications (e.g., methanol, other organic chemicals). The vast gas resources of Alaska's North Slope (ANS) offer more than 22 Tcf of gas and GTL production in this application alone, and could account for as much as 300,000 to 700,000 bpd for 20 to 30+ years. We developed a new sorbent, which is an essential part of the High Efficiency Oxygen Process (HOP). We tested

  7. Automotive fuel efficiency

    International Nuclear Information System (INIS)

    Abelson, P.H.

    1992-01-01

    For at least the remainder of this century, the United States faces a growing dependence on imported oil. Costs are substantial, and they will mount. In June 1992, net imports provided nearly 50% of supplies, and their cost was $4.3 billion. Cost of net imports of motor vehicles and parts amounted to $3.0 billion. The two items combined totaled more than the negative trade balance of $6.6 billion. The light-duty highway fleet alone accounted for 38.2% of U.S. oil consumption in 1988. Correspondingly, the fleet was a substantial emitter of air pollutants - NO x , CO, and nonmethane hydrocarbons. In addition, it was a major source of CO 2 . The twin problems of oil imports and pollution would be ameliorated if the fuel economy if cars and trucks could be improved and their emissions were also reduced. In principle, the mileage of US automobiles could be substantially improved. But on purchasing a car, U.S. buyers rank fuel efficiency eight when making their choice. They are attracted to options that lower mileage. Consumers also tend to prefer large cars over small ones for reasons of safety. Increasingly, buyers are purchasing light trucks and vans that have inferior fuel efficiency. As a result of the above trends, the average mileage of the US automotive fleet has been diminishing. As long as fuel is available at comparatively low prices and there is no federal requirement for better mileage, improvement is unlikely. Moreover, even if improvements were mandated, change would be slow

  8. Efficiency of contactors

    International Nuclear Information System (INIS)

    Orth, D.A.; Graham, F.R.; Holt, D.L.

    1986-01-01

    The Savannah River Plant has two separations plants that began Purex operations in 1954 and 1955 with pump-mix mixer-settlers as contactors to process nuclear fuels. The only changes to the extraction equipment were replacement of most of the mixer-settlers in one plant with larger units in 1959, and the further replacement of the large 1A bank with a bank of rapid-contact centrifugal contactors in 1966. Improved performance of the old units has become highly desirable, and an experimental program is underway. Good contact between the phases, and adequate settling without entrainment of the opposite phase are required for high efficiency operation of the mixer-settlers. Factors that determine efficiency are mixer design, drop size generated, and phase coalescence properties. The original development work and accumulated plant data confirm that the tip speed of a given impeller design determines the throughput capacity and extraction performance. An experimental unit with three full-scale stages has been constructed and is being utilized to test different impeller designs; reduced pumping and better mixing with lower speeds appear to be the key factors for improvement. Decontamination performance of the rapid-contact centrifugal contactors is limited by the number of scrub contacts and the time of contact because of slowly equilibrating fission product species. Where solvent degradation is not a factor, the longer scrub contact of mixer-settlers gives better decontamination than the centrifugals. This kinetic effect can be overcome with long scrub contacts that follow the initial short extraction and short scrub contacts in the centrifugal contactors. A hybrid experimental unit with both rapid contact sections and longer contact scrub sections is under development to establish the degree of improvement that might be attained

  9. Shrew trap efficiency

    DEFF Research Database (Denmark)

    Gambalemoke, Mbalitini; Mukinzi, Itoka; Amundala, Drazo

    2008-01-01

    We investigated the efficiency of four trap types (pitfall, Sherman LFA, Victor snap and Museum Special snap traps) to capture shrews. This experiment was conducted in five inter-riverine forest blocks in the region of Kisangani. The total trapping effort was 6,300, 9,240, 5,280 and 5,460 trap......-nights for the pitfall, Sherman, Victor and Museum Special traps, respectively. In total, we captured 366 shrews. The use of pitfall traps yielded the highest trapping success (4.1) with at least 18 shrew species identified. Trapping success and the number of species collected was lower for the Sherman (0.6, at least 11...... species), Victor (0.6, at least 8 species) and Museum Special (0.5, at least 6 species) traps. Although Crocidura olivieri and C. denti were caught using all four trap types, captures with different trap types did not produce a sample with the same taxonomic composition. In agreement with previous studies...

  10. Impacts of appliance efficiency standards

    Energy Technology Data Exchange (ETDEWEB)

    Hummel, P.; Kesselring, J.

    1992-12-01

    Federal efficiency standards for appliances limit the range of models available on the market. Utility energy efficiency programs often encourage consumers to purchase the most efficient of the remaining options, but more-stringent efficiency standards could have a significant impact on such utility efforts. Using long-run technology scenario forecasting, EPRI-sponsored researchers studied ways in which the interplay of technological change, federal standards, utility efficiency programs, and market forces could affect future energy markets for three major residential end uses: refrigeration, central air conditioning, and water heating. The researchers reached several important conclusions about utility demand-side management efforts. 2 figs., 1 tab.

  11. Efficiency Evaluation of Energy Systems

    CERN Document Server

    Kanoğlu, Mehmet; Dinçer, İbrahim

    2012-01-01

    Efficiency is one of the most frequently used terms in thermodynamics, and it indicates how well an energy conversion or process is accomplished. Efficiency is also one of the most frequently misused terms in thermodynamics and is often a source of misunderstanding. This is because efficiency is often used without being properly defined first. This book intends to provide a comprehensive evaluation of various efficiencies used for energy transfer and conversion systems including steady-flow energy devices (turbines, compressors, pumps, nozzles, heat exchangers, etc.), various power plants, cogeneration plants, and refrigeration systems. The book will cover first-law (energy based) and second-law (exergy based) efficiencies and provide a comprehensive understanding of their implications. It will help minimize the widespread misuse of efficiencies among students and researchers in energy field by using an intuitive and unified approach for defining efficiencies. The book will be particularly useful for a clear ...

  12. Encapsulation Efficiency, Oscillatory Rheometry

    Directory of Open Access Journals (Sweden)

    Z. Mohammad Hassani

    2014-01-01

    Full Text Available Nanoliposomes are one of the most important polar lipid-based nanocarriers which can be used for encapsulation of both hydrophilic and hydrophobic active compounds. In this research, nanoliposomes based on lecithin-polyethylene glycol-gamma oryzanol were prepared by using a modified thermal method. Only one melting peak in DSC curve of gamma oryzanol bearing liposomes was observed which could be attributed to co-crystallization of both compounds. The addition of gamma oryzanol, caused to reduce the melting point of 5% (w/v lecithin-based liposome from 207°C to 163.2°C. At high level of lecithin, increasing of liposome particle size (storage at 4°C for two months was more obvious and particle size increased from 61 and 113 to 283 and 384 nanometers, respectively. The encapsulation efficiency of gamma oryzanol increased from 60% to 84.3% with increasing lecithin content. The encapsulation stability of oryzanol in liposome was determined at different concentrations of lecithin 3, 5, 10, 20% (w/v and different storage times (1, 7, 30 and 60 days. In all concentrations, the encapsulation stability slightly decreased during 30 days storage. The scanning electron microscopy (SEM images showed relatively spherical to elliptic particles which indicated to low extent of particles coalescence. The oscillatory rheometry showed that the loss modulus of liposomes were higher than storage modulus and more liquid-like behavior than solid-like behavior. The samples storage at 25°C for one month, showed higher viscoelastic parameters than those having been stored at 4°C which were attributed to higher membrane fluidity at 25°C and their final coalescence.Nanoliposomes are one of the most important polar lipid based nanocarriers which can be used for encapsulation of both hydrophilic and hydrophobic active compounds. In this research, nanoliposomes based on lecithin-polyethylene glycol-gamma oryzanol were prepared by using modified thermal method. Only one

  13. Efficient reactive Brownian dynamics

    Science.gov (United States)

    Donev, Aleksandar; Yang, Chiao-Yu; Kim, Changho

    2018-01-01

    We develop a Split Reactive Brownian Dynamics (SRBD) algorithm for particle simulations of reaction-diffusion systems based on the Doi or volume reactivity model, in which pairs of particles react with a specified Poisson rate if they are closer than a chosen reactive distance. In our Doi model, we ensure that the microscopic reaction rules for various association and dissociation reactions are consistent with detailed balance (time reversibility) at thermodynamic equilibrium. The SRBD algorithm uses Strang splitting in time to separate reaction and diffusion and solves both the diffusion-only and reaction-only subproblems exactly, even at high packing densities. To efficiently process reactions without uncontrolled approximations, SRBD employs an event-driven algorithm that processes reactions in a time-ordered sequence over the duration of the time step. A grid of cells with size larger than all of the reactive distances is used to schedule and process the reactions, but unlike traditional grid-based methods such as reaction-diffusion master equation algorithms, the results of SRBD are statistically independent of the size of the grid used to accelerate the processing of reactions. We use the SRBD algorithm to compute the effective macroscopic reaction rate for both reaction-limited and diffusion-limited irreversible association in three dimensions and compare to existing theoretical predictions at low and moderate densities. We also study long-time tails in the time correlation functions for reversible association at thermodynamic equilibrium and compare to recent theoretical predictions. Finally, we compare different particle and continuum methods on a model exhibiting a Turing-like instability and pattern formation. Our studies reinforce the common finding that microscopic mechanisms and correlations matter for diffusion-limited systems, making continuum and even mesoscopic modeling of such systems difficult or impossible. We also find that for models in which

  14. Identification of variables contributing to superovulation efficiency for production of transgenic prairie voles (Microtus ochrogaster

    Directory of Open Access Journals (Sweden)

    Keebaugh Alaine C

    2012-07-01

    Full Text Available Abstract Background The prairie vole (Microtus ochrogaster is an emerging animal model for biomedical research because of its rich sociobehavioral repertoire. Recently, lentiviral transgenic technology has been used to introduce the gene encoding the green fluorescent protein (GFP into the prairie vole germline. However, the efficiency of transgenesis in this species is limited by the inability to reliably produce large numbers of fertilized embryos. Here we examined several factors that may contribute to variability in superovulation success including, age and parentage of the female, and latency to mating after being placed with the male. Methods Females produced from 5 genetically distinct breeder lines were treated with 100 IU of pregnant mare serum gonadotrophin (PMSG and immediately housed with a male separated by a perforated Plexiglas divider. Ovulation was induced 72 hr later with 30 IU of human chorionic gonadotropin (hCG and 2 hrs later mating was allowed. Results Superovulation was most efficient in young females. For example, females aged 6-11 weeks produced more embryos (14 +/- 1.4 embryos as compared to females aged 12-20 weeks (4 +/- 1.6 embryos. Females aged 4-5 weeks did not produce embryos. Further, females that mated within 15 min of male exposure produced significantly more embryos than those that did not. Interestingly, there was a significant effect of parentage. For example, 12 out of 12 females from one breeder pair superovulated (defined as producing 5 or more embryos, while only 2 out of 10 females for other lines superovulated. Conclusions The results of this work suggest that age and genetic background of the female are the most important factors contributing to superovulation success and that latency to mating is a good predictor of the number of embryos to be recovered. Surprisingly we found that cohabitation with the male prior to mating is not necessary for the recovery of embryos but is necessary to recover

  15. USE Efficiency -- Universities and Students for Energy Efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Melandri, Daniela

    2010-09-15

    Universities and Student for Energy Efficiency is a European Project within the Intelligent Energy Programme. It intends to create a common stream for energy efficiency systems in university buildings. Universities and students are proposed as shining examples for energy efficiency solutions and behaviour. The Project involves 10 countries and has the aim to improve energy efficiency in university buildings. Students are the main actors of the project together with professors and technicians. To act on students means to act on direct future market players in diffusion of public opinions. A strong communication action supports the succeeding of the action.

  16. Improving Gas Flooding Efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Reid Grigg; Robert Svec; Zheng Zeng; Alexander Mikhalin; Yi Lin; Guoqiang Yin; Solomon Ampir; Rashid Kassim

    2008-03-31

    This study focuses on laboratory studies with related analytical and numerical models, as well as work with operators for field tests to enhance our understanding of and capabilities for more efficient enhanced oil recovery (EOR). Much of the work has been performed at reservoir conditions. This includes a bubble chamber and several core flood apparatus developed or modified to measure interfacial tension (IFT), critical micelle concentration (CMC), foam durability, surfactant sorption at reservoir conditions, and pressure and temperature effects on foam systems.Carbon dioxide and N{sub 2} systems have been considered, under both miscible and immiscible conditions. The injection of CO2 into brine-saturated sandstone and carbonate core results in brine saturation reduction in the range of 62 to 82% brine in the tests presented in this paper. In each test, over 90% of the reduction occurred with less than 0.5 PV of CO{sub 2} injected, with very little additional brine production after 0.5 PV of CO{sub 2} injected. Adsorption of all considered surfactant is a significant problem. Most of the effect is reversible, but the amount required for foaming is large in terms of volume and cost for all considered surfactants. Some foams increase resistance to the value beyond what is practical in the reservoir. Sandstone, limestone, and dolomite core samples were tested. Dissolution of reservoir rock and/or cement, especially carbonates, under acid conditions of CO2 injection is a potential problem in CO2 injection into geological formations. Another potential change in reservoir injectivity and productivity will be the precipitation of dissolved carbonates as the brine flows and pressure decreases. The results of this report provide methods for determining surfactant sorption and can be used to aid in the determination of surfactant requirements for reservoir use in a CO{sub 2}-foam flood for mobility control. It also provides data to be used to determine rock permeability

  17. Efficient Metropolitan Resource Allocation

    Directory of Open Access Journals (Sweden)

    Richard Arnott

    2016-05-01

    Full Text Available Over the past 30 years Calgary has doubled in size, from a population of 640,645 in 1985 to 1,230,915 in 2015. During that time the City has had five different mayors, hosted the Winter Olympics, and expanded the C-Train from 25 platforms to 45. Calgary’s Metropolitan Area has grown too, with Airdrie, Chestermere, Okotoks and Cochrane growing into full-fledged cities, ripe with inter-urban commuters.* And with changes to provincial legislation in the mid-’90s, rural Rocky View County and the Municipal District of Foothills are now real competitors for residential, commercial and industrial development that in the past would have been considered urban. In this metropolitan system, where people live, their household structure, and their place of work informs the services they need to conduct their daily lives, and directly impacts the spatial character of the City and the broader region. In sum, Metropolitan Calgary is increasingly complex. Calgary and the broader metropolitan area will continue to grow, even with the current economic slowdown. Frictions within Calgary, between the various municipalities in the metropolitan area, and the priorities of other local authorities (such as the School Boards and Alberta Health Services will continue to impact the agendas of local politicians and their ability to answer to the needs of their residents. How resources – whether it is hard infrastructure, affordable housing, classrooms, or hospital beds – are allocated over space and how these resources are funded, directly impacts these relationships. This technical paper provides my perspective as an urban economist on the efficient allocation of resources within a metropolitan system in general, with reference to Calgary where appropriate, and serves as a companion to the previously released “Reflections on Calgary’s Spatial Structure: An Urban Economists Critique of Municipal Planning in Calgary.” It is hoped that the concepts reviewed

  18. Socially-Efficient Tax Reforms

    OpenAIRE

    Duclos, Jean-Yves; Makdissi, Paul; Wodon, Quentin

    2002-01-01

    We propose graphical methods to determine whether commodity-tax changes are "socially efficient", in the sense of improving social welfare or decreasing poverty for large classes of social welfare and poverty indices. We also derive estimators of critical poverty lines and economic efficiency ratios which can be used to characterize socially-efficient tax reforms. The statistical properties of the various estimators are derived in order to make the method implementable using survey data. The ...

  19. Energy efficiency policies and measures

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-07-01

    This document makes a review of the energy efficiency and demand side management (DSM) policies and measures in European Union countries and Norway in 1999: institutional changes, measures and programmes, budget, taxation, existence of a national DSM programme, national budgets for DSM programmes, electricity pricing: energy/environment tax, national efficiency standards and regulation for new electrical appliances, implementation of Commission directives, efficiency requirements, labelling, fiscal and economic incentives. (J.S.)

  20. Stochastic efficiency: five case studies

    International Nuclear Information System (INIS)

    Proesmans, Karel; Broeck, Christian Van den

    2015-01-01

    Stochastic efficiency is evaluated in five case studies: driven Brownian motion, effusion with a thermo-chemical and thermo-velocity gradient, a quantum dot and a model for information to work conversion. The salient features of stochastic efficiency, including the maximum of the large deviation function at the reversible efficiency, are reproduced. The approach to and extrapolation into the asymptotic time regime are documented. (paper)

  1. Efficiency, sustainability and global warming

    International Nuclear Information System (INIS)

    Woodward, Richard T.; Bishop, Richard C.

    1995-01-01

    Economic analyses of global warming have typically been grounded in the theory of economic efficiency. Such analyses may be inappropriate because many of the underlying concerns about climate change are rooted not in efficiency, but in the intergenerational allocation of economic endowments. A simple economic model is developed which demonstrates that an efficient economy is not necessarily a sustainable economy. This result leads directly to questions about the policy relevance of several economic studies of the issue. We then consider policy alternatives to address global warming in the context of economies with the dual objectives of efficiency and sustainability, with particular attention to carbon-based taxes

  2. From energy efficiency towards resource efficiency within the Ecodesign Directive

    DEFF Research Database (Denmark)

    Bundgaard, Anja Marie; Mosgaard, Mette; Remmen, Arne

    2017-01-01

    The article examines the integration of resource efficiency into the European Ecodesign Directive. The purpose is to analyse the processes and stakeholder interactions, which formed the basis for integrating resource efficiency requirements into the implementing measure for vacuum cleaners and th...

  3. Retargeting Polyomavirus-Like Particles to Cancer Cells by Chemical Modification of Capsid Surface

    Czech Academy of Sciences Publication Activity Database

    Žáčková Suchanová, J.; Neburková, Jitka; Španielová, Hana; Forstová, J.; Cígler, Petr

    2017-01-01

    Roč. 28, č. 2 (2017), s. 307-313 ISSN 1043-1802 Institutional support: RVO:61388963 Keywords : transferrin * sialic acid * targeting Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.818, year: 2016

  4. Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas

    NARCIS (Netherlands)

    Helfrich, Wijnand; Kroesen, Bart-Jan; Roovers, R.C; Westers, L; Molema, Ingrid; Hoogenboom, H.R; de Leij, Lou

    1998-01-01

    We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO 17-1A antigen or KSA) and the CD3 epsilon chain of human

  5. Retargeting transposon insertions by the adeno-associated virus Rep protein

    Science.gov (United States)

    Ammar, Ismahen; Gogol-Döring, Andreas; Miskey, Csaba; Chen, Wei; Cathomen, Toni; Izsvák, Zsuzsanna; Ivics, Zoltán

    2012-01-01

    The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein–protein and protein–DNA interactions. PMID:22523082

  6. On-Line Trajectory Retargeting for Alternate Landing Sites, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Barron Associates, Inc. proposes to develop a novel on-line trajectory optimization approach for Reusable Launch Vehicles (RLVs) under failure scenarios, targeting...

  7. Virotherapy of ovarian cancer with polymer-cloaked adenovirus retargeted to the epidermal growth factor receptor

    Czech Academy of Sciences Publication Activity Database

    Morrison, J.; Briggs, S. S.; Green, N.; Fisher, K.; Šubr, Vladimír; Ulbrich, Karel; Kehoe, S.; Seymour, L. W.

    2008-01-01

    Roč. 16, č. 2 (2008), s. 244-251 ISSN 1525-0016 EU Projects: European Commission(XE) 512087 - GIANT Institutional research plan: CEZ:AV0Z40500505 Keywords : adenovirus * gene delivery * N-(2-hydroxypropyl)methacrylamide Subject RIV: CD - Macromolecular Chemistry Impact factor: 5.970, year: 2008

  8. Producing energy efficiently; Energieeffizient produzieren

    Energy Technology Data Exchange (ETDEWEB)

    Blass, H.W.; Schumacher, M.; Coy, C; Pfister, J.; Finkbeiner, M.; Kuehnle, J.; Kiel, E.; Duerrschmidt, R.; Diede, J.; Scheffels, G.; Kranz, H.; Simon, J.; Diebold, K.; Koch, H.R.; Lehnhardt, M.

    2008-04-07

    Producing energy efficiently means saving much energy and money. Drives are responsible for the accounting of power for companies. The fair shows examples on the complete field of producing sector, hits the advantages of heat recovery and an efficient cooling. (GL)

  9. Market Competition and Efficient Cooperation

    NARCIS (Netherlands)

    Brandts, J.; Riedl, Arno

    2016-01-01

    We find that in market-partners, market experience has adverse effects on the efficiency of cooperation on both market-winner and market-loser pairs. In market-strangers, pairs of market-winners manage to cooperate more efficiently. These results indicate that it is not market experience per se that

  10. Motor-operated gearbox efficiency

    Energy Technology Data Exchange (ETDEWEB)

    DeWall, K.G.; Watkins, J.C.; Bramwell, D. [Idaho National Engineering Lab., Idaho Falls, ID (United States); Weidenhamer, G.H.

    1996-12-01

    Researchers at the Idaho National Engineering Laboratory recently conducted tests investigating the operating efficiency of the power train (gearbox) in motor-operators typically used in nuclear power plants to power motor-operated valves. Actual efficiency ratios were determined from in-line measurements of electric motor torque (input to the operator gearbox) and valve stem torque (output from the gearbox) while the operators were subjected to gradually increasing loads until the electric motor stalled. The testing included parametric studies under reduced voltage and elevated temperature conditions. As part of the analysis of the results, the authors compared efficiency values determined from testing to the values published by the operator manufacturer and typically used by the industry in calculations for estimating motor-operator capabilities. The operators they tested under load ran at efficiencies lower than the running efficiency (typically 50%) published by the operator manufacturer.

  11. Motor-operator gearbox efficiency

    International Nuclear Information System (INIS)

    DeWall, K.G.; Watkins, J.C.; Bramwell, D.

    1996-01-01

    Researchers at the Idaho National Engineering Laboratory recently conducted tests investigating the operating efficiency of the power train (gearbox) in motor-operators typically used in nuclear power plants to power motor-operated valves. Actual efficiency ratios were determined from in-line measurements of electric motor torque (input to the operator gearbox) and valve stem torque (output from the gearbox) while the operators were subjected to gradually increasing loads until the electric motor stalled. The testing included parametric studies under reduced voltage and elevated temperature conditions. As part of the analysis of the results, we compared efficiency values determined from testing to the values published by the operator manufacturer and typically used by the industry in calculations for estimating motor-operator capabilities. The operators we tested under load ran at efficiencies lower than the running efficiency (typically 50%) published by the operator manufacturer

  12. Gold, currencies and market efficiency

    Science.gov (United States)

    Kristoufek, Ladislav; Vosvrda, Miloslav

    2016-05-01

    Gold and currency markets form a unique pair with specific interactions and dynamics. We focus on the efficiency ranking of gold markets with respect to the currency of purchase. By utilizing the Efficiency Index (EI) based on fractal dimension, approximate entropy and long-term memory on a wide portfolio of 142 gold price series for different currencies, we construct the efficiency ranking based on the extended EI methodology we provide. Rather unexpected results are uncovered as the gold prices in major currencies lay among the least efficient ones whereas very minor currencies are among the most efficient ones. We argue that such counterintuitive results can be partly attributed to a unique period of examination (2011-2014) characteristic by quantitative easing and rather unorthodox monetary policies together with the investigated illegal collusion of major foreign exchange market participants, as well as some other factors discussed in some detail.

  13. Motor-operated gearbox efficiency

    International Nuclear Information System (INIS)

    DeWall, K.G.; Watkins, J.C.; Bramwell, D.; Weidenhamer, G.H.

    1996-01-01

    Researchers at the Idaho National Engineering Laboratory recently conducted tests investigating the operating efficiency of the power train (gearbox) in motor-operators typically used in nuclear power plants to power motor-operated valves. Actual efficiency ratios were determined from in-line measurements of electric motor torque (input to the operator gearbox) and valve stem torque (output from the gearbox) while the operators were subjected to gradually increasing loads until the electric motor stalled. The testing included parametric studies under reduced voltage and elevated temperature conditions. As part of the analysis of the results, the authors compared efficiency values determined from testing to the values published by the operator manufacturer and typically used by the industry in calculations for estimating motor-operator capabilities. The operators they tested under load ran at efficiencies lower than the running efficiency (typically 50%) published by the operator manufacturer

  14. Material Efficiency of Building Construction

    Directory of Open Access Journals (Sweden)

    Antti Ruuska

    2014-07-01

    Full Text Available Better construction and use of buildings in the European Union would influence 42% of final energy consumption, about 35% of our greenhouse gas emissions and more than 50% of all extracted materials. It could also help to save up to 30% of water consumption. This paper outlines and draws conclusions about different aspects of the material efficiency of buildings and assesses the significance of different building materials on the material efficiency. The research uses an extensive literature study and a case-study in order to assess: should the depletion of materials be ignored in the environmental or sustainability assessment of buildings, are the related effects on land use, energy use and/or harmful emissions significant, should related indicators (such as GHGs be used to indicate the material efficiency of buildings, and what is the significance of scarce materials, compared to the use of other building materials. This research suggests that the material efficiency should focus on the significant global impacts of material efficiency; not on the individual factors of it. At present global warming and greenhouse gas emissions are among the biggest global problems on which material efficiency has a direct impact on. Therefore, this paper suggests that greenhouse gas emissions could be used as an indicator for material efficiency in building.

  15. Polish Foundation for Energy Efficiency

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The Polish Foundation for Energy Efficiency (FEWE) was established in Poland at the end of 1990. FEWE, as an independent and non-profit organization, has the following objectives: to strive towards an energy efficient national economy, and to show the way and methods by use of which energy efficiency can be increased. The activity of the Foundation covers the entire territory of Poland through three regional centers: in Warsaw, Katowice and Cracow. FEWE employs well-known and experienced specialists within thermal and power engineering, civil engineering, economy and applied sciences. The organizer of the Foundation has been Battelle Memorial Institute - Pacific Northwest Laboratories from the USA.

  16. The Efficiency of Educational Production

    DEFF Research Database (Denmark)

    Bogetoft, Peter; Heinesen, Eskil; Tranæs, Torben

    and maintain the same graduation/completion rates as today if the country could achieve the same level of cost effectiveness as its most efficient counterparts. Comparing Denmark to a group of the richest OECD countries reveals that potential savings lie between 12 and 34 percent. Figures that fall to between...... to explain the higher costs of upper secondary education. On the output side, if earnings and levels of employment among those who complete their education are taken into account, then Denmark is in fact found to be efficient. However, this high level of efficiency has become less clear-cut in recent years...

  17. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    Science.gov (United States)

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Is energy efficiency environmentally friendly?

    Energy Technology Data Exchange (ETDEWEB)

    Herring, H. [Open University, Milton Keynes (United Kingdom). Energy and Environment Research Unit

    2000-07-01

    The paper challenges the view that improving the efficiency of energy use will lead to a reduction in national energy consumption, and hence is an effective policy for reducing CO{sub 2} emissions. It argues that improving energy efficiency lowers the implicit price of energy and hence makes its use more affordable, thus leading to greater use. The paper presents the views of economists, as well as green critics of 'efficiency' and the 'dematerialization' thesis. It argues that a more effective CO{sub 2} policy is to concentrate on shifting to non-fossil fuel, like renewables, subsidized through a carbon tax. Ultimately what is needed, to limit energy consumption is energy conservation not energy efficiency. 44 refs.

  19. EFFICIENCY OF POMEGRANATE SEED OIL

    Directory of Open Access Journals (Sweden)

    Sukru Karatas

    2014-01-01

    Full Text Available In this paper pomegranate kinds that reach to harvest maturity which are produced in commercial scales in Adana, Antalya, Finike and Kilis regions in Turkey and also Hicaz and Eksilik kinds are chosen randomly to be investigated. Seed efficiencies, grain ratios, oil efficiency of these chosen pomegranate kinds were studied. According to this study, pomegranate kind taken from Kilis region is found to have the highest grain efficieny with a ratio of 71% and pomegranate kind taken from Hicaz region is found to have the highest seed oil with a ratio of 18%. Also various solvent efficiencies including Hexan, Diethylether and petroleum ether on pomegranate seed oil is examined and most efficient one is obtained with Diethylether.

  20. The Efficiency of Educational Production

    DEFF Research Database (Denmark)

    Bogetoft, Peter; Heinesen, Eskil; Tranæs, Torben

    2015-01-01

    of graduation/completion rates and expected earnings after completed education. We use data envelopment analysis (DEA) to compare (benchmark) the Nordic countries with a relevant group of rich OECD countries and calculate input efficiency scores for each country. We estimate a wide range of specifications...... in order to analyse different aspects of efficiency. In purely quantitative models (where inputs and outputs are expenditure and number of students at different levels of the educational system) and in models where graduation or completion rates are included as indicators of output quality, Finland...... is the most efficient Nordic country (often fully efficient), whereas Sweden and especially Norway and Denmark are clearly inefficient. However, using PISA test scores as indicators of student input quality in upper secondary education reduces the inefficiencies of these three countries. Also, when expected...

  1. Efficiency principles of consulting entrepreneurship

    OpenAIRE

    Moroz Yustina S.; Drozdov Igor N.

    2015-01-01

    The article reviews the primary goals and problems of consulting entrepreneurship. The principles defining efficiency of entrepreneurship in the field of consulting are generalized. The special attention is given to the importance of ethical principles of conducting consulting entrepreneurship activity.

  2. Planning for resource efficient cities

    DEFF Research Database (Denmark)

    Fertner, Christian; Groth, Niels Boje

    2016-01-01

    development from energy consumption are crucial for a city’s future vulnerability and resilience against changes in general resource availability. The challenge gets further complex, as resource and energy efficiency in a city is deeply interwoven with other aspects of urban development such as social...... structures and the geographical context. As cities are the main consumer of energy and resources, they are both problem and solution to tackle issues of energy efficiency and saving. Cities have been committed to this agenda, especially to meet the national and international energy targets. Increasingly......, cities act as entrepreneurs of new energy solutions acknowledging that efficient monitoring of energy and climate policies has become important to urban branding and competitiveness. This special issue presents findings from the European FP7 project ‘Planning for Energy Efficient Cities’ (PLEEC...

  3. Energy efficiency: utopia or reality?

    International Nuclear Information System (INIS)

    Anon.

    2006-01-01

    In its 2006 allocution the world council on the energy WEC, analyzes the role of the energy efficiency in the energy life cycle. In spite of different objectives followed by the developing and developed countries, implement a world energy efficiency economy is a challenge possible by the cooperation.The WEC is an ideal forum for the information and experience exchange. (A.L.B.)

  4. Efficient computation of argumentation semantics

    CERN Document Server

    Liao, Beishui

    2013-01-01

    Efficient Computation of Argumentation Semantics addresses argumentation semantics and systems, introducing readers to cutting-edge decomposition methods that drive increasingly efficient logic computation in AI and intelligent systems. Such complex and distributed systems are increasingly used in the automation and transportation systems field, and particularly autonomous systems, as well as more generic intelligent computation research. The Series in Intelligent Systems publishes titles that cover state-of-the-art knowledge and the latest advances in research and development in intelligen

  5. Energy end-use efficiency

    OpenAIRE

    Jochem, E.

    2000-01-01

    Since the 1970s more efficient energy use in OECD countries has weakened or eliminated the link between economic growth and energy use. At the global level just 37 percent of primary energy is converted to useful energy - meaning that nearly two-thirds is lost. the next 20 years percent in most industrialised countries and more than 40 percent transition economies. Dematerialization and recycling will further reduce energy intensity. Thus energy efficiency is one of the main technological dri...

  6. Energy management and energy efficiency

    International Nuclear Information System (INIS)

    Anon.

    2013-01-01

    This bibliographical note presents an official publication which contains the latest standards and standard projects dealing with energy audits (general requirements, buildings, process, transport) as well as energy efficiency (definitions and requirements, benchmarking methodology) and energy management and its terminology in relationship with the new European directive 2012/27/UE on energy efficiency. Regulatory texts associated with the national and European energy policies are also proposed

  7. Energy Efficient Hydraulic Hybrid Drives

    OpenAIRE

    Rydberg, Karl-Erik

    2009-01-01

    Energy efficiency of propulsion systems for cars, trucks and construction machineries has become one of the most important topics in today’s mobile system design, mainly because of increased fuel costs and new regulations about engine emissions, which is needed to save the environment. To meet the increased requirements on higher efficiency and better functionality, components and systems have been developed over the years. For the last ten years the development of hybrid systems can be divid...

  8. Energy Efficient Drivepower: An Overview.

    Energy Technology Data Exchange (ETDEWEB)

    Ula, Sadrul; Birnbaum, Larry E.; Jordan, Don

    1993-05-01

    This report examines energy efficiency in drivepower systems. Only systems where the prime movers are electrical motors are discussed. A systems approach is used to examine all major aspects of drivepower, including motors, controls, electrical tune-ups, mechanical efficiency, maintenance, and management. Potential annual savings to the US society of $25 to $50 billion are indicated. The report was written for readers with a semi-technical background.

  9. Advanced high efficiency concentrator cells

    Energy Technology Data Exchange (ETDEWEB)

    Gale, R. (Varian Associates, Inc., Palo Alto, CA (United States). Varian Research Center)

    1992-06-01

    This report describes research to develop the technology needed to demonstrate a monolithic, multijunction, two-terminal, concentrator solar cell with a terrestrial power conversion efficiency greater than 35%. Under three previous subcontracts, Varian developed many of the aspects of a technology needed to fabricate very high efficiency concentrator cells. The current project was aimed at exploiting the new understanding of high efficiency solar cells. Key results covered in this report are as follows. (1) A 1.93-eV AlGaAs/1.42-eV GaAs metal-interconnected cascade cell was manufactured with a one-sun efficiency at 27.6% at air mass 1.5 (AM1.5) global. (2) A 1.0eV InGaAs cell was fabricated on the reverse'' side of a low-doped GaAs substrate with a one-sun efficiency of 2.5% AM1.5 diffuse and a short-circuit current of 14.4 mA/cm{sup 2}. (3) Small-scale manufacturing of GaAs p/n concentrator cells was attempted and obtained an excellent yield of high-efficiency cells. (4) Grown-in tunnel junction cell interconnects that are transparent and thermally stable using C and Si dopants were developed. 10 refs.

  10. Transformer Efficiency Assessment - Okinawa, Japan

    Energy Technology Data Exchange (ETDEWEB)

    Thomas L. Baldwin; Robert J. Turk; Kurt S. Myers; Jake P. Gentle; Jason W. Bush

    2012-05-01

    The US Army Engineering & Support Center, Huntsville (USAESCH), and the US Marine Corps Base (MCB), Okinawa, Japan retained Idaho National Laboratory (INL) to conduct a Transformer Efficiency Assessment of “key” transformers located at multiple military bases in Okinawa, Japan. The purpose of this assessment is to support the Marine Corps Base, Okinawa in evaluating medium voltage distribution transformers for potential efficiency upgrades. The original scope of work included the MCB providing actual transformer nameplate data, manufacturer’s factory test sheets, electrical system data (kWh), demand data (kWd), power factor data, and electricity cost data. Unfortunately, the MCB’s actual data is not available and therefore making it necessary to de-scope the original assessment. Note: Any similar nameplate data, photos of similar transformer nameplates, and basic electrical details from one-line drawings (provided by MCB) are not a replacement for actual load loss test data. It is recommended that load measurements are performed on the high and low sides of transformers to better quantify actual load losses, demand data, and power factor data. We also recommend that actual data, when available, be inserted by MCB Okinawa where assumptions have been made and then the LCC analysis updated. This report covers a generalized assessment of modern U.S. transformers in a three level efficiency category, Low-Level efficiency, Medium-Level efficiency, and High-Level efficiency.

  11. Transformer Efficiency Assessment - Okinawa, Japan

    Energy Technology Data Exchange (ETDEWEB)

    Thomas L. Baldwin; Robert J. Turk; Kurt S. Myers; Jake P. Gentle; Jason W. Bush

    2012-08-01

    The US Army Engineering & Support Center, Huntsville (USAESCH), and the US Marine Corps Base (MCB), Okinawa, Japan retained Idaho National Laboratory (INL) to conduct a Transformer Efficiency Assessment of “key” transformers located at multiple military bases in Okinawa, Japan. The purpose of this assessment is to support the Marine Corps Base, Okinawa in evaluating medium voltage distribution transformers for potential efficiency upgrades. The original scope of work included the MCB providing actual transformer nameplate data, manufacturer’s factory test sheets, electrical system data (kWh), demand data (kWd), power factor data, and electricity cost data. Unfortunately, the MCB’s actual data is not available and therefore making it necessary to de-scope the original assessment. Note: Any similar nameplate data, photos of similar transformer nameplates, and basic electrical details from one-line drawings (provided by MCB) are not a replacement for actual load loss test data. It is recommended that load measurements are performed on the high and low sides of transformers to better quantify actual load losses, demand data, and power factor data. We also recommend that actual data, when available, be inserted by MCB Okinawa where assumptions have been made and then the LCC analysis updated. This report covers a generalized assessment of modern U.S. transformers in a three level efficiency category, Low-Level efficiency, Medium-Level efficiency, and High-Level efficiency.

  12. Benefits for whom? Energy efficiency within the efficient market

    International Nuclear Information System (INIS)

    Chello, Dario

    2015-01-01

    How should the lack of an efficient energy market affect the design of energy efficiency policies and their implementation? What the consequences of an inefficient energy market on end users’ behaviour? This article tries to give an answer to such questions, by considering the decision making of domestic users following a few fundamental concepts of behavioural economics. The mechanism of price formation in the market, with particular reference to the internal energy market in Europe, will be examined and we will show that price remains the inflexible attribute in making an energy choice. Then, some conclusions will be addressed to policy makers on how to overcome the barriers illustrated.

  13. Humid free efficient solar panel

    Science.gov (United States)

    Panjwani, Manoj Kumar; Panjwani, Suresh Kumar; Mangi, Fareed Hussain; Khan, Danish; Meicheng, Li

    2017-09-01

    The paper examines the impact of the humidity on the Solar panels which makes a space for the drastic variation in the power generated and makes the device less efficient. Humidity readily affects the efficiency of the solar cells and creates a minimal layer of water on its surface. It also decreases the efficiency by 10-20% of the total power output produced. Moreover, to handle this issue, all around characterized measures are required to be taken to guarantee the smooth working of the solar panels utilized in humid areas. In connection with this issue, Karachi, the biggest city of Pakistan which is located near the costal line touching Arabian Sea, was taken as a reference city to measure the humidity range. In Karachi, the average humidity lies between 25-70% (as per Pakistan Meteorological Department PMD), that indirectly leads in decreasing power acquired from a Solar Panel and develops various complexities for the solar system. The system on average experiences stability issues, such as those of power fluctuations etc., due to which, the whole solar system installed observes abnormal variations in acquired power. Silica Gel was used as a desiccant material in order to assure dryness over the solar panel. More than four experiments were conducted with the usage of water absorbent to improve the efficiency and to make system more power efficient.

  14. Improving efficiency in meat production.

    Science.gov (United States)

    Brameld, John M; Parr, Tim

    2016-08-01

    Selective breeding and improved nutritional management over the past 20-30 years has resulted in dramatic improvements in growth efficiency for pigs and poultry, particularly lean tissue growth. However, this has been achieved using high-quality feed ingredients, such as wheat and soya that are also used for human consumption and more recently biofuels production. Ruminants on the other hand are less efficient, but are normally fed poorer quality ingredients that cannot be digested by human subjects, such as grass or silage. The challenges therefore are to: (i) maintain the current efficiency of growth of pigs and poultry, but using more ingredients not needed to feed the increasing human population or for the production of biofuels; (ii) improve the efficiency of growth in ruminants; (iii) at the same time produce animal products (meat, milk and eggs) of equal or improved quality. This review will describe the use of: (a) enzyme additives for animal feeds, to improve feed digestibility; (b) known growth promoting agents, such as growth hormone, β-agonists and anabolic steroids, currently banned in the European Union but used in other parts of the world; (c) recent transcriptomic studies into molecular mechanisms for improved growth efficiency via low residual feed intake. In doing so, the use of genetic manipulation in animals will also be discussed.

  15. Mobilising Investment in Energy Efficiency

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2012-07-01

    Taxes, loans and grants, trading schemes and white certificates, public procurement and investment in R&D or infrastructure: known collectively as 'economic instruments', these tools can be powerful means of mobilising the finances needed to achieve policy goals by implementing energy efficiency measures. The role of economic instruments is to kick-start the private financial markets and to motivate private investors to fund EE measures. They should reinforce and promote energy performance regulations. This IEA analysis addresses the fact that, to date, relatively little effort has been directed toward evaluating how well economic instruments work. Using the buildings sector to illustrate how such measures can support energy efficiency, this paper can help policy makers better select and design economic instruments appropriate to their policy objectives and national contexts. This report’s three main aims are to: 1) Examine how economic instruments are currently used in energy efficiency policy; 2) Consider how economic instruments can be more effective and efficient in supporting low-energy buildings; and 3) Assess how economic instruments should be funded, where public outlay is needed. Detailed case studies in this report assess examples of economic instruments for energy efficiency in the buildings sector in Canada (grants), France (tax relief and loans), Germany (loans and grants), Ireland (grants) and Italy (white certificates and tax relief).

  16. From energy efficiency towards resource efficiency within the Ecodesign Directive

    DEFF Research Database (Denmark)

    Bundgaard, Anja Marie; Mosgaard, Mette; Remmen, Arne

    2017-01-01

    and dishwashers. The role of DG Environment in the Ecodesign Directive could be strengthen in order to include broader environ- mental aspects and drive the resource efficiency and circular economy agenda. A final recommendation is to continue to expand the work on developing standards defining test methods...

  17. Cleanroom Energy Efficiency Workshop Proceedings

    Energy Technology Data Exchange (ETDEWEB)

    Tschudi, Bill

    1999-03-15

    On March 15, 1999, Lawrence Berkeley National Laboratory hosted a workshop focused on energy efficiency in Cleanroom facilities. The workshop was held as part of a multiyear effort sponsored by the California Institute for Energy Efficiency, and the California Energy Commission. It is part of a project that concentrates on improving energy efficiency in Laboratory type facilities including cleanrooms. The project targets the broad market of laboratory and cleanroom facilities, and thus cross-cuts many different industries and institutions. This workshop was intended to raise awareness by sharing case study success stories, providing a forum for industry networking on energy issues, contributing LBNL expertise in research to date, determining barriers to implementation and possible solutions, and soliciting input for further research.

  18. Green corridor : energy efficiency initiatives

    Energy Technology Data Exchange (ETDEWEB)

    Bartlett, M.; Strickland, R.; Harding, N. [Windsor Univ., ON (Canada)

    2005-07-01

    This presentation discussed environmental sustainability using alternative energy technologies. It discussed Ecohouse, which is a house designed using conventional and inventive products and techniques to represent an eco-efficient model for living, a more sustainable house, demonstrating sustainable technologies in action and setting a new standard for resource efficiency in Windsor. The presentation provided a building analysis and discussed the following: geothermal heating; distributive power; green roof; net metering; grey water plumbing; solar water heating; passive lighting; energy efficient lighting and geothermal heating and cooling. It also discussed opportunities for innovation, namely: greenhouse; composting toilets; alternative insulation; net metering; solar arrays; hydroponics; and expansion of the house. Also discussed were a nature bridge, an underwater electric kite, and a vertically aerodynamic turbine. The benefits of renewable energy, small hydro power potential, and instream energy generation technology were presented. 9 refs., figs.

  19. GATE: Improving the computational efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Staelens, S. [UGent-ELIS, St-Pietersnieuwstraat, 41, B-9000 Gent (Belgium)]. E-mail: steven.staelens@ugent.be; De Beenhouwer, J. [UGent-ELIS, St-Pietersnieuwstraat, 41, B-9000 Gent (Belgium); Kruecker, D. [Institute of Medicine-Forschungszemtrum Juelich, D-52425 Juelich (Germany); Maigne, L. [Departement de Curietherapie-Radiotherapie, Centre Jean Perrin, F-63000 Clermont-Ferrand (France); Rannou, F. [Departamento de Ingenieria Informatica, Universidad de Santiago de Chile, Santiago (Chile); Ferrer, L. [INSERM U601, CHU Nantes, F-44093 Nantes (France); D' Asseler, Y. [UGent-ELIS, St-Pietersnieuwstraat, 41, B-9000 Gent (Belgium); Buvat, I. [INSERM U678 UPMC, CHU Pitie-Salpetriere, F-75634 Paris (France); Lemahieu, I. [UGent-ELIS, St-Pietersnieuwstraat, 41, B-9000 Gent (Belgium)

    2006-12-20

    GATE is a software dedicated to Monte Carlo simulations in Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET). An important disadvantage of those simulations is the fundamental burden of computation time. This manuscript describes three different techniques in order to improve the efficiency of those simulations. Firstly, the implementation of variance reduction techniques (VRTs), more specifically the incorporation of geometrical importance sampling, is discussed. After this, the newly designed cluster version of the GATE software is described. The experiments have shown that GATE simulations scale very well on a cluster of homogeneous computers. Finally, an elaboration on the deployment of GATE on the Enabling Grids for E-Science in Europe (EGEE) grid will conclude the description of efficiency enhancement efforts. The three aforementioned methods improve the efficiency of GATE to a large extent and make realistic patient-specific overnight Monte Carlo simulations achievable.

  20. GATE: Improving the computational efficiency

    International Nuclear Information System (INIS)

    Staelens, S.; De Beenhouwer, J.; Kruecker, D.; Maigne, L.; Rannou, F.; Ferrer, L.; D'Asseler, Y.; Buvat, I.; Lemahieu, I.

    2006-01-01

    GATE is a software dedicated to Monte Carlo simulations in Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET). An important disadvantage of those simulations is the fundamental burden of computation time. This manuscript describes three different techniques in order to improve the efficiency of those simulations. Firstly, the implementation of variance reduction techniques (VRTs), more specifically the incorporation of geometrical importance sampling, is discussed. After this, the newly designed cluster version of the GATE software is described. The experiments have shown that GATE simulations scale very well on a cluster of homogeneous computers. Finally, an elaboration on the deployment of GATE on the Enabling Grids for E-Science in Europe (EGEE) grid will conclude the description of efficiency enhancement efforts. The three aforementioned methods improve the efficiency of GATE to a large extent and make realistic patient-specific overnight Monte Carlo simulations achievable

  1. Efficiency of Capacitively Loaded Converters

    DEFF Research Database (Denmark)

    Andersen, Thomas; Huang, Lina; Andersen, Michael A. E.

    2012-01-01

    is introduced as a definition of efficiency. The calculated and measured efficiency curves for charging DEAP actuator, polypropylene film capacitor and X7R MLCC are provided and compared. The attention has to be paid for the voltage dependent capacitive load, like X7R MLCC, when evaluating the charging......This paper explores the characteristic of capacitance versus voltage for dielectric electro active polymer (DEAP) actuator, 2kV polypropylene film capacitor as well as 3kV X7R multi layer ceramic capacitor (MLCC) at the beginning. An energy efficiency for capacitively loaded converters...... polypropylene film capacitor can be the equivalent capacitive load. Because of the voltage dependent characteristic, X7R MLCC cannot be used to replace the DEAP actuator. However, this type of capacitor can be used to substitute the capacitive actuator with voltage dependent property at the development phase....

  2. Historical Analysis of Champion Photovoltaic Module Efficiencies

    Energy Technology Data Exchange (ETDEWEB)

    Kurtz, Sarah [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Repins, Ingrid L [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Metzger, Wyatt K [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Emery, Keith A [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Kazmerski, Lawrence L [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Levi, Dean H [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Tappan, Ian A [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Verlinden, Pierre J. [Trina Solar; Huang, Susan [Mantech International; Bowden, Stuart [Arizona State University

    2018-02-14

    Champion photovoltaic (PV) cell and module efficiencies have been reported in Progress in PV since 1993. Following the evolution of these efficiencies enables researchers to track the progress of various technologies. National Renewable Energy Laboratory has maintained a historical chart of the champion cell efficiencies, but has not published a similar chart of champion module efficiencies. Here, we analyze champion module efficiencies and compare them to champion cell efficiencies to better understand technology trends over the last three decades, highlighting that, in some cases, module efficiencies exceed 90% of cell efficiencies. Recommendations are provided on how to change the data collection and reporting for champion efficiencies to increase the value of these records.

  3. Energy Efficiency in Future PONs

    DEFF Research Database (Denmark)

    Reschat, Halfdan; Laustsen, Johannes Russell; Wessing, Henrik

    2012-01-01

    There is a still increasing tendency to give energy efficiency a high priority, even in already low energy demanding systems. This is also the case for Passive Optical Networks (PONs) for which many different methods for saving energy are proposed. This paper uses simulations to evaluate three...... proposed power saving solutions for PONs which use sleep mechanisms for saving power. The discovered advantages and disadvantages of these methods are then used as a basis for proposing a new solution combining different techniques in order to increase the energy efficiency further. This novel solution...

  4. Information efficiency in visual communication

    Science.gov (United States)

    Alter-Gartenberg, Rachel; Rahman, Zia-Ur

    1993-01-01

    This paper evaluates the quantization process in the context of the end-to-end performance of the visual-communication channel. Results show that the trade-off between data transmission and visual quality revolves around the information in the acquired signal, not around its energy. Improved information efficiency is gained by frequency dependent quantization that maintains the information capacity of the channel and reduces the entropy of the encoded signal. Restorations with energy bit-allocation lose both in sharpness and clarity relative to restorations with information bit-allocation. Thus, quantization with information bit-allocation is preferred for high information efficiency and visual quality in optimized visual communication.

  5. The RAE and University Efficiency.

    Science.gov (United States)

    Mace, John

    2000-01-01

    Evaluates effects of the new British funding formula for universities, based on the research assessment exercise (RAE). Compares effects of the RAE on two contrasting universities and finds the RAE has dramatically affected university organization, teaching, and research. RAE may have increased efficiency in teaching and research but encourages…

  6. Efficient synthesis of substituted dihydrotetraazapentacenes.

    Science.gov (United States)

    Seillan, Claire; Brisset, Hugues; Siri, Olivier

    2008-09-18

    We describe a versatile and very efficient synthesis of previously unknown substituted 5,14-dihydro-5,7,12,14-tetraazapentacenes (DHTAPs). A structural study by NMR spectroscopy showed that the conjugated pi-system of the pentacyclic skeleton rearranges depending on the electronic effect of the substituent(s).

  7. Energy Efficiency Indicators Methodology Booklet

    Energy Technology Data Exchange (ETDEWEB)

    Sathaye, Jayant; Price, Lynn; McNeil, Michael; de la rue du Can, Stephane

    2010-05-01

    This Methodology Booklet provides a comprehensive review and methodology guiding principles for constructing energy efficiency indicators, with illustrative examples of application to individual countries. It reviews work done by international agencies and national government in constructing meaningful energy efficiency indicators that help policy makers to assess changes in energy efficiency over time. Building on past OECD experience and best practices, and the knowledge of these countries' institutions, relevant sources of information to construct an energy indicator database are identified. A framework based on levels of hierarchy of indicators -- spanning from aggregate, macro level to disaggregated end-use level metrics -- is presented to help shape the understanding of assessing energy efficiency. In each sector of activity: industry, commercial, residential, agriculture and transport, indicators are presented and recommendations to distinguish the different factors affecting energy use are highlighted. The methodology booklet addresses specifically issues that are relevant to developing indicators where activity is a major factor driving energy demand. A companion spreadsheet tool is available upon request.

  8. Efficient simulation of semiflexible polymers

    NARCIS (Netherlands)

    Panja, Deb; Barkema, Gerard T.; van Leeuwen, J. M. J.

    2015-01-01

    Using a recently developed bead-spring model for semiflexible polymers that takes into account their natural extensibility, we report an efficient algorithm to simulate the dynamics for polymers like double-stranded DNA (dsDNA) in the absence of hydrodynamic interactions. The dsDNA is modeled with

  9. Efficient GPS Position Determination Algorithms

    National Research Council Canada - National Science Library

    Nguyen, Thao Q

    2007-01-01

    ... differential GPS algorithm for a network of users. The stand-alone user GPS algorithm is a direct, closed-form, and efficient new position determination algorithm that exploits the closed-form solution of the GPS trilateration equations and works...

  10. Design-Efficiency in Security

    DEFF Research Database (Denmark)

    Yuksel, Ender; Nielson, Hanne Riis; Nielson, Flemming

    In this document, we present our applied results on balancing security and performance using a running example, which is based on sensor networks. These results are forming a basis for a new approach to balance security and performance, and therefore provide design-­efficiency of key updates. We...

  11. Energy sustainability: consumption, efficiency, and ...

    Science.gov (United States)

    One of the critical challenges in achieving sustainability is finding a way to meet the energy consumption needs of a growing population in the face of increasing economic prosperity and finite resources. According to ecological footprint computations, the global resource consumption began exceeding planetary supply in 1977 and by 2030, global energy demand, population, and gross domestic product are projected to greatly increase over 1977 levels. With the aim of finding sustainable energy solutions, we present a simple yet rigorous procedure for assessing and counterbalancing the relationship between energy demand, environmental impact, population, GDP, and energy efficiency. Our analyses indicated that infeasible increases in energy efficiency (over 100 %) would be required by 2030 to return to 1977 environmental impact levels and annual reductions (2 and 3 %) in energy demand resulted in physical, yet impractical requirements; hence, a combination of policy and technology approaches is needed to tackle this critical challenge. This work emphasizes the difficulty in moving toward energy sustainability and helps to frame possible solutions useful for policy and management. Based on projected energy consumption, environmental impact, human population, gross domestic product (GDP), and energy efficiency, for this study, we explore the increase in energy-use efficiency and the decrease in energy use intensity required to achieve sustainable environmental impact le

  12. Improving efficiency in pig production

    African Journals Online (AJOL)

    made, are that each system is 100% efficient, and that the levels of production have at least been achieved in some of the experimental work. No allowance has been made for any penalty which the processor may apply to the carcass, the assumption being entirely that the objective is to produce lean meat independently of ...

  13. EYES -- Energy Efficient Sensor Networks

    NARCIS (Netherlands)

    Havinga, Paul J.M.; Etalle, Sandro; Karl, Holger; Petrioli, Chiara; Zorzi, Michele; Kip, Harry; Lentsch, Thomas; Conti, M.; Giordano, S.; Gregori, E.; Olariu, S.

    The EYES project (IST-2001-34734) is a three years European research project on self-organizing and collaborative energy-efficient sensor networks. It will address the convergence of distributed information processing, wireless communications, and mobile computing. The goal of the project is to

  14. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  15. Budgeting for Efficiency and Effectiveness

    Science.gov (United States)

    Pereus, Steven C.

    2012-01-01

    For most districts, budgeting has become a cost-cutting exercise designed to close the gap between revenues and expenses. During this process, decision makers inherently assume that existing operations are efficient and effective--an assumption that is rarely validated by facts. Cutting programs and services balances budgets but does not…

  16. Homothetic Efficiency: Theory and Applications

    NARCIS (Netherlands)

    J. Heufer (Jan); Hjertstrand, P. (Per)

    2017-01-01

    textabstractWe provide a nonparametric revealed preference approach to demand analysis based on homothetic efficiency. Homotheticity is widely assumed (often implicitly) because it is a convenient and often useful restriction. However, this assumption is rarely tested, and data rarely satisfy

  17. Simple and Practical Efficiency Lessons

    Science.gov (United States)

    Kolpin, Van

    2018-01-01

    The derivation of conditions necessary for Pareto efficient production and exchange is a lesson frequently showcased in microeconomic theory textbooks. Traditional delivery of this lesson is, however, limited in its scope of application and can be unnecessarily convoluted. The author shows that the universe of application is greatly expanded and a…

  18. Efficiency of participation in planning

    Directory of Open Access Journals (Sweden)

    Ghada Farouk Hassan

    2011-06-01

    A comparison between the two processes will take place in order to indentify the participation activities and their real efficiency. The comparison will be focused on the parameter of participation realized in each case in order to find gaps that have negative effects and needs to be filled.

  19. Efficient Audio Power Amplification - Challenges

    DEFF Research Database (Denmark)

    Andersen, Michael Andreas E.

    2005-01-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...... extensive research and development are needed is covered....

  20. Testing for Stochastic Dominance Efficiency

    NARCIS (Netherlands)

    G.T. Post (Thierry); O. Linton; Y-J. Whang

    2005-01-01

    textabstractWe propose a new test of the stochastic dominance efficiency of a given portfolio over a class of portfolios. We establish its null and alternative asymptotic properties, and define a method for consistently estimating critical values. We present some numerical evidence that our

  1. Soap with increased deactivation efficiency

    International Nuclear Information System (INIS)

    Severa, J.; Knajfl, J.; Cmolik, J.

    1980-01-01

    The decontamination effect of new types of toilet soap was studied in experiments with rat skin contaminated with an aqueous solution of fission products (pH=2.6). Most suitable for decontamination are soaps containing complex-forming substances; 1 hour after contamination their decontamination efficiency was 94.9+-0.6%. (author)

  2. Efficiently learning simple timed automata

    NARCIS (Netherlands)

    Verwer, S.E.; De Weerdt, M.M.; Witteveen, C.

    2008-01-01

    We describe an efficient algorithm for learning deterministic real-time automata (DRTA) from positive data. This data can be obtained from observations of the process to be modeled. The DRTA model we learn from such data can be used reason and gain knowledge about realtime systems such as network

  3. Declining Efficiency in the Economy

    DEFF Research Database (Denmark)

    Nørgaard, Jørgen

    1995-01-01

    with a high material standard of living. Examples are presented as are suggestions for how to improve the efficiency. These improvements, however, have a tendency to reduce the gross domestic product and hence are conflicting with the conventional political goals of growing GDP....

  4. China's energy efficiency target 2010

    International Nuclear Information System (INIS)

    Yang Ming

    2008-01-01

    The Chinese government has set an ambitious target: reducing China's energy intensity by 20%, or 4.36% each year between 2006 and 2010 on the 2005 level. Real data showed that China missed its target in 2006, having reduced its energy intensity only by 1.3%. The objective of this study is to evaluate the feasibility and potential of the Chinese to achieve the target. This paper presents issues of macro-economy, population migration, energy savings, and energy efficiency policy measures to achieve the target. A top-down approach was used to analyse the relationship between the Chinese economic development and energy demand cycles and to identify the potentials of energy savings in sub-sectors of the Chinese economy. A number of factors that contribute to China's energy intensity are identified in a number of energy-intensive sectors. This paper concludes that China needs to develop its economy at its potential GDP growth rate; strengthen energy efficiency auditing, monitoring and verification; change its national economy from a heavy-industry-dominated mode to a light industry or a commerce-dominated mode; phase out inefficient equipment in industrial sectors; develop mass and fast railway transportation; and promote energy-efficient technologies at the end use. This paper transfers key messages to policy makers for designing their policy to achieve China's energy efficiency target

  5. Energy efficiency through energy audit

    International Nuclear Information System (INIS)

    Esan, A. A.

    2000-08-01

    Energy is an essential factor to economic and social development and improved standards of living in developing countries. Nigeria in particular. There is a strong need for greater energy efficiency in every sector of economy in order to reduce costs. enhance competitiveness, conserve energy resources and reduce environmental impacts associated with production, distribution and use of energy. Energy auditing and monitoring has a significant role in any energy management and conservation project. Energy auditing as an important part of industrial energy management on plant level, represents a complex of activities aiming at the efficient use of energy. The activities are undertaken by a team of experts who use a set of measuring instruments to monitor and evaluate all the necessary data to elaborate a package of recommendations on improvements in the field of energy efficiency and possible product quality. The inefficient conversion and use of energy have been identified as a central problem for all developing countries, Nigeria inclusive, since they all consume significantly higher amounts of energy per unit of GDP than OECD countries. This aggravates energy-related environmental problems and is also a burden on domestic resources and foreign exchange. Energy prices have risen drastically in many developing countries, while energy intensities remain high. Price changes alone are not rapidly translating energy efficiency improvements. Identifying and removing the obstacles to greater energy efficiency should be priority for government in developing countries. This is why the Energy Commission, an apex organ of government on Energy matters in all its ramifications is out to encourage relatively low-cost energy audits for the Textile industries - such audits can identify ''good house-keeping's' measures, such as simply process improvements, that reduce energy consumption and operating costs. This will be followed by the training of plant workers/energy managers

  6. THEORETICAL FOUNDATIONS EFFICIENT MASS VALUATION

    Directory of Open Access Journals (Sweden)

    Koshel A.

    2016-08-01

    Full Text Available In the article the theoretical basis for determining the effectiveness of mass valuation of land in present-day conditions are described. The concept defenitsy effect and effectiveness as economic categories and their classification values for mass valuation of land are presented. The effectiveness of mass valuation of land in the settlements defines the structure of local budget and economic activities undertaken by local authorities on the basis of the results of the mass appraisal of real estate. Mass valuation is regular and it is characterized by high degree of standardization of procedures and a significant increase in the role of statistical methods for processing data related to the use of the most significant factors influencing the parameters of the object to its cost, as well as the need to determine the value of the objective laws change equivalent, which is only possible when using economic and mathematical methods and statistical analysis. Quality control results of mass valuation carried out principally in other ways, as obtained by applying statistical machine results can be checked as soon statistical methods. This shows the relevance of research topic and lack of elaboration for Ukraine problems of efficiency of mass land valuation. Scientific research conducted through the use of the dialectical method and techniques of abstraction, comparative analysis and synthesis, the article various models and methods of valuation of land for taxation purposes are analyzed. In addition, the group explored methods used, comparisons, and more. In economic theory and practice problems and determine the effect of efficiency aimed at profit is quite relevant. Economists consider cost-effectiveness, such as economic efficiency. In this case, the production can be attributed to the activities to conduct and organization of mass valuation of land. This pushes many different positions on criteria and indicators of economic efficiency, the

  7. Detection and control of lentiviral infections in sheep and goats

    NARCIS (Netherlands)

    Brinkhof, J.M.A.

    2009-01-01

    Infections caused by the small ruminant lentiviruses (SRLV) of sheep (maedi visna virus) and goats (caprine arthritis encephalitis virus) are a serious economical threat to small ruminant farming particularly in the more intensive settings like dairy farms. Revenue is ultimately negatively

  8. Construction and Screening of a Lentiviral Secretome Library.

    Science.gov (United States)

    Liu, Tao; Jia, Panpan; Ma, Huailei; Reed, Sean A; Luo, Xiaozhou; Larman, H Benjamin; Schultz, Peter G

    2017-06-22

    Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. 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

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

    African Journals Online (AJOL)

    ajl yemi

    2011-10-26

    Oct 26, 2011 ... The essence of cancer development and growth is that mutant cells unlimitedly proliferate and cannot ... big enzyme family that can act on ester bonds between phosphoglyceric acetic substituents and .... extracted using small plasmid extractive kit (Takara, Japan). The absorbance and concentrations of the ...

  11. Efficient transformer for electromagnetic waves

    Science.gov (United States)

    Miller, R.B.

    A transformer structure for efficient transfer of electromagnetic energy from a transmission line to an unmatched load provides voltage multiplication and current division by a predetermined constant. Impedance levels are transformed by the square of that constant. The structure includes a wave splitter, connected to an input transmission device and to a plurality of output transmission devices. The output transmission devices are effectively connected in parallel to the input transmission device. The output transmission devices are effectively series connected to provide energy to a load. The transformer structure is particularly effective in increasing efficiency of energy transfer through an inverting convolute structure by capturing and transferring energy losses from the inverter to the load.

  12. The Efficiency of Educational Production

    DEFF Research Database (Denmark)

    Bogetoft, Peter; Heinesen, Eskil; Tranæs, Torben

    the educational sector, both in terms of a large number of students enrolled in educational programmes and a high completion rate. The methodology used is to compare (benchmark) Denmark with a relevant group of countries and to calculate how much cheaper Denmark could teach the same number of students...... to explain the higher costs of upper secondary education. On the output side, if earnings and levels of employment among those who complete their education are taken into account, then Denmark is in fact found to be efficient. However, this high level of efficiency has become less clear-cut in recent years......Denmark, Norway, New Zealand, Canada and the USA are the OECD countries that spend most on education, measured in relation to GDP. Focusing in particular on upper secondary education, this paper examines whether the heavy expenditure on education in Denmark is matched by high output from...

  13. Hadoop Tutorial - Efficient data ingestion

    CERN Multimedia

    CERN. Geneva; Baranowski, Zbigniew

    2016-01-01

    The Hadoop ecosystem is the leading opensource platform for distributed storage and processing of "big data". The Hadoop platform is available at CERN as a central service provided by the IT department. Real-time data ingestion to Hadoop ecosystem due to the system specificity is non-trivial process and requires some efforts (which is often underestimated) in order to make it efficient (low latency, optimize data placement, footprint on the cluster). In this tutorial attendees will learn about: The important aspects of storing the data in Hadoop Distributed File System (HDFS).  Data ingestion techniques and engines that are capable of shipping data to Hadoop in an efficient way. Setting up a full data ingestion flow into a Hadoop Distributed Files System from various sources (streaming, log files, databases) using the best practices and components available around the ecosystem (including Sqoop, Kite, Flume, Kafka...

  14. Worst Case Efficient Data Structures

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting

    We study the design of efficient data structures. In particular we focus on the design of data structures where each operation has a worst case efficient implementations. The concrete problems we consider are partial persistence, implementation of priority queues, and implementation of dictionaries....... The first problem we consider is how to make bounded in-degree and out-degree data structures partially persistent, i.e., how to remember old versions of a data structure for later access. A node copying technique of Driscoll et al. supports update steps in amortized constant time and access steps in worst...... case constant time. The worst case time for an update step can be linear in the size of the structure. We show how to extend the technique of Driscoll et al. such that update steps can be performed in worst case constant time on the pointer machine model. We present two new comparison based priority...

  15. Southern Energy Efficiency Center (SEEC)

    Energy Technology Data Exchange (ETDEWEB)

    Vieira, Robin; Sonne, Jeffrey; Withers, Charles; Cummings, James; Verdict, Malcolm; Roberts, Sydney

    2009-09-30

    The Southern Energy Efficiency Center (SEEC) builds collaborative partnerships with: state and local governments and their program support offices, the building delivery industry (designers, contractors, realtors and commissioning agents), product manufacturers and their supply chains, utilities and their program implementers, consumers and other stakeholders in order to forge a strong regional network of building energy efficiency allies. Through a project Steering Committee composed of the state energy offices and building industry stakeholders, the SEEC works to establish consensus-based goals, priorities and strategies at the regional, state and local levels that will materially advance the deployment of high-performance “beyond code” buildings. In its first Phase, SEEC will provide limited technical and policy support assistance, training, certification and education to a wide spectrum of the building construction, codes and standards, and the consumer marketplace.

  16. Effective education for energy efficiency

    International Nuclear Information System (INIS)

    Zografakis, Nikolaos; Menegaki, Angeliki N.; Tsagarakis, Konstantinos P.

    2008-01-01

    A lot of today's world vices can be eliminated if certain targeted modules and adapted curricula are introduced in the schooling system. One of these vices is energy squandering with all its negative consequences for the planet (e.g. depletion of finite energy sources and the subsequent climate change). This paper describes the results of an energy-thrift information and education project taking place in different levels of education in Crete-Greece, which records 321 students' and their parents' routine energy-related behavior and proves that this behavior changes to a more energy efficient one, after the dissemination of relevant information and the participation into the energy education projects. Namely, response percentages indicating the energy-efficient behavior increased after project participation while the ones indicating an energy-squandering behavior decreased. The Wilcoxon signed rank test was statistically significant in all energy behavior questions related to students and to most questions related to parents

  17. The Efficiency of Educational Production

    DEFF Research Database (Denmark)

    Bogetoft, Peter; Heinesen, Eskil; Tranæs, Torben

    2015-01-01

    in order to analyse different aspects of efficiency. In purely quantitative models (where inputs and outputs are expenditure and number of students at different levels of the educational system) and in models where graduation or completion rates are included as indicators of output quality, Finland......Focusing in particular on upper secondary education, this paper examines whether the relatively high level of expenditure on education in the Nordic countries is matched by high output from the educational sector, both in terms of student enrolment and indicators of output quality in the form...... of graduation/completion rates and expected earnings after completed education. We use data envelopment analysis (DEA) to compare (benchmark) the Nordic countries with a relevant group of rich OECD countries and calculate input efficiency scores for each country. We estimate a wide range of specifications...

  18. The Efficiency of Educational Production

    DEFF Research Database (Denmark)

    Bogetoft, Peter; Heinesen, Eskil; Tranæs, Torben

    in order to analyse different aspects of efficiency. In purely quantitative models (where inputs and outputs are expenditure and number of students at different levels of the educational system) and in models where graduation or completion rates are included as an indicator of output quality, Finland......Focusing in particular on upper secondary education, this paper examines whether the relatively high level of expenditure on education in the Nordic countries is matched by high output from the educational sector, both in terms of student enrolment and indicators of output quality in the form...... of graduation/completion rates and expected earnings after completed education. We use Data Envelopment Analysis (DEA) to compare (benchmark) the Nordic countries with a relevant group of rich OECD countries and calculate input efficiency scores for each country. We estimate a wide range of specifications...

  19. Selected Banks Economic Efficiency Indicators

    Directory of Open Access Journals (Sweden)

    Adam Szafarczyk

    2007-04-01

    Full Text Available There are a lot of economic efficiency indicators in theory. It is necessary to choice some of them for concrete purposes. ROA, ROE and C/I play important role due to last experience of polish banks.Cost to Income Ratio plays very important role affecting not only efficiency monitoring process in the banks but also becoming a very important target in strategy or finance plan. There are several methodological approaches to construct this index in detail. Results are differing due to implementing methodology. This article describes trends C/I concerning both polish and foreign banks. It is difficult to compare banks in emerging country and real giants in finance mature economies.

  20. Increased energy efficiency of hobs

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-01-01

    The objective of the project is to save energy when cooking food on hobs. A great part of the total energy consumption used for cooking is consumed by hobs. The amount of energy depends on the temperature used for cooking and energy used for evaporation of liquid, focussing especially on the latter in this project. CHEC B is a method for controlling the supply of energy to the zone, so that a minimum of energy is used for reaching a set temperature of the food/liquid in the pot and maintaining this temperature. Today the efficiency of hobs is between 50 - 75%. Using CHEC B the energy efficiency is expected to be higher. (au)

  1. Efficient Electrotransformation of Bacteroides fragilis▿

    OpenAIRE

    Ichimura, Minoru; Nakayama-Imaohji, Haruyuki; Wakimoto, Shin; Morita, Hidetoshi; Hayashi, Tetsuya; Kuwahara, Tomomi

    2010-01-01

    This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 co...

  2. High Efficiency Room Air Conditioner

    Energy Technology Data Exchange (ETDEWEB)

    Bansal, Pradeep [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2015-01-01

    This project was undertaken as a CRADA project between UT-Battelle and Geberal Electric Company and was funded by Department of Energy to design and develop of a high efficiency room air conditioner. A number of novel elements were investigated to improve the energy efficiency of a state-of-the-art WAC with base capacity of 10,000 BTU/h. One of the major modifications was made by downgrading its capacity from 10,000 BTU/hr to 8,000 BTU/hr by replacing the original compressor with a lower capacity (8,000 BTU/hr) but high efficiency compressor having an EER of 9.7 as compared with 9.3 of the original compressor. However, all heat exchangers from the original unit were retained to provide higher EER. The other subsequent major modifications included- (i) the AC fan motor was replaced by a brushless high efficiency ECM motor along with its fan housing, (ii) the capillary tube was replaced with a needle valve to better control the refrigerant flow and refrigerant set points, and (iii) the unit was tested with a drop-in environmentally friendly binary mixture of R32 (90% molar concentration)/R125 (10% molar concentration). The WAC was tested in the environmental chambers at ORNL as per the design rating conditions of AHAM/ASHRAE (Outdoor- 95F and 40%RH, Indoor- 80F, 51.5%RH). All these modifications resulted in enhancing the EER of the WAC by up to 25%.

  3. Gold, currencies and market efficiency

    Czech Academy of Sciences Publication Activity Database

    Krištoufek, Ladislav; Vošvrda, Miloslav

    2016-01-01

    Roč. 449, č. 1 (2016), s. 27-34 ISSN 0378-4371 R&D Projects: GA ČR(CZ) GBP402/12/G097 Institutional support: RVO:67985556 Keywords : Efficient market hypothesis, * Gold * Currencies, * Fractal dimension * Entropy * Long-Term memory Subject RIV: AH - Economics Impact factor: 2.243, year: 2016 http://library.utia.cas.cz/separaty/2016/E/kristoufek-0455876.pdf

  4. Commodity futures and market efficiency

    Czech Academy of Sciences Publication Activity Database

    Krištoufek, Ladislav; Vošvrda, Miloslav

    2014-01-01

    Roč. 42, č. 1 (2014), s. 50-57 ISSN 0140-9883 R&D Projects: GA ČR GA402/09/0965 Grant - others:GA ČR(CZ) GAP402/11/0948 Program:GA Institutional support: RVO:67985556 Keywords : commodities * efficiency * entropy * long-term memory * fractal dimension Subject RIV: AH - Economics Impact factor: 2.708, year: 2014 http://library.utia.cas.cz/separaty/2013/E/kristoufek-0420811.pdf

  5. Efficiency of tax bodies' performance

    OpenAIRE

    Fedorovskaya, M.

    2011-01-01

    The problems of assessment of tax bodies' activities efficiency which tax administration faces are touched upon. Suggestions made provide an opportunity for bodies of legislative and executive power to take specific measures aimed at enhancing the system of tax administration in the country. Suggested criteria for assessing tax bodies' performance can be put into practice by tax authorities. A new approach to aspects of solving tasks of control over tax compliance, preventing and excluding ta...

  6. SOUR graphs for efficient completion

    OpenAIRE

    Lynch, Christopher; Strogova, Polina

    1998-01-01

    International audience; We introduce a data structure called \\emphSOUR graphs and present an efficient Knuth-Bendix completion procedure based on it. \\emphSOUR graphs allow for a maximal structure sharing of terms in rewriting systems. The term representation is a dag representation, except that edges are labelled with equational constraints and variable renamings. The rewrite rules correspond to rewrite edges, the unification problems to unification edges. The Critical Pair and Simplificatio...

  7. Energy efficiency opportunities in Hotels

    Directory of Open Access Journals (Sweden)

    Dina Said

    2017-03-01

    Full Text Available According to the statistics in Egypt (2013, the number of hotels is 1193, about 407 of them have contracted power greater than 500 kW.Air conditioning, lighting, water heating and refrigeration represent the main activities demanding electrical energy in hotel business.The energy consumption per night spend changes a lot, depending on various factors; facilities provided, category of hotel, occupancy , geographical situation, weather conditions, nationality of clients, design and control of the installations.Energy benchmarking is an internal management tool designed to provide ongoing, reliable and verifiable tracking on the hotels performance. The most useful performance indicator (or Energy Efficiency Benchmarking of hotels are: Lighting Power Density (LPD in W (for lighting/m2, and energy intensity (kWh/m2/ y.There are multiple benefits for improving energy in hotel business; reduces the hotel's operating cost, reduces climate change risks and promotes green tourism.Energy efficiency opportunities are low-cost measures and cost- effective investments.   There are many energy saving opportunities for lighting in hotel's guest rooms as well as the more obvious savings in lobbies and exterior lighting areas. Behavior campaigns can yield substantial energy savings, both through the guests and housekeeper behavior. Encouraging housekeepers to use natural light during room cleaning is a simple first step to implement energy saving program.This paper presents the energy efficiency guidelines and energy benchmarking for hotels. Also a case study showing how the energy efficiency program implemented is presented. 

  8. USSR Energy Efficiency and Prospects

    OpenAIRE

    Sinyak, Y.

    1991-01-01

    The USSR is the largest energy producer and the second largest energy consumer in the world. Its share of global energy reached above 17% in 1988. The Soviet energy system is characterized by low efficiency and high "per capita" energy consumption, although there are some reasons justifying the greater USSR energy use per unit of product output than in other industrialized countries. The present energy savings potential is approximately equal to one-half of the domestic energy consumption. Im...

  9. Efficiency of antlion trap construction.

    Science.gov (United States)

    Fertin, Arnold; Casas, Jérôme

    2006-09-01

    Assessing the architectural optimality of animal constructions is in most cases extremely difficult, but is feasible for antlion larvae, which dig simple pits in sand to catch ants. Slope angle, conicity and the distance between the head and the trap bottom, known as off-centring, were measured using a precise scanning device. Complete attack sequences in the same pits were then quantified, with predation cost related to the number of behavioural items before capture. Off-centring leads to a loss of architectural efficiency that is compensated by complex attack behaviour. Off-centring happened in half of the cases and corresponded to post-construction movements. In the absence of off-centring, the trap is perfectly conical and the angle is significantly smaller than the crater angle, a physical constant of sand that defines the steepest possible slope. Antlions produce efficient traps, with slopes steep enough to guide preys to their mouths without any attack, and shallow enough to avoid the likelihood of avalanches typical of crater angles. The reasons for the paucity of simplest and most efficient traps such as theses in the animal kingdom are discussed.

  10. USSR energy efficiency and prospects

    International Nuclear Information System (INIS)

    Sinyak, Y.

    1991-06-01

    The U.S.S.R. is the largest energy producer and the second largest energy consumer in the world. Its share of global energy use reached above 17% in 1988. The soviet energy system is characterized by low efficiency and high per capita energy consumption, although there are some reasons justifying the greater U.S.S.R. energy use per unit of product output than in other industrialized countries. The present energy-savings potential is approximately equal to one-half of the domestic energy consumption. Improvements in energy efficiency at all levels of the national economy are now considered to be the primary goal of national energy policy for the next couple of decades. Being endowed with abundant natural gas resources, the U.S.S.R. will count on this energy source in the future to improve its energy efficiency, reduce expenses and cope with air pollution. After 2005-2010, stabilized primary energy consumption may be reached or there may even be a decline of total energy use. The U.S.S.R. could reduce CO 2 emissions by 20% by 2030 but with substantial negative impacts on GNP growth. Required improvements in the Soviet energy system depend on changes in energy management, including reduction of the role of centralized planning, decentralization and privatization of energy-producing facilities, energy-price reforms, reshaping of investment patterns, reduction in military expenditures, etc. (author)

  11. Efficient flapping flight of pterosaurs

    Science.gov (United States)

    Strang, Karl Axel

    In the late eighteenth century, humans discovered the first pterosaur fossil remains and have been fascinated by their existence ever since. Pterosaurs exploited their membrane wings in a sophisticated manner for flight control and propulsion, and were likely the most efficient and effective flyers ever to inhabit our planet. The flapping gait is a complex combination of motions that sustains and propels an animal in the air. Because pterosaurs were so large with wingspans up to eleven meters, if they could have sustained flapping flight, they would have had to achieve high propulsive efficiencies. Identifying the wing motions that contribute the most to propulsive efficiency is key to understanding pterosaur flight, and therefore to shedding light on flapping flight in general and the design of efficient ornithopters. This study is based on published results for a very well-preserved specimen of Coloborhynchus robustus, for which the joints are well-known and thoroughly described in the literature. Simplifying assumptions are made to estimate the characteristics that can not be inferred directly from the fossil remains. For a given animal, maximizing efficiency is equivalent to minimizing power at a given thrust and speed. We therefore aim at finding the flapping gait, that is the joint motions, that minimize the required flapping power. The power is computed from the aerodynamic forces created during a given wing motion. We develop an unsteady three-dimensional code based on the vortex-lattice method, which correlates well with published results for unsteady motions of rectangular wings. In the aerodynamic model, the rigid pterosaur wing is defined by the position of the bones. In the aeroelastic model, we add the flexibility of the bones and of the wing membrane. The nonlinear structural behavior of the membrane is reduced to a linear modal decomposition, assuming small deflections about the reference wing geometry. The reference wing geometry is computed for

  12. Role of Appraisals in Energy Efficiency Financing

    Energy Technology Data Exchange (ETDEWEB)

    Doyle, V.; Bhargava, A.

    2012-05-01

    This research identifies barriers and challenges and current industry status including several key appraisal industry developments for identifying and valuing energy efficiency, critical obstacles to documenting and assessing the potential added value from energy efficiency improvements, current opportunities to support and standardize reporting on energy efficiency and to ensure proper valuation, and next steps towards enabling energy efficiency financing market transformation.

  13. Energy Efficiency Policy in Slovenia

    International Nuclear Information System (INIS)

    Beravs, F.

    1998-01-01

    When Slovenia gained its independence in 1991, its energy sector was characterised by largely centralised state planning and artificially low prices maintained by widespread subsidies. Supply side considerations tended to dominate the energy policy and sectoral planning. As a result the final energy intensity in Slovenia was (still albeit declining) considerably higher than the EU average. In order to support economic growth and transition to a modern market economy, integrated and competitive in the European and world market structures, the National Assembly of the Republic of Slovenia adopted a resolution on the Strategy of Energy Use and Supply of Slovenia in early 1996. In the field of energy use, the long-term strategic orientation is to increase energy efficiency in all sectors of energy consumption. The main objective can be summarised as to secure the provision of reliable and environmentally friendly energy services at least costs. In quantitative terms the Strategy attaches a high priority to energy efficiency and environmental protection and sets the target of improving the overall energy efficiency by 2% p.a. over the next 10 to 15 years. To achieve the target mentioned above the sectoral approach and a number of policy instruments have been foreseen. Besides market based energy prices which will, according to the European Energy Charter, gradually incorporate the cost of environment and social impacts, the following policy instruments will be intensified and budget-supported: education and awareness building, energy consultation, regulations and agreements, financial incentives, innovation and technology development. The ambitious energy conservation objectives represent a great challenge to the whole society. (author)

  14. Efficient exploration of multiplex networks

    Science.gov (United States)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2016-04-01

    Efficient techniques to navigate networks with local information are fundamental to sample large-scale online social systems and to retrieve resources in peer-to-peer systems. Biased random walks, i.e. walks whose motion is biased on properties of neighbouring nodes, have been largely exploited to design smart local strategies to explore a network, for instance by constructing maximally mixing trajectories or by allowing an almost uniform sampling of the nodes. Here we introduce and study biased random walks on multiplex networks, graphs where the nodes are related through different types of links organised in distinct and interacting layers, and we provide analytical solutions for their long-time properties, including the stationary occupation probability distribution and the entropy rate. We focus on degree-biased random walks and distinguish between two classes of walks, namely those whose transition probability depends on a number of parameters which is extensive in the number of layers, and those whose motion depends on intrinsically multiplex properties of the neighbouring nodes. We analyse the effect of the structure of the multiplex network on the steady-state behaviour of the walkers, and we find that heterogeneous degree distributions as well as the presence of inter-layer degree correlations and edge overlap determine the extent to which a multiplex can be efficiently explored by a biased walk. Finally we show that, in real-world multiplex transportation networks, the trade-off between efficient navigation and resilience to link failure has resulted into systems whose diffusion properties are qualitatively different from those of appropriately randomised multiplex graphs. This fact suggests that multiplexity is an important ingredient to include in the modelling of real-world systems.

  15. Biofuels Drying Process Efficiency Research

    Directory of Open Access Journals (Sweden)

    Osvaldas Šlepikas

    2014-02-01

    Full Text Available The paper deals with biofuel drying process efficiency opportunities.Research was carried out with a special stand and performingexperiments. Experimental rig consists of an ultrasonic generator,ultrasonic transducer, a drying chamber and the humidity,temperature gauge. Tests were used for wood pellets. During theexperiment, they were irrigated with water, dried with hot air andadditionally exposed to different frequency ultrasonic vibrations.The tests results have showed that the convective drying processis combined with the ultrasonic vibrations, the drying time isreduced, which means a positive impact on the ultrasonic process.Studies have confirmed that the effectiveness of convectivedrying method combined with operating ultrasonic vibrationsincreases.

  16. Efficient separations & processing crosscutting program

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-08-01

    The Efficient Separations and Processing Crosscutting Program (ESP) was created in 1991 to identify, develop, and perfect chemical and physical separations technologies and chemical processes which treat wastes and address environmental problems throughout the DOE complex. The ESP funds several multiyear tasks that address high-priority waste remediation problems involving high-level, low-level, transuranic, hazardous, and mixed (radioactive and hazardous) wastes. The ESP supports applied research and development (R & D) leading to the demonstration or use of these separations technologies by other organizations within the Department of Energy (DOE), Office of Environmental Management.

  17. ICT applications enhancing energy efficiency

    Directory of Open Access Journals (Sweden)

    A. G. Matani

    2016-06-01

    Full Text Available Computers, laptops and mobile devices – information technology (IT accounts for 2% of human greenhouse gas emissions worldwide, as evidenced in a study by Global Action Plan, a UK based environmental organization. This figure can be reduced if the green segment, or Green IT, continues to grow. Energy can also be saved through cloud computing, namely the principle of outsourcing the programs and functions of one’s own computer to service providers over the internet. This also means sharing storage capacity with others. This paper highlights the impact of information technology applications towards enhancing energy efficiency of the systems.

  18. Power-Efficient Design Challenges

    Science.gov (United States)

    Pangrle, Barry

    Design teams find themselves facing decreasing power budgets while simultaneously the products that they design continue to require the integration of increasingly complex levels of functionality. The market place (driven by consumer preferences) and new regulations and guidelines on energy efficiency and environmental impact are the key drivers. This in turn has generated new approaches in all IC and electronic system design domains from the architecture to the physical layout of ICs, to design-for-test, as well as for design verification to insure that the design implementation actually meets the intended requirements and specifications. This chapter covers key aspects of these forces from a technological and market perspective that are driving designers to produce more energy-efficient products. Observations by significant industry leaders from AMD, ARM, IBM, Intel, nVidia and TSMC are cited, and the emerging techniques and technologies used to address these issues now and into the future are explored. Topic areas include: System level: Architectural analysis and transaction-level modeling. How architectural decisions can dramatically reduce the design power and the importance of modeling hardware and software together. IC (Chip) level: The impact of creating on-chip power domains for selectively turning power off and/or multi-voltage operation on: (1) chip verification, (2) multi-corner multi-mode analysis during placement and routing of logic cells and (3) changes to design-for-test, all in order to accommodate for power-gating and multi-voltage control logic, retention registers, isolation cells and level shifters needed to implement these power saving techniques. Process level: The disappearing impact of body-bias techniques on leakage control and why new approaches like High-K Metal Gate (HKMG) technology help but don't eliminate power issues. Power-efficient design is impacting the way chip designers work today, and this chapter focuses on where the most

  19. Efficiency factors in Mie scattering

    International Nuclear Information System (INIS)

    Nussenzveig, H.M.

    1980-04-01

    Asymptotic approximation to the Mie efficiency factors for extinction, absorption and radiation pressure, derived from complex angular momentum theory and averaged over Δβ approximately π (β = size parameter), are given and compared with the exact results. For complex refractive indices N = n + i kappa with 1.1 -2 - 10 -3 % between β = 10 and β = 1000, and computing time is reduced by a factor of order β, so that the Mie formulae can advantageously by replaced by the asymptotic ones in most applications. (Author) [pt

  20. Efficient Intra-Household Allocations

    DEFF Research Database (Denmark)

    Browning, Martin

    for households which have more than one member. We make minimal assumptions about how the individual members of the household resolve conflicts. All we assume is that however decisions are made, outcomes are efficient. We refer to this as the collective setting. We show that in the collective setting household....... The tests of the usual symmetry conditions are rejected for two person households but not for one person households. We also show that income pooling is rejected for two person households. We then test for our collective setting conditions on the couples data. None of the collective setting restrictions...

  1. The development of efficient sunscreens

    Directory of Open Access Journals (Sweden)

    Dominique Moyal

    2012-01-01

    Full Text Available Skin exposure to acute or repetitive ultraviolet light induces risks which are now well identified. An efficient photoprotection is thus required for both UVB and UVA radiation. In particular, increasing evidence of the detrimental effects of UVA on skin has led to the development of a new generation of sunscreens that provide effective protection throughout the whole UV radiation spectrum. Many new UV filters have been introduced in the last decade, particularly UVA filters, with improved efficacy and safety. Sunscreen filters must be carefully combined to achieve esthetically pleasing products offering photostable and well-balanced photoprotection.

  2. Efficient Type Representation in TAL

    Science.gov (United States)

    Chen, Juan

    2009-01-01

    Certifying compilers generate proofs for low-level code that guarantee safety properties of the code. Type information is an essential part of safety proofs. But the size of type information remains a concern for certifying compilers in practice. This paper demonstrates type representation techniques in a large-scale compiler that achieves both concise type information and efficient type checking. In our 200,000-line certifying compiler, the size of type information is about 36% of the size of pure code and data for our benchmarks, the best result to the best of our knowledge. The type checking time is about 2% of the compilation time.

  3. CEE Energy Efficiency Report - Slovakia

    International Nuclear Information System (INIS)

    Hecl, V.

    2005-01-01

    A review of future trends of energy consumption shows that, in the absence of an active energy policy which promotes energy efficiency, energy consumption will increase as a whole by approximately 6.8% by 2012 continuing to raise after this period.. This result hides large differences between the different sources of energy (mainly heat, fuels and electricity) and between the different sectors - transport, industry, buildings etc. It is therefore clear that a strong energy policy is needed to counterbalance the expected increase in energy consumption in all sectors, with emphasis on measures in the building sector (both residential and tertiary) and in the transport sector. Furthermore improvements in the district heating sector are also essential to prevent further disconnection from district heating and a shift to other means of heating. A review of the main barriers to energy efficiency leads to the conclusion that while significant changes are needed in the regulatory framework, the lack of access to finance and the general lack of awareness about existing technologies and best practice represent the greatest barriers. In order to evaluate the success of energy. In a few studies available from past 2-3 years the calculation of low and high targets for energy policy was elaborated. The low targets would represent about 11% - 12% reduction in overall energy consumption. The high targets would represent a 13% - 15% reduction in overall energy consumption. Policy instruments have been identified which can turn energy efficiency into one of the driving forces of the overall economic and development strategy of the country. Some of these instruments deal with general issues such as general policy issues, regulatory and legal aspects, the institutional framework and fiscal, taxation and pricing policy. They are designed to improve the present conditions and would use only a limited part of the available public budget. The state budget dedicated to energy issues will

  4. Urban liveability versus economic efficiency

    DEFF Research Database (Denmark)

    Farné Fratini, Chiara; Jensen, Jens Stissing

    2013-01-01

    development. Against this agenda a national strategy is currently being enforced which addresses water as a context-independent functional sector. This agenda operates with a narrow definition of economic efficiency in service provision through a benchmarking system focusing only on technical performances...... agenda was formulated and advocated as a response to specific tension and ambiguities within and among the socio-material actor-worlds of the established water management regime, and how the agenda was translated into new institutional arrangements. We then analyse the navigations by which...

  5. Electrorheology Leads to Efficient Combustion

    Science.gov (United States)

    Tao, R.; Huang, K.; Tang, H.; Bell, D.

    2009-03-01

    Improving engine efficiency and reducing pollutant emissions are important. Since combustion starts at the interface between fuel and air and most harmful emissions come from incomplete burning, reducing the size of fuel droplets for the fuel injection would increase the total surface area to start burning, leading to a cleaner and more efficient engine. While most efforts are focused on ultra-dilute mixtures at extremely high pressure to produce much finer mist of fuel for combustion, the new technology is still under development and only for next generation vehicles. Here we report our fuel injection technology based on new physics principle that proper application of electrorheology can reduce the viscosity of petroleum fuels. A small device is thus introduced just before the fuel injection for the engine, producing a strong electric field to reduce the fuel viscosity, resulting in much smaller fuel droplets in atomization. Both lab tests and road tests confirm our theory and indicate that such a device improves fuel mileage significantly and reduces emission. The technology is expected to have broad applications, applicable to current internal combustion engines and future engines as well. Supported by STWA and RAND.

  6. The Challenge of Energy Efficiency

    International Nuclear Information System (INIS)

    Alonso Gonzalez, J. A.

    2009-01-01

    Recent Directive 2009/28/EC on the promotion of the use of renewable energies sets some binding targets for the contribution of renewable energies in 2020 to total consumption, setting the share at 20% of final energy demand, with a particularisation of 10% for the transport sector, and also a 20% reduction of greenhouse gases Together with these targets, it also sets another target relative to energy efficiency, aiming for a 20% improvement, under the terms set down by the Commission in its announcement dated 19 October 2006. This energy saving target is going to have a decisive influence on the achievement of the other two. In order to quantify the degree of difficulty of achieving the saving target and determine the policies and measures to be taken, we are going to analyze the evolution of energy efficiency (energy consumption energy units per unit of GDP - economic unit) in Spain from 1980 to date and the value of energy intensity that we should have in 2020 to achieve the targets. This will give us an idea of the magnitude of the challenge and, therefore, of the efforts we will have to make to achieve the target. (Author)

  7. Efficient functionalization of alginate biomaterials.

    Science.gov (United States)

    Dalheim, Marianne Ø; Vanacker, Julie; Najmi, Maryam A; Aachmann, Finn L; Strand, Berit L; Christensen, Bjørn E

    2016-02-01

    Peptide coupled alginates obtained by chemical functionalization of alginates are commonly used as scaffold materials for cells in regenerative medicine and tissue engineering. We here present an alternative to the commonly used carbodiimide chemistry, using partial periodate oxidation followed by reductive amination. High and precise degrees of substitution were obtained with high reproducibility, and without formation of by-products. A protocol was established using l-Tyrosine methyl ester as a model compound and the non-toxic pic-BH3 as the reducing agent. DOSY was used to indirectly verify covalent binding and the structure of the product was further elucidated using NMR spectroscopy. The coupling efficiency was to some extent dependent on alginate composition, being most efficient on mannuronan. Three different bioactive peptide sequences (GRGDYP, GRGDSP and KHIFSDDSSE) were coupled to 8% periodate oxidized alginate resulting in degrees of substitution between 3.9 and 6.9%. Cell adhesion studies of mouse myoblasts (C2C12) and human dental stem cells (RP89) to gels containing various amounts of GRGDSP coupled alginate demonstrated the bioactivity of the material where RP89 cells needed higher peptide concentrations to adhere. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Efficient quantum memory for light.

    Science.gov (United States)

    Hedges, Morgan P; Longdell, Jevon J; Li, Yongmin; Sellars, Matthew J

    2010-06-24

    Storing and retrieving a quantum state of light on demand, without corrupting the information it carries, is an important challenge in the field of quantum information processing. Classical measurement and reconstruction strategies for storing light must necessarily destroy quantum information as a consequence of the Heisenberg uncertainty principle. There has been significant effort directed towards the development of devices-so-called quantum memories-capable of avoiding this penalty. So far, successful demonstrations of non-classical storage and on-demand recall have used atomic vapours and have been limited to low efficiencies, of less than 17 per cent, using weak quantum states with an average photon number of around one. Here we report a low-noise, highly efficient (up to 69 per cent) quantum memory for light that uses a solid-state medium. The device allows the storage and recall of light more faithfully than is possible using a classical memory, for weak coherent states at the single-photon level through to bright states of up to 500 photons. For input coherent states containing on average 30 photons or fewer, the performance exceeded the no-cloning limit. This guaranteed that more information about the inputs was retrieved from the memory than was left behind or destroyed, a feature that will provide security in communications applications.

  9. Efficient robust conditional random fields.

    Science.gov (United States)

    Song, Dongjin; Liu, Wei; Zhou, Tianyi; Tao, Dacheng; Meyer, David A

    2015-10-01

    Conditional random fields (CRFs) are a flexible yet powerful probabilistic approach and have shown advantages for popular applications in various areas, including text analysis, bioinformatics, and computer vision. Traditional CRF models, however, are incapable of selecting relevant features as well as suppressing noise from noisy original features. Moreover, conventional optimization methods often converge slowly in solving the training procedure of CRFs, and will degrade significantly for tasks with a large number of samples and features. In this paper, we propose robust CRFs (RCRFs) to simultaneously select relevant features. An optimal gradient method (OGM) is further designed to train RCRFs efficiently. Specifically, the proposed RCRFs employ the l1 norm of the model parameters to regularize the objective used by traditional CRFs, therefore enabling discovery of the relevant unary features and pairwise features of CRFs. In each iteration of OGM, the gradient direction is determined jointly by the current gradient together with the historical gradients, and the Lipschitz constant is leveraged to specify the proper step size. We show that an OGM can tackle the RCRF model training very efficiently, achieving the optimal convergence rate [Formula: see text] (where k is the number of iterations). This convergence rate is theoretically superior to the convergence rate O(1/k) of previous first-order optimization methods. Extensive experiments performed on three practical image segmentation tasks demonstrate the efficacy of OGM in training our proposed RCRFs.

  10. Efficiency of quantum volume hologram

    Science.gov (United States)

    Vasilyev, D. V.; Sokolov, I. V.

    2012-11-01

    We discuss storage and retrieval efficiency of parallel spatially multimode quantum memory for light - quantum volume hologram. The introduced in [D.V. Vasyliev, I.V. Sokolov, E.S. Polzik, Phys. Rev. A 81, 020302(R) (2010)] scheme is based on the counter-propagating (non-collinear in general case) quantum signal wave and strong classical reference wave in presence of the Raman-type off-resonant interaction with atomic spins rotating in the magnetic field. By the forward-propagating retrieval the quantum volume hologram is less sensitive to diffraction [D.V. Vasyliev, I.V. Sokolov, E.S. Polzik, Phys. Rev. A 81, 020302(R) (2010)] and therefore is capable of achieving high density of storage of spatial modes. We propose to use for the forward-propagating retrieval the signal temporal eigenmodes of the whole write-in and readout memory cycle. As compared to the approach when there are used the eigenmodes optimal only for the write-in stage of the memory, our proposal allows for better efficiencies for given physical parameters of the scheme, and, hence, for higher quantum capacity of parallel quantum memory. We also demonstrate that for the backward-propagating retrieval of quantum volume hologram the collective spin wave momentum inversion is needed, which is achieved by means of the π-pulse of stimulated Raman scattering of counter-propagating classical waves.

  11. Efficiency of fermionic quantum distillation

    Energy Technology Data Exchange (ETDEWEB)

    Herbrych, Jacek W. [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Feiguin, Adrian E. [Northeastern Univ., Boston, MA (United States); Dagotto, Elbio R. [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Heidrich-Meisner, F. [Ludwig-Maximilians-Univ. Munchen, Munchen (Germany)

    2017-09-13

    Here, we present a time-dependent density-matrix renormalization group investigation of the quantum distillation process within the Fermi-Hubbard model on a quasi-one-dimensional ladder geometry. The term distillation refers to the dynamical, spatial separation of singlons and doublons in the sudden expansion of interacting particles in an optical lattice, i.e., the release of a cloud of atoms from a trapping potential. Remarkably, quantum distillation can lead to a contraction of the doublon cloud, resulting in an increased density of the doublons in the core region compared to the initial state. As a main result, we show that this phenomenon is not limited to chains that were previously studied. Interestingly, there are additional dynamical processes on the two-leg ladder such as density oscillations and self-trapping of defects that lead to a less efficient distillation process. An investigation of the time evolution starting from product states provides an explanation for this behavior. Initial product states are also considered since in optical lattice experiments, such states are often used as the initial setup. We propose configurations that lead to a fast and efficient quantum distillation.

  12. Efficient transfer of francium atoms

    Science.gov (United States)

    Aubin, Seth; Behr, John; Gorelov, Alexander; Pearson, Matt; Tandecki, Michael; Collister, Robert; Gwinner, Gerald; Shiells, Kyle; Gomez, Eduardo; Orozco, Luis; Zhang, Jiehang; Zhao, Yanting; FrPNC Collaboration

    2016-05-01

    We report on the progress of the FrPNC collaboration towards Parity Non Conservation Measurements (PNC) using francium atoms at the TRIUMF accelerator. We demonstrate efficient transfer (higher than 40%) to the science vacuum chamber where the PNC measurements will be performed. The transfer uses a downward resonant push beam from the high-efficiency capture magneto optical trap (MOT) towards the science chamber where the atoms are recaptured in a second MOT. The transfer is very robust with respect to variations in the parameters (laser power, detuning, alignment, etc.). We accumulate a growing number of atoms at each transfer pulse (limited by the lifetime of the MOT) since the push beam does not eliminate the atoms already trapped in the science MOT. The number of atoms in the science MOT is on track to meet the requirements for competitive PNC measurements when high francium rates (previously demonstrated) are delivered to our apparatus. The catcher/neutralizer for the ion beam has been tested reliably to 100,000 heating/motion cycles. We present initial tests on the direct microwave excitation of the ground hyperfine transition at 45 GHz. Support from NSERC and NRC from Canada, NSF and Fulbright from USA, and CONACYT from Mexico.

  13. State ownership and efficiency characteristics

    Directory of Open Access Journals (Sweden)

    Alexander Abramov

    2017-06-01

    Full Text Available This study examines the influence of state participation in the ownership structure of companies on their financial efficiency using a sample of 114 largest companies in Russia. As an indirect indicator of efficiency, we used a variety of financial indicators: revenue per employee (gross margin, return on equity, profit margin and debt burden. The effects of direct and indirect state ownership are considered separately. Using econometric analysis, we conclude that the dominance of the block of shares owned by the state has a negative effect on the performance characteristics, and its increase is associated with an increase in the debt burden of the companies. According to our criteria, state-owned enterprises (SOEs perform worse on average than private companies. The mechanism of how changes in the “real sector” affect profitability is examined particularly closely. The study shows that a change in the profitability of private companies is characterized by a significant dependence on the movement of labor productivity characteristics. At the same time, for SOEs, a similar correlation was not revealed. These companies demonstrated no visible relationship between their profitability and performance characteristics. The study shows that increases in the size of direct government ownership lead to lower labor productivity and profitability; the impact of indirect ownership is, seemingly, more complicated.

  14. Quantitative Efficiency Evaluation Method for Transportation Networks

    Directory of Open Access Journals (Sweden)

    Jin Qin

    2014-11-01

    Full Text Available An effective evaluation of transportation network efficiency/performance is essential to the establishment of sustainable development in any transportation system. Based on a redefinition of transportation network efficiency, a quantitative efficiency evaluation method for transportation network is proposed, which could reflect the effects of network structure, traffic demands, travel choice, and travel costs on network efficiency. Furthermore, the efficiency-oriented importance measure for network components is presented, which can be used to help engineers identify the critical nodes and links in the network. The numerical examples show that, compared with existing efficiency evaluation methods, the network efficiency value calculated by the method proposed in this paper can portray the real operation situation of the transportation network as well as the effects of main factors on network efficiency. We also find that the network efficiency and the importance values of the network components both are functions of demands and network structure in the transportation network.

  15. Efficiency of osmotic pipe flows

    DEFF Research Database (Denmark)

    Haaning, Louise Sejling; Jensen, Kaare Hartvig; Helix Nielsen, Claus

    2013-01-01

    on the relative magnitude of radial diffusion and advection as well as the ratio of the osmotic velocity to pumping velocity, in very good agreement with experiments and with no adjustable parameters. Our analysis provides criteria that are useful for optimizing osmotic flow processes in, e.g., water purification......We present experiments and theory for flows of sugar or salt solutions in cylindrical tubes with semipermeable walls (hollow fiber membranes) immersed in water, quantifying the strength of the osmotic driving force in relation to the dimensionless parameters that specify the system. The pumping...... efficiency of these flows is limited by the presence of “unstirred” concentration boundary layers near the tube walls, and our primary aim is to understand and quantify these layers and their effect on the flow. We measure the outlet flow rate Qout while varying the inlet flow rate Q*, concentration c...

  16. Efficient universal blind quantum computation.

    Science.gov (United States)

    Giovannetti, Vittorio; Maccone, Lorenzo; Morimae, Tomoyuki; Rudolph, Terry G

    2013-12-06

    We give a cheat sensitive protocol for blind universal quantum computation that is efficient in terms of computational and communication resources: it allows one party to perform an arbitrary computation on a second party's quantum computer without revealing either which computation is performed, or its input and output. The first party's computational capabilities can be extremely limited: she must only be able to create and measure single-qubit superposition states. The second party is not required to use measurement-based quantum computation. The protocol requires the (optimal) exchange of O(Jlog2(N)) single-qubit states, where J is the computational depth and N is the number of qubits needed for the computation.

  17. The Efficiency of Educational Production

    DEFF Research Database (Denmark)

    Bogetoft, Peter; Heinesen, Eskil; Tranæs, Torben

    Denmark, Norway, New Zealand, Canada and the USA are the OECD countries that spend most on education, measured in relation to GDP. Focusing in particular on upper secondary education, this paper examines whether the heavy expenditure on education in Denmark is matched by high output from...... the educational sector, both in terms of a large number of students enrolled in educational programmes and a high completion rate. The methodology used is to compare (benchmark) Denmark with a relevant group of countries and to calculate how much cheaper Denmark could teach the same number of students...... and maintain the same graduation/completion rates as today if the country could achieve the same level of cost effectiveness as its most efficient counterparts. Comparing Denmark to a group of the richest OECD countries reveals that potential savings lie between 12 and 34 percent. Figures that fall to between...

  18. Transparent Electrodes for Efficient Optoelectronics

    KAUST Repository

    Morales-Masis, Monica

    2017-03-30

    With the development of new generations of optoelectronic devices that combine high performance and novel functionalities (e.g., flexibility/bendability, adaptability, semi or full transparency), several classes of transparent electrodes have been developed in recent years. These range from optimized transparent conductive oxides (TCOs), which are historically the most commonly used transparent electrodes, to new electrodes made from nano- and 2D materials (e.g., metal nanowire networks and graphene), and to hybrid electrodes that integrate TCOs or dielectrics with nanowires, metal grids, or ultrathin metal films. Here, the most relevant transparent electrodes developed to date are introduced, their fundamental properties are described, and their materials are classified according to specific application requirements in high efficiency solar cells and flexible organic light-emitting diodes (OLEDs). This information serves as a guideline for selecting and developing appropriate transparent electrodes according to intended application requirements and functionality.

  19. Efficiency of some spectrochemical carriers

    International Nuclear Information System (INIS)

    Gomes, R.P.

    1978-01-01

    A comparative study of the efficiency of some spectrochemical carriers for the quantitative spectrographic analysis of Ag, Al, B, Bi, Cd, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, Pb, Si, Sn, V and Zn in uranium-base materials is presented. The volatility behavior of the eighteen elements is verified by means of the moving plate technique and each of the mentioned carriers. The best results are obtained with 4% In 2 O 3 , 6% AgCl and 5% NaF in a U 3 O 8 matrix. The sensitivities for some elements were extended to fractions of p.p.m. The precision, accuracy and acceptability of the method are calculated for all elements. The total error values as approximately in the range of 16-45% [pt

  20. Incentives – Effectiveness and efficiency

    Directory of Open Access Journals (Sweden)

    Björn Hinderlich

    2014-03-01

    Full Text Available This paper covers the question if and how incentive schemes work evaluated by their impact on company performance (market capitalization and profit before tax. Based on a unique data set for German executive directors of DAX companies it can be proved that neither short (STI nor long term incentives (LTI plans necessarily support the company success. It rather depends on the efficiency of each plan, i. e. on its design. Special attention has to be paid on target setting. Short term focused objectives often miss their targets, whereas long term oriented objectives significantly support the company success. To solve the prisoner’s dilemma between employers and employees by a quasi-endless game, additional measures may be helpful, such as share ownership guidelines.

  1. Efficient classification using salient regions

    Science.gov (United States)

    Yang, Bing; Xu, Duanqing

    2012-07-01

    Classification of images in many categorized datasets has rapidly improved in recent years. However, methods that perform well on particular datasets typically have one or more limitations, such as insufficient image-transformation invariance or significant performance degradation as the number of classes is increased. We attempt to overcome these challenges by extracting and matching visual features only at the focuses of visual saliency instead of the entire scene. First, we propose a visual-saliency detection method that combines the respective merits of color-saliency boosting and global-region-based contrast schemes to achieve more accurate saliency maps. Using a single feature type, we then obtain good performance on three public datasets when compared to other state-of-the-art approaches. Overall, our results prove that robust and efficient fixation-based classification, in terms of reducing the complexity of feature extraction, is possible.

  2. Method for Refrigerators Efficiency Increasing

    Science.gov (United States)

    Suchilin, V. A.; Sumzina, L. V.; Maksimov, A. V.

    2017-11-01

    The article shows that in condenser-type domestic refrigerators the condenser is one of the main parts of the refrigeration system where the refrigerant (freon) compressed by the compressor condenses from the vapor state to the liquid. The condensation process is an exothermic process, i.e., the condensation of steam generates thermal energy. The condenser is heated and, to perform the refrigeration cycle, this heat must be removed from the refrigeration system. Heat removal is carried out in household refrigerators due to the natural circulation of ambient air around the condenser, which must have a significant heat-reflecting area to do this. Although the condenser is most often made of thin tubes that occupy almost the entire rear wall of the refrigerator, the cooling process is not effective enough. A new method for the condenser cooling is proposed which will improve the efficiency of the condenser and household refrigerators as a whole.

  3. Energy efficient sensor network implementations

    Energy Technology Data Exchange (ETDEWEB)

    Frigo, Janette R [Los Alamos National Laboratory; Raby, Eric Y [Los Alamos National Laboratory; Brennan, Sean M [Los Alamos National Laboratory; Kulathumani, Vinod [WEST VIRGINIA UNIV.; Rosten, Ed [CAMBRIDGE UNIV.; Wolinski, Christophe [IRISA; Wagner, Charles [IRISA; Charot, Francois [IRISA

    2009-01-01

    In this paper, we discuss a low power embedded sensor node architecture we are developing for distributed sensor network systems deployed in a natural environment. In particular, we examine the sensor node for energy efficient processing-at-the-sensor. We analyze the following modes of operation; event detection, sleep(wake-up), data acquisition, data processing modes using low power, high performance embedded technology such as specialized embedded DSP processors and a low power FPGAs at the sensing node. We use compute intensive sensor node applications: an acoustic vehicle classifier (frequency domain analysis) and a video license plate identification application (learning algorithm) as a case study. We report performance and total energy usage for our system implementations and discuss the system architecture design trade offs.

  4. Energy efficiency in the foreground

    International Nuclear Information System (INIS)

    Baettig, I.

    2006-01-01

    In this interview with Eberhard Jochem, professor at the Centre for Energy Policy and Economics at the Federal Institute of Science and Technology (ETH) in Zurich, Switzerland, several energy-relevant topics are discussed. These include high oil prices, possible power shortages and binding commitments in the climate-protection area. The question is asked, how, in consideration of such general conditions, energy use and energy supply should develop in Switzerland. Options for increasing efficiency or the tapping of new energy sources is discussed, as is Switzerland's increasing energy consumption. The '2000 Watt' concept being worked on at the ETH and the activities needed for its realisation are discussed. The effects of this concept on economical and business development are discussed. The potential of renewable forms of energy and the possibility of building combined gas and steam power stations are looked at. Ways of promoting renewable energy and questions concerning the extent of the state intervention in the energy business are considered

  5. Energy Efficiency in Manufacturing Systems

    CERN Document Server

    Thiede, Sebastian

    2012-01-01

    Energy consumption is of great interest to manufacturing companies. Beyond considering individual processes and machines, the perspective on process chains and factories as a whole holds major potentials for energy efficiency improvements. To exploit these potentials, dynamic interactions of different processes as well as auxiliary equipment (e.g. compressed air generation) need to be taken into account. In addition, planning and controlling manufacturing systems require  balancing technical, economic and environmental objectives. Therefore, an innovative and comprehensive methodology – with a generic energy flow-oriented manufacturing simulation environment as a core element – is developed and embedded into a step-by-step application cycle. The concept is applied in its entirety to a wide range of case studies such as aluminium die casting, weaving mills, and printed circuit board assembly in order to demonstrate the broad applicability and the benefits that can be achieved.

  6. Motor Carrier Efficiency Study Phase I

    Science.gov (United States)

    2009-02-01

    The Safe, Accountable, Flexible, Efficient Transportation Equity Act: A Legacy for Users (SAFETEA-LU, Public Law 109-59), Section 5503, set aside funding to examine the application of wireless technology to improve the safety and efficiency of trucki...

  7. Improving pumping system efficiency at coal plants

    Energy Technology Data Exchange (ETDEWEB)

    Livoti, W.C.; McCandless, S.; Poltorak, R. [Baldor Electric Co. (United States)

    2009-03-15

    The industry must employ ultramodern technologies when building or upgrading power plant pumping systems thereby using fuels more efficiently. The article discusses the uses and efficiencies of positive displacement pumps, centrifugal pumps and multiple screw pumps. 1 ref., 4 figs.

  8. LiDAR for data efficiency.

    Science.gov (United States)

    2011-09-30

    This report documents the AHMCT research project: LiDAR for Data Efficiency for the Washington State Department of Transportation (WSDOT). The research objective was to evaluate mobile LiDAR technology to enhance safety, determine efficiency ga...

  9. The ways of SOFC systems efficiency increasing

    Energy Technology Data Exchange (ETDEWEB)

    Demin, A.K.; Timofeyeva, N.

    1996-04-01

    The efficiency of solid oxide fuel cells (SOFCs) is described. This paper considers methods to lift the fuel utilization and/or the average cell voltage with the goal of increasing the cell efficiency by improved cell designs.

  10. Frontiers in the economics of energy efficiency

    International Nuclear Information System (INIS)

    Miguel, Carlos de; Labandeira, Xavier; Löschel, Andreas

    2015-01-01

    Energy efficiency has become an essential instrument to obtain effective greenhouse gas mitigation and reduced energy dependence. This introductory article contextualizes the contributions of the supplemental issue by showing the new setting for energy efficiency economics and policy; discussing the role of price instruments to promote energy savings; presenting new approaches for energy efficiency policies; and placing energy efficiency within a wider energy and environmental framework.

  11. Power-efficient computer architectures recent advances

    CERN Document Server

    Själander, Magnus; Kaxiras, Stefanos

    2014-01-01

    As Moore's Law and Dennard scaling trends have slowed, the challenges of building high-performance computer architectures while maintaining acceptable power efficiency levels have heightened. Over the past ten years, architecture techniques for power efficiency have shifted from primarily focusing on module-level efficiencies, toward more holistic design styles based on parallelism and heterogeneity. This work highlights and synthesizes recent techniques and trends in power-efficient computer architecture.Table of Contents: Introduction / Voltage and Frequency Management / Heterogeneity and Sp

  12. Global status report on energy efficiency 2008

    OpenAIRE

    Blok, K.; van Breevoort, P.; Roes, A.L.; Coenraads, R.; Müller, N.

    2008-01-01

    There is wide agreement that energy efficiency improvement is one of the key strategies to achieve greater sustainability of the energy system. In the past, the contribution of energy efficiency has already been considerable.Without the energy efficiency improvements achieved since the 1970s, current energy use would have been much higher. However, the potential for energy efficiency improvement is much larger than has already been implemented. In the period leading to 2050, it is possible to...

  13. Thermoelectric efficiency of nanowires in contact

    Science.gov (United States)

    Akguc, Gursoy B.

    2018-02-01

    Divergence of density of state near Fermi level at low dimensional systems helps to increase efficiency of thermoelectric materials. It is shown here two contacting nanowires in the middle results in electron conduction resonances that enhance the efficiency of overall thermoelectric output of the system. A non-linear response approach as well as efficiency at max power are necessary ingredients for the analysis.

  14. Global status report on energy efficiency 2008

    NARCIS (Netherlands)

    Blok, K.; van Breevoort, P.; Roes, A.L.; Coenraads, R.; Müller, N.

    2008-01-01

    There is wide agreement that energy efficiency improvement is one of the key strategies to achieve greater sustainability of the energy system. In the past, the contribution of energy efficiency has already been considerable.Without the energy efficiency improvements achieved since the 1970s,

  15. High efficiency, long life terrestrial solar panel

    Science.gov (United States)

    Chao, T.; Khemthong, S.; Ling, R.; Olah, S.

    1977-01-01

    The design of a high efficiency, long life terrestrial module was completed. It utilized 256 rectangular, high efficiency solar cells to achieve high packing density and electrical output. Tooling for the fabrication of solar cells was in house and evaluation of the cell performance was begun. Based on the power output analysis, the goal of a 13% efficiency module was achievable.

  16. Consumer Efficiency in Conflict with GDP Growth

    DEFF Research Database (Denmark)

    Nørgaard, Jørgen

    2006-01-01

    During the last couple of decades some attention has been devoted to the potential for utilizing energy more efficiently. Most of this debate has not concentrated on the energy efficiency of the economy as a whole, but merely addressed options for implementing more energy efficient technology. Th...

  17. Efficiency of Health Investment: Education or Intelligence?

    NARCIS (Netherlands)

    Bijwaard, G.E.; van Kippersluis, H.

    2016-01-01

    In this paper, we hypothesize that education is associated with a higher efficiency of health investment, yet that this efficiency advantage is solely driven by intelligence. We operationalize efficiency of health investment as the probability of dying conditional on a certain hospital diagnosis and

  18. Efficiency of Health Investment: Education or Intelligence?

    NARCIS (Netherlands)

    Bijwaard, G.E.; van Kippersluis, H.

    2015-01-01

    In this paper we hypothesize that education is associated with a higher efficiency of health investment, yet that this efficiency advantage is solely driven by intelligence. We operationalize efficiency of health investment as the probability of dying conditional on a certain hospital diagnosis, and

  19. Energy efficiency public service advertising campaign

    Energy Technology Data Exchange (ETDEWEB)

    Gibson-Grant, Amanda [Advertising Council, New York, NY (United States)

    2015-06-12

    The Advertising Council (“the Ad Council”) and The United States Department of Energy (DOE) created and launched a national public service advertising campaign designed to promote energy efficiency. The objective of the Energy Efficiency campaign was to redefine how consumers approach energy efficiency by showing that saving energy can save homeowners money.

  20. Efficiency of Health Investment: Education or Intelligence?

    NARCIS (Netherlands)

    G.E. Bijwaard (Govert); J.L.W. van Kippersluis (Hans)

    2016-01-01

    textabstractIn this paper, we hypothesize that education is associated with a higher efficiency of health investment, yet that this efficiency advantage is solely driven by intelligence. We operationalize efficiency of health investment as the probability of dying conditional on a certain hospital