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

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

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.

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.

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

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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.

Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors.

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Galimi, Francesco; Noll, Meenakshi; Kanazawa, Yoshiyuki; Lax, Timothy; Chen, Cindy; Grompe, Markus; Verma, Inder M

2002-10-15

Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.

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

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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.

[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.

Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

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Anne-Christine Field

Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

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 ...

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

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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.

Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.

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Pavlik, Benjamin J; Hruska, Elizabeth J; Van Cott, Kevin E; Blum, Paul H

2016-03-30

Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.

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

Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing

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Pavel I. Ortinski

2017-06-01

Full Text Available The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs present an attractive means for delivery of CRISPR/Cas9 components because: (1 they are capable of transducing a broad range of cells and tissues, (2 have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors, and (3 they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

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

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

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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.

Design and Potential of Non-Integrating Lentiviral Vectors

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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.

Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs.

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Molina-Estevez, F Javier; Nowrouzi, Ali; Lozano, M Luz; Galy, Anne; Charrier, Sabine; von Kalle, Christof; Guenechea, Guillermo; Bueren, Juan A; Schmidt, Manfred

2015-01-01

Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.

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

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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.

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

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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.

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

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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.

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

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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.

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector

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Lauro Augusto de Oliveira

2010-10-01

Full Text Available PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS. We evaluated the transduction efficiency (TE over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells. RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1% and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

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

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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.

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 ...

Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy.

Science.gov (United States)

Cartier, Nathalie; Hacein-Bey-Abina, Salima; Bartholomae, Cynthia C; Bougnères, Pierre; Schmidt, Manfred; Kalle, Christof Von; Fischer, Alain; Cavazzana-Calvo, Marina; Aubourg, Patrick

2012-01-01

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic demyelinating disease caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. When performed at an early stage of the disease, allogeneic hematopoietic stem cell transplantation (HCT) can arrest the progression of cerebral demyelinating lesions. To overcome the limitations of allogeneic HCT, hematopoietic stem cell (HSC) gene therapy strategy aiming to perform autologous transplantation of lentivirally corrected cells was developed. We demonstrated the preclinical feasibility of HSC gene therapy for ALD based on the correction of CD34+ cells from X-ALD patients using an HIV1-derived lentiviral vector. These results prompted us to initiate an HSC gene therapy trial in two X-ALD patients who had developed progressive cerebral demyelination, were candidates for allogeneic HCT, but had no HLA-matched donors or cord blood. Autologous CD34+ cells were purified from the peripheral blood after G-CSF stimulation, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1 cDNA, and then reinfused into the patients after they had received full myeloablative conditioning. Over 3 years of follow-up, the hematopoiesis remained polyclonal in the two patients treated with 7-14% of granulocytes, monocytes, and T and B lymphocytes expressing the lentivirally encoded ALD protein. There was no evidence of clonal dominance or skewing based on the retrieval of lentiviral insertion repertoire in different hematopoietic lineages by deep sequencing. Cerebral demyelination was arrested 14 and 16months, respectively, in the two treated patients, without further progression up to the last follow-up, a clinical outcome that is comparable to that observed after allogeneic HCT. Longer follow-up of these two treated patients and HSC gene therapy performed in additional ALD patients are however needed to evaluate the safety and efficacy of lentiviral HSC

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions

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Kenta Kobayashi

2017-08-01

Full Text Available Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1 with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G and vesicular stomatitis virus glycoprotein (VSV-G enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E, which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E–pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson’s disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E–pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson’s disease.

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.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent.

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Lévy, Camille; Amirache, Fouzia; Girard-Gagnepain, Anais; Frecha, Cecilia; Roman-Rodríguez, Francisco J; Bernadin, Ornellie; Costa, Caroline; Nègre, Didier; Gutierrez-Guerrero, Alejandra; Vranckx, Lenard S; Clerc, Isabelle; Taylor, Naomi; Thielecke, Lars; Cornils, Kerstin; Bueren, Juan A; Rio, Paula; Gijsbers, Rik; Cosset, François-Loïc; Verhoeyen, Els

2017-10-24

Hematopoietic stem cell (HSC)-based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein-displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34 + (hCD34 + ) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34 + cells, whereas conventional vesicular stomatitis virus G (VSV-G)-LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc -/- (NSG) mice with H/F-LV transduced prestimulated or resting hCD34 + cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34 + cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34 + progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34 + cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.

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

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Xianqi Zhao

2015-06-01

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

Efficient and sustained IGF-1 expression in the adipose tissue-derived stem cells mediated via a lentiviral vector.

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Chen, Ting; Huang, Dangsheng; Chen, Guanghui; Yang, Tingshu; Yi, Jun; Tian, Miao

2015-02-01

The adipose tissue-derived stem cells (ADSCs) represent a significant area of the cell therapy. Genetic modification of ADSCs may further improve their therapeutic potential. Here, we aimed to generate a lentiviral vector expressing insulin-like growth factor-I (IGF-1) and investigate the impact of IGF-1 transduction on the properties of cultured ADSCs. Isolated rat ADSCs were assessed by flow cytometric analysis. IGF-1 was cloned and inserted into the pLenO-DCE plasmid to acquire pLenO-DCE-IGF-1 plasmid. Lentivirus was enveloped with pRsv-REV, pMDlg-pRRE and pMD2G plasmids in 293T cells. The ADSCs were transfected with the vectors. And then IGF-1-induced anti-apoptosis was evaluated by annexin V-FITC. Besides, proliferation of cells was detected by MTT assay and EdU. Moreover, Akt phosphorylation was evaluated by Western blotting analysis. Stable expression of IGF-1 in ADSCs was confirmed. ADSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45. The transduction of IGF-1 to the ADSCs caused a dramatic increase in P-Akt expression. Over-expression of IGF-1 in ADSCs could improve the paracine of IGF-1 in a time-dependent manner, but could not promote the proliferation of ADSCs. This study indicated that lentiviral vectors offered a promising mean of delivering IGF-1 to the ADSCs. Lentiviral-mediated over-expression of therapeutic IGF-1 gene in ADSCs could prolong the anti-apoptosis effect of IGF-1, which might be induced by the activation of the PI3K/Akt pathway. And our data would improve the efficacy of ADSC-based therapies.

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

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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.

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

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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.

The feasibility of incorporating Vpx into lentiviral gene therapy vectors

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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.

Improved content aware scene retargeting for retinitis pigmentosa patients

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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

A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.

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Stephen Hare

2009-01-01

Full Text Available Lens epithelium derived growth factor (LEDGF, also known as PC4 and SFRS1 interacting protein 1 (PSIP1 and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD of HIV-2 IN in complex with the IN binding domain (IBD of LEDGF. The structure extends the known IN-LEDGF interface, elucidating primarily charge-charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD-IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN-LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

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Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-08-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

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

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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.

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

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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.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

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Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

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

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Margison Geoffrey P

2006-03-01

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

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

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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.

Construction of lentiviral shRNA expression vector targeting ...

African Journals Online (AJOL)

ajl yemi

2011-10-26

Oct 26, 2011 ... was then selected, while the titer of lentiviral packing PLD2-shRNA was 3.47 × 104 TU/ml and the virus was successfully ... MATERIALS AND METHODS .... such as: transfecting cells not only in mitotic active phase but also in ...

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

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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.

Non-Primate Lentiviral Vectors and Their Applications in Gene Therapy for Ocular Disorders

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Vincenzo Cavalieri

2018-06-01

Full Text Available Lentiviruses have a number of molecular features in common, starting with the ability to integrate their genetic material into the genome of non-dividing infected cells. A peculiar property of non-primate lentiviruses consists in their incapability to infect and induce diseases in humans, thus providing the main rationale for deriving biologically safe lentiviral vectors for gene therapy applications. In this review, we first give an overview of non-primate lentiviruses, highlighting their common and distinctive molecular characteristics together with key concepts in the molecular biology of lentiviruses. We next examine the bioengineering strategies leading to the conversion of lentiviruses into recombinant lentiviral vectors, discussing their potential clinical applications in ophthalmological research. Finally, we highlight the invaluable role of animal organisms, including the emerging zebrafish model, in ocular gene therapy based on non-primate lentiviral vectors and in ophthalmology research and vision science in general.

A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis.

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Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

2008-04-01

Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

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

International Nuclear Information System (INIS)

Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung; Kim, Yeon Soo

2004-01-01

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

Lentiviral Vectors for Cancer Immunotherapy and Clinical Applications

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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.

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.

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

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.

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

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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.

Large-scale production of lentiviral vector in a closed system hollow fiber bioreactor

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Jonathan Sheu

Full Text Available Lentiviral vectors are widely used in the field of gene therapy as an effective method for permanent gene delivery. While current methods of producing small scale vector batches for research purposes depend largely on culture flasks, the emergence and popularity of lentiviral vectors in translational, preclinical and clinical research has demanded their production on a much larger scale, a task that can be difficult to manage with the numbers of producer cell culture flasks required for large volumes of vector. To generate a large scale, partially closed system method for the manufacturing of clinical grade lentiviral vector suitable for the generation of induced pluripotent stem cells (iPSCs, we developed a method employing a hollow fiber bioreactor traditionally used for cell expansion. We have demonstrated the growth, transfection, and vector-producing capability of 293T producer cells in this system. Vector particle RNA titers after subsequent vector concentration yielded values comparable to lentiviral iPSC induction vector batches produced using traditional culture methods in 225 cm2 flasks (T225s and in 10-layer cell factories (CF10s, while yielding a volume nearly 145 times larger than the yield from a T225 flask and nearly three times larger than the yield from a CF10. Employing a closed system hollow fiber bioreactor for vector production offers the possibility of manufacturing large quantities of gene therapy vector while minimizing reagent usage, equipment footprint, and open system manipulation.

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

African Journals Online (AJOL)

USER

2010-03-29

Mar 29, 2010 ... Construction of the lentiviral expression vector. Both self-inactivating (SIN) ..... activated partial thromboplastin time. Figure 4. Expression of ... 1 entry to an endocytic pathway and suppresses both the requirement for Nef and ...

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 ...

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.

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

Science.gov (United States)

De Meyer, Simon F.; Vanhoorelbeke, Karen; Chuah, Marinee K.; Pareyn, Inge; Gillijns, Veerle; Hebbel, Robert P.; Collen, Désiré; Deckmyn, Hans; VandenDriessche, Thierry

2006-01-01

Von Willebrand disease (VWD) is an inherited bleeding disorder, caused by quantitative (type 1 and 3) or qualitative (type 2) defects in von Willebrand factor (VWF). Gene therapy is an appealing strategy for treatment of VWD because it is caused by a single gene defect and because VWF is secreted into the circulation, obviating the need for targeting specific organs or tissues. However, development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA (8.4 kb [kilobase]) and the inherent complexity of the VWF protein that requires extensive posttranslational processing. In this study, a gene-based approach for VWD was developed using lentiviral transduction of blood-outgrowth endothelial cells (BOECs) to express functional VWF. A lentiviral vector encoding complete human VWF was used to transduce BOECs isolated from type 3 VWD dogs resulting in high-transduction efficiencies (95.6% ± 2.2%). Transduced VWD BOECs efficiently expressed functional vector-encoded VWF (4.6 ± 0.4 U/24 hour per 106 cells), with normal binding to GPIbα and collagen and synthesis of a broad range of multimers resulting in phenotypic correction of these cells. These results indicate for the first time that gene therapy of type 3 VWD is feasible and that BOECs are attractive target cells for this purpose. PMID:16478886

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

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Emanuela Chiarella

Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Science.gov (United States)

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

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.

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.

Prolonged liver-specific transgene expression by a non-primate lentiviral vector

International Nuclear Information System (INIS)

Condiotti, Reba; Curran, Michael A.; Nolan, Garry P.; Giladi, Hilla; Ketzinel-Gilad, Mali; Gross, Eitan; Galun, Eithan

2004-01-01

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease

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.

Construction Of An Optimized Lentiviral Vector Containing Pdx-1 Gene For Transduction Of Stem Cells Towards Gene Therapy Diabetes Type 1

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S Rahmati

2013-02-01

Full Text Available Abstract Background & aim: Nowadays, most of gene therapy protocols are performed by lentiviral vectors. One of the most important factors which is involved in pancreas development and transcription of insulin gene is pancreatic & duodenal homeobox 1 (PDX-1 transcription factor. The goal of this study was to optimize a lentiviral construct, containing pdx-1 gene, to transfect stem cells towards gene therapy of type-1 diabetes. Methods: In this experimental study, first, the pdx-1 gene was multiplied by PCR from pcDNA3.1-pdx-1 and cloned into pTG19-T vector. Then, pdx-1 was subcloned on upstream of IRES-EGFP gene into IRES2-EGFP vector. At the next step, the cloned parts of IRES-EGFP and pdx-1 were isolated and cloned into the lentiviral expression vector pSINTREM in upstream of TRE-CMV gene. After sequencing, final construct was transfected into HEK 293 cells and gene expression of pdx-1 was evaluated using flow cytometry analysis and reverse fluorescent microscopy. Results: Flow cytometry results and inverted fluorescent microscopy observing showed that pdx-1 and GFP genes are expressed in cells transfected with final recombinant construct. Conclusion: Regarding the design of this construct, to ensure long time expression with higher in vivo and in vitro expression efficiency for stem cells and also use of Tet on induced optimized system, it seems that the current construct can be among the best ones to transfect stem cells. Key words: Gene therapy, Diabetes, Stem cells

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

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

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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.

Production of lentiviral vectors

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Otto-Wilhelm Merten

2016-01-01

Full Text Available Lentiviral vectors (LV have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.

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.

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.

Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction.

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Paul Lin

Full Text Available Human mesenchymal stem cells (hMSCs can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1-8 µg/mL negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr. Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging

NARCIS (Netherlands)

Pfeifer, A.; Kessler, T.; Yang, M.; Baranov, E.; Kootstra, N.; Cheresh, D. A.; Hoffman, R. M.; Verma, I. M.

2001-01-01

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for

Evaluation of Lentiviral-Mediated Expression of Sodium Iodide Symporter in Anaplastic Thyroid Cancer and the Efficacy of In Vivo Imaging and Therapy

Directory of Open Access Journals (Sweden)

Chien-Chih Ke

2011-01-01

Full Text Available Anaplastic thyroid carcinoma (ATC is one of the most deadly cancers. With intensive multimodalities of treatment, the survival remains low. ATC is not sensitive to 131I therapy due to loss of sodium iodide symporter (NIS gene expression. We have previously generated a stable human NIS-expressing ATC cell line, ARO, and the ability of iodide accumulation was restored. To make NIS-mediated gene therapy more applicable, this study aimed to establish a lentiviral system for transferring hNIS gene to cells and to evaluate the efficacy of in vitro and in vivo radioiodide accumulation for imaging and therapy. Lentivirus containing hNIS cDNA were produced to transduce ARO cells which do not concentrate iodide. Gene expression, cell function, radioiodide imaging and treatment were evaluated in vitro and in vivo. Results showed that the transduced cells were restored to express hNIS and accumulated higher amount of radioiodide than parental cells. Therapeutic dose of 131I effectively inhibited the tumor growth derived from transduced cells as compared to saline-treated mice. Our results suggest that the lentiviral system efficiently transferred and expressed hNIS gene in ATC cells. The transduced cells showed a promising result of tumor imaging and therapy.

Rapid lentiviral transduction preserves the engraftment potential of Fanca(-/-) hematopoietic stem cells.

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Müller, Lars U W; Milsom, Michael D; Kim, Mi-Ok; Schambach, Axel; Schuesler, Todd; Williams, David A

2008-06-01

Fanconi anemia (FA) is a rare recessive syndrome, characterized by congenital anomalies, bone marrow failure, and predisposition to cancer. Two earlier clinical trials utilizing gamma-retroviral vectors for the transduction of autologous FA hematopoietic stem cells (HSCs) required extensive in vitro manipulation and failed to achieve detectable long-term engraftment of transduced HSCs. As a strategy for minimizing ex vivo manipulation, we investigated the use of a "rapid" lentiviral transduction protocol in a murine Fanca(-/-) model. Importantly, while this and most murine models of FA fail to completely mimic the human hematopoietic phenotype, we observed a high incidence of HSC transplant engraftment failure and low donor chimerism after conventional transduction (CT) of Fanca(-/-) donor cells. In contrast, rapid transduction (RT) of Fanca(-/-) HSCs preserved engraftment to the level achieved in wild-type cells, resulting in long-term multilineage engraftment of gene-modified cells. We also demonstrate the correction of the characteristic hypersensitivity of FA cells against the cross-linking agent mitomycin C (MMC), and provide evidence for the advantage of using pharmacoselection as a means of further increasing gene-modified cells after RT. Collectively, these data support the use of rapid lentiviral transduction for gene therapy in FA.

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

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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.

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 boundary...

Reliable and inexpensive expression of large, tagged, exogenous proteins in murine bone marrow-derived macrophages using a second generation lentiviral system

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Matthew R. Miller

2015-08-01

Full Text Available Over the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection. This method utilizes laboratory-propagated second generation plasmids to produce efficient virus that can be stored for later use. The expression of proteins up to 150 kDa in size is achieved in 30–70% of cells while maintaining normal macrophage differentiation and morphology as determined by fluorescence microscopy and Western blot analysis. This manuscript delineates the reagents and methods used to produce lentivirus to express exogenous DNA in murine bone marrow-derived macrophages sufficient for single cell microscopy as well as functional assays requiring large numbers of murine bone marrow-derived macrophages.

Neuron-specific RNA interference using lentiviral vectors

DEFF Research Database (Denmark)

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

2009-01-01

BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise...... transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding...... the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We...

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

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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.

Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.

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Merten, Otto-Wilhelm; Charrier, Sabine; Laroudie, Nicolas; Fauchille, Sylvain; Dugué, Céline; Jenny, Christine; Audit, Muriel; Zanta-Boussif, Maria-Antonietta; Chautard, Hélène; Radrizzani, Marina; Vallanti, Giuliana; Naldini, Luigi; Noguiez-Hellin, Patricia; Galy, Anne

2011-03-01

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

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Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

2015-01-01

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers.

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

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Gideon Hen

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

Inhibition of experimental lung metastasis by systemic lentiviral delivery of kallistatin

International Nuclear Information System (INIS)

Shiau, Ai-Li; Wu, Chao-Liang; Lee, Che-Hsin; Teo, Min-Li; Chen, Shin-Yao; Wang, Chrong-Reen; Hsieh, Jeng-Long; Chang, Meng-Ya; Chang, Chih-Jui; Chao, Julie; Chao, Lee

2010-01-01

Angiogenesis plays an important role in the development and progression of tumors. Kallistatin exerts anti-angiogenic and anti-inflammatory activities that may be effective in inhibiting tumor metastasis. We investigated the antitumor effect of lentivirus-mediated kallistatin gene transfer in a syngeneic murine tumor model. Lentiviral vector encoding kallistatin (LV-Kallistatin) was constructed. The expression of kallistatin was verified by enzyme-linked immunosorbent assay (ELISA), and the bioactivity of kallistatin was determined by using cell proliferation, migration, and invasion assays. In addition, antitumor effects of LV-Kallistatin were evaluated by the intravenous injection of virus into tumor-bearing mice. The conditioned medium from LV-Kallistatin-treated cells inhibited the migration and proliferation of endothelial cells. Meanwhile, it also reduced the migration and invasion of tumor cells. In the experimental lung metastatic model, tumor-bearing mice receiving LV-Kallistatin had lower tumor nodules and longer survival than those receiving control virus or saline. Moreover, the microvessel densities, the levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, and nuclear factor κB (NF-κB) transcriptional activity were reduced in the LV-Kallistatin-treated mice. Results of this study showed that systemic administration of lentiviral vectors encoding kallistatin inhibited the growth of metastatic tumor and prolonged the survival of tumor-bearing mice. These results suggest that gene therapy using lentiviruses carrying the kallistatin gene, which exerts anti-angiogenic and anti-inflammatory activities, represents a promising strategy for the treatment of lung cancer

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

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.

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

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L. Ding

2014-06-01

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

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.

Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling▿ †

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Stolp, Bettina; Abraham, Libin; Rudolph, Jochen M.; Fackler, Oliver T.

2010-01-01

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2 (HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo. PMID:20147394

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

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Zulema Romero

Full Text Available Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

[Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer].

Science.gov (United States)

FENG, Yan-ming; WU, Yi-ming; TU, Xin-ming; XU, Zheng-shun; WU, Wei-dong

2010-02-01

To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer. First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM. It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (Ppathways may lead to new targeted therapies for non-small cell lung cancer.

[Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

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Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

2013-04-01

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

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

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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.

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

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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.

Inhibition of experimental lung metastasis by systemic lentiviral delivery of kallistatin

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Chao Julie

2010-05-01

Full Text Available Abstract Background Angiogenesis plays an important role in the development and progression of tumors. Kallistatin exerts anti-angiogenic and anti-inflammatory activities that may be effective in inhibiting tumor metastasis. We investigated the antitumor effect of lentivirus-mediated kallistatin gene transfer in a syngeneic murine tumor model. Methods Lentiviral vector encoding kallistatin (LV-Kallistatin was constructed. The expression of kallistatin was verified by enzyme-linked immunosorbent assay (ELISA, and the bioactivity of kallistatin was determined by using cell proliferation, migration, and invasion assays. In addition, antitumor effects of LV-Kallistatin were evaluated by the intravenous injection of virus into tumor-bearing mice. Results The conditioned medium from LV-Kallistatin-treated cells inhibited the migration and proliferation of endothelial cells. Meanwhile, it also reduced the migration and invasion of tumor cells. In the experimental lung metastatic model, tumor-bearing mice receiving LV-Kallistatin had lower tumor nodules and longer survival than those receiving control virus or saline. Moreover, the microvessel densities, the levels of vascular endothelial growth factor (VEGF, tumor necrosis factor (TNF-α, and nuclear factor κB (NF-κB transcriptional activity were reduced in the LV-Kallistatin-treated mice. Conclusion Results of this study showed that systemic administration of lentiviral vectors encoding kallistatin inhibited the growth of metastatic tumor and prolonged the survival of tumor-bearing mice. These results suggest that gene therapy using lentiviruses carrying the kallistatin gene, which exerts anti-angiogenic and anti-inflammatory activities, represents a promising strategy for the treatment of lung cancer.

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

DEFF Research Database (Denmark)

GLUD, AN; Hedegaard, Claus; Nielsen, Mette Slot

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...... per 2.5 microliters lentivirus capable of transducing cells and mediating expression of recombinant EGFP. The animals were euthanized after four (n=3) or twenty weeks (n=6). Fresh brain tissue from three animals was used for PCR. The remaining six brains were cryo- or paraffin...

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

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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.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

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Stephanie Friedrichs

2015-01-01

Full Text Available Disease-specific induced pluripotent stem (iPS cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

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

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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.

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...

Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils.

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Zhu, Xin-Hua; Liao, Bing; Xu, Yi; Liu, Ke; Huang, Yun; Huang, Quan-Long; Liu, Yue-Hui

2017-02-01

RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

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.

[Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

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Zhu, Jing; Chen, Bo-Jiang; Huang, Na; Li, Wei-Min

2014-03-01

To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells. Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot. AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot. The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.

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

, including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase...

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

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

2013-10-18

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

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

International Nuclear Information System (INIS)

Cheng, Lei; Liu, Yi; Zhao, Hua; Zhang, Wen; Guo, Ying-Jun; Nie, Lin

2013-01-01

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

Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

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Alaín González Pose

2014-09-01

Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

Characterization of complete particles (VSV-G/SIN-GFP) and empty particles (VSV-G/EMPTY) in human immunodeficiency virus type 1-based lentiviral products for gene therapy: potential applications for improvement of product quality and safety.

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Zhao, Yuan; Keating, Kenneth; Dolman, Carl; Thorpe, Robin

2008-05-01

Lentiviral vectors persist in the host and are therefore ideally suited for long-term gene therapy. To advance the use of lentiviral vectors in humans, improvement of their production, purification, and characterization has become increasingly important and challenging. In addition to cellular contaminants derived from packaging cells, empty particles without therapeutic function are the major impurities that compromise product safety and efficacy. Removal of empty particles is difficult because of their innate similarity in particle size and protein composition to the complete particles. We propose that comparison of the properties of lentiviral products with those of purposely expressed empty particles may reveal potential differences between empty and complete particles. For this, three forms of recombinant lentiviral samples, that is, recombinant vesicular stomatitis virus glycoprotein (VSV-G) proteins, empty particles (VSV-G/Empty), and complete particles (VSV-G/SIN-GFP) carrying viral RNA, were purified by size-exclusion chromatography (SEC). The SEC-purified samples were further analyzed by immunoblotting with six antibodies to examine viral and cellular proteins associated with the particles. This study has demonstrated, for the first time, important differences between VSV-G/Empty particles and complete VSV-G/SIN-GFP particles. Differences include the processing of Gag protein and the inclusion of cellular proteins in the particles. Our findings support the development of improved production, purification, and characterization methods for lentiviral products.

Lentiviral Delivery of a Vesicular Glutamate Transporter 1 (VGLUT1)-Targeting Short Hairpin RNA Vector Into the Mouse Hippocampus Impairs Cognition

NARCIS (Netherlands)

King, Madeleine V.; Kurian, Nisha; Qin, Si; Papadopoulou, Nektaria; Westerink, Ben H. C.; Cremers, Thomas I.; Epping-Jordan, Mark P.; Le Poul, Emmanuel; Ray, David E.; Fone, Kevin C. F.; Kendall, David A.; Marsden, Charles A.; Sharp, Tyson V.

Glutamate is the principle excitatory neurotransmitter in the mammalian brain, and dysregulation of glutamatergic neurotransmission is implicated in the pathophysiology of several psychiatric and neurological diseases. This study utilized novel lentiviral short hairpin RNA (shRNA) vectors to target

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

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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.

Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

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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.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia.

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Heckl, Dirk; Wicke, Daniel C; Brugman, Martijn H; Meyer, Johann; Schambach, Axel; Büsche, Guntram; Ballmaier, Matthias; Baum, Christopher; Modlich, Ute

2011-04-07

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

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

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.

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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Shaoxing YANG

2012-12-01

Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy.

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Almarza, Elena; Río, Paula; Meza, Nestor W; Aldea, Montserrat; Agirre, Xabier; Guenechea, Guillermo; Segovia, José C; Bueren, Juan A

2007-08-01

Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.

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

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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.

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

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Swati Singh

2017-03-01

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

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

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

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Andrew H. Baker

2010-10-01

Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

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

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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.

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.

Efficient in vivo gene transfer to xenotransplanted human skin by lentivirus-mediated, but not by AAV-directed, gene delivery

DEFF Research Database (Denmark)

Jakobsen, Maria Vad; Askou, Anne Louise; Dokkedahl, Karin Stenderup

skin graft, and firefly luciferase expression was observed primarily in neighboring tissue beneath or surrounding the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin...... graft only. The study demonstrates limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo....

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

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Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

2018-06-15

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

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

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

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Desclaux, Mathieu; Teigell, Marisa; Amar, Lahouari; Vogel, Roland; Gimenez Y Ribotta, Minerva; Privat, Alain; Mallet, Jacques

2009-07-14

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). 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. 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 increasing glial permissiveness and permitting axonal regeneration

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

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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.

Packaging of HCV-RNA into lentiviral vector

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Caval, Vincent; Piver, Eric; Ivanyi-Nagy, Roland; Darlix, Jean-Luc; Pagès, Jean-Christophe

2011-01-01

Highlights: ► Description of HCV-RNA Core-D1 interactions. ► In vivo evaluation of the packaging of HCV genome. ► Determination of the role of the three basic sub-domains of D1. ► Heterologous system involving HIV-1 vector particles to mobilise HCV genome. ► 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.

Packaging of HCV-RNA into lentiviral vector

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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.

CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

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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. PMID:25541967

Atomic Stretch: Optimally bounded real-time stretching and beyond

DEFF Research Database (Denmark)

Jensen, Rasmus Ramsbøl; Nielsen, Jannik Boll

2016-01-01

Atomic Stretch is a plugin for your preferred Adobe video editing tool, allowing real-time smooth and optimally bounded retarget-ting from and to any aspect ratio. The plugin allows preserving of high interest pixels through a protected region, attention redirection through color-modification, co......Atomic Stretch is a plugin for your preferred Adobe video editing tool, allowing real-time smooth and optimally bounded retarget-ting from and to any aspect ratio. The plugin allows preserving of high interest pixels through a protected region, attention redirection through color...

Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

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Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I.; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

2014-01-01

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options. PMID:24925206

Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

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Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

2014-10-01

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

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Plumas Joel

2009-01-01

Full Text Available Abstract Background Gene modified dendritic cells (DC are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG, the gibbon ape leukaemia virus envelope (GaLV or the feline endogenous virus envelope (RD114. At the same time, we evaluated transgene expression (E-GFP reporter gene under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV under the control of phoshoglycerate kinase (PGK and elongation factor-1 (EF1α promoters (28% to 90% of E-GFP+ cells, respectively in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12 described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

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

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

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Kruithof Egbert KO

2004-08-01

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

Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

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Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun [Seoul National University College of Medicine, Seoul (Korea, Republic of); Kim, Sung Jin; Lee, Heui Ran [Medical Research Center, Seoul National University, Seoul (Korea, Republic of)

2008-10-15

Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. 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 hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning 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 transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1{+-}4.7%, 54.0{+-}6.4%, 85.7{+-}8.7%, and 98.4{+-}1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704{+-}6,659 picomole/10{sup 6} cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168{+-}2,134 picomole/10{sup 6} cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

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

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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

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.

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

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Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

2007-07-30

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

Partial Correction of Psoriasis upon Genetic Knock-Down of Human TNF-α by Lentivirus-Encoded shRNAs in a Xenograft Mouse Model

DEFF Research Database (Denmark)

Jakobsen, Maria; Stenderup, Karin; Rosada, Cecilia

samples treated with irrelevant shRNAs, were selected and cloned into lentiviral vectors. The lentiviral vectors expressing TNF- shRNAs were used to transduce HEK-293 cells and verify vector-derived knock-down of stable TNF- expression in vitro. The most efficient TNF- -directed shRNA, which in cell lines...

Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

Science.gov (United States)

Piao, Yulan; Hung, Sandy Shen-Chi; Lim, Shiang Y; Wong, Raymond Ching-Bong; Ko, Minoru S H

2014-07-01

Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes. ©AlphaMed Press.

Lentiviral-mediated targeted NF-kappaB blockade in dorsal spinal cord glia attenuates sciatic nerve injury-induced neuropathic pain in the rat.

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Meunier, Alice; Latrémolière, Alban; Dominguez, Elisa; Mauborgne, Annie; Philippe, Stéphanie; Hamon, Michel; Mallet, Jacques; Benoliel, Jean-Jacques; Pohl, Michel

2007-04-01

Neuropathic pain developing after peripheral nerve injury is associated with altered neuronal and glial cell functions in the spinal cord. Activated glia produces algogenic mediators, exacerbating pain. Among the different intracellular pathways possibly involved in the modified glial function, the nuclear factor kappaB (NF-kappaB) system is of particular interest, as numerous genes encoding inflammation- and pain-related molecules are controlled by this transcription factor. NF-kappaB is a pleiotropic factor also involved in central nervous system homeostasy. To study its role in chronic pain, it is thus essential to inhibit the NF-kappaB pathway selectively in activated spinal glial cells. Here, we show that when restricted to spinal cord and targeted to glial cells, lentiviral vector-mediated delivery of NF-kappaB super- repressor IkappaBalpha resulted in an inhibition of the NF-kappaB pathway activated in the rat spinal cord after sciatic nerve injury (chronic constriction injury, CCI). Concomitantly, IkappaBalpha overproduction prevented the enhanced expression of interleukin-6 and of inducible nitric oxide synthase associated with chronic constriction injury and resulted in prolonged antihyperalgesic and antiallodynic effects. These data show that targeted blockade of NF-kappaB activity in spinal glia efficiently alleviates pain behavior in CCI rats, demonstrating the active participation of the glial NF-kappaB pathway in the development of neuropathic pain after peripheral nerve injury.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts.

Science.gov (United States)

Gupta, Manoj K; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F; Windmueller, Rebecca; Wagers, Amy J; Kulkarni, Rohit N

2015-10-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. ©AlphaMed Press.

CLEC4M慢病毒载体的构建及其在K562细胞中的表达

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WANG Yuanyuan

2014-06-01

Full Text Available ObjectiveTo construct the lentiviral vector encoding CLEC4M and prepare K-562 cells with stable overexpression of CLEC4M. MethodsThe gene sequence of normal CLEC4M was cloned by RT-PCR and then inserted into GV166 vector to construct GV166-CLEC4M lentiviral expression vector, and then lentiviral packaging was performed by transfection of 293T cells. The obtained lentiviral liquid was used to infect human leukemia cell line K-562. Real-time PCR and Western blot were used to detect the overexpression of CLEC4M in K-562 cells. ResultsSequencing showed that the recombinant lentiviral expression plasmid GV166-CLEC4M was successfully constructed. Lentiviruses could efficiently infect K-562 cells, according to real-time PCR. CLEC4M was successfully over-expressed in K-562 cells at mRNA and protein levels. ConclusionThe construction of lentiviral vector encoding CLEC4M lays a foundation for further study of CLEC4M gene involved in HCV entry into host cells.

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.

A designated centre for people with disabilities operated by COPE Foundation, Cork

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

Gene delivery to pancreatic exocrine cells in vivo and in vitro

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Houbracken Isabelle

2012-10-01

Full Text Available Abstract Background Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas. Results For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression. Conclusions In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.

Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

International Nuclear Information System (INIS)

Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun; Kim, Sung Jin; Lee, Heui Ran

2008-01-01

Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. 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 hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning 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 transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1±4.7%, 54.0±6.4%, 85.7±8.7%, and 98.4±1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704±6,659 picomole/10 6 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168±2,134 picomole/10 6 cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression

Lentiviral-mediated Targeted NF-κB Blockade in Dorsal Spinal Cord Glia Attenuates Sciatic Nerve Injury-induced Neuropathic Pain in the Rat.

Science.gov (United States)

Meunier, Alice; Latrémolière, Alban; Dominguez, Elisa; Mauborgne, Annie; Philippe, Stéphanie; Hamon, Michel; Mallet, Jacques; Benoliel, Jean-Jacques; Pohl, Michel

2007-04-01

Neuropathic pain developing after peripheral nerve injury is associated with altered neuronal and glial cell functions in the spinal cord. Activated glia produces algogenic mediators, exacerbating pain. Among the different intracellular pathways possibly involved in the modified glial function, the nuclear factor κB (NF-κB) system is of particular interest, as numerous genes encoding inflammation- and pain-related molecules are controlled by this transcription factor. NF-κB is a pleiotropic factor also involved in central nervous system homeostasy. To study its role in chronic pain, it is thus essential to inhibit the NF-κB pathway selectively in activated spinal glial cells. Here, we show that when restricted to spinal cord and targeted to glial cells, lentiviral vector-mediated delivery of NF-κB super- repressor IκBα resulted in an inhibition of the NF-κB pathway activated in the rat spinal cord after sciatic nerve injury (chronic constriction injury, CCI). Concomitantly, IκBα overproduction prevented the enhanced expression of interleukin-6 and of inducible nitric oxide synthase associated with chronic constriction injury and resulted in prolonged antihyperalgesic and antiallodynic effects. These data show that targeted blockade of NF-κB activity in spinal glia efficiently alleviates pain behavior in CCI rats, demonstrating the active participation of the glial NF-κB pathway in the development of neuropathic pain after peripheral nerve injury. Copyright © 2007 The American Society of Gene Therapy. Published by Elsevier Inc. All rights reserved.

Joint sparse learning for 3-D facial expression generation.

Science.gov (United States)

Song, Mingli; Tao, Dacheng; Sun, Shengpeng; Chen, Chun; Bu, Jiajun

2013-08-01

3-D facial expression generation, including synthesis and retargeting, has received intensive attentions in recent years, because it is important to produce realistic 3-D faces with specific expressions in modern film production and computer games. In this paper, we present joint sparse learning (JSL) to learn mapping functions and their respective inverses to model the relationship between the high-dimensional 3-D faces (of different expressions and identities) and their corresponding low-dimensional representations. Based on JSL, we can effectively and efficiently generate various expressions of a 3-D face by either synthesizing or retargeting. Furthermore, JSL is able to restore 3-D faces with holes by learning a mapping function between incomplete and intact data. Experimental results on a wide range of 3-D faces demonstrate the effectiveness of the proposed approach by comparing with representative ones in terms of quality, time cost, and robustness.

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

Science.gov (United States)

Weber, K; Mock, U; Petrowitz, B; Bartsch, U; Fehse, B

2010-04-01

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

The base excision repair pathway is required for efficient lentivirus integration.

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Kristine E Yoder

Full Text Available An siRNA screen has identified several proteins throughout the base excision repair (BER pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.

Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals

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Clare Pridans

2014-01-01

Full Text Available 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.

Aspects of the epidemiology, research, and control of lentiviral infections of small ruminants and their relevance to Dutch sheep and goat farming.

Science.gov (United States)

van Maanen, C; Brinkhof, J M A; Moll, L; Colenbrander, B; Houwers, D J

2010-08-15

In 1862, the veterinarian Loman reported the first sheep in The Netherlands with symptoms associated with lentiviral infection, although at the time the symptoms were ascribed to ovine progressive pneumonia. In the following century, similar cases were reported by South African, French, American, and Icelandic researchers. Extensive research into the pathology, aetiology, and epidemiology of this slowly progressive and ultimately fatal disease was initiated in several countries, including the Netherlands. Studies of the causative agents--maedi visna virus (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats, comprising the heterogeneous group of the small ruminant lentiviruses (SRLV)--prompted the development of diagnostic methods and the initiation of disease control programmes in many European countries including the Netherlands, as a pioneer in 1982, and in the U.S.A. and Canada.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

Science.gov (United States)

Becker, P S; Taylor, J A; Trobridge, G D; Zhao, X; Beard, B C; Chien, S; Adair, J; Kohn, D B; Wagner, J E; Shimamura, A; Kiem, H-P

2010-10-01

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (Pgene-corrected cells in patients with FANCA.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

Science.gov (United States)

Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

2017-05-31

Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

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Rezakhanlou Alireza

2010-03-01

Full Text Available Abstract Indoleamine 2,3 dioxygenase (IDO is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

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

Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1.

Science.gov (United States)

Allahverdi, Amir; Abroun, Saied; Jafarian, Arefeh; Soleimani, Masoud; Taghikhani, Mohammad; Eskandari, Fatemeh

2015-01-01

Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.

Modification of an impulse-factoring orbital transfer technique to account for orbit determination and maneuver execution errors

Science.gov (United States)

Kibler, J. F.; Green, R. N.; Young, G. R.; Kelly, M. G.

1974-01-01

A method has previously been developed to satisfy terminal rendezvous and intermediate timing constraints for planetary missions involving orbital operations. The method uses impulse factoring in which a two-impulse transfer is divided into three or four impulses which add one or two intermediate orbits. The periods of the intermediate orbits and the number of revolutions in each orbit are varied to satisfy timing constraints. Techniques are developed to retarget the orbital transfer in the presence of orbit-determination and maneuver-execution errors. Sample results indicate that the nominal transfer can be retargeted with little change in either the magnitude (Delta V) or location of the individual impulses. Additonally, the total Delta V required for the retargeted transfer is little different from that required for the nominal transfer. A digital computer program developed to implement the techniques is described.

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

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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.

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

Science.gov (United States)

Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

2003-10-01

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells.

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Dai-tze Wu

Full Text Available The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular, in the identification of induced pluripotent stem (iPS cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies.

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

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Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector

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Santhosh Chakkaramakkil Verghese

2016-11-01

Full Text Available Abstract Fanconi anemia (FA is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

DEFF Research Database (Denmark)

Staunstrup, Nicklas H; Moldt, Brian; Mátés, Lajos

2009-01-01

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral...... Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase......-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans...

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

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Sue VandeWoude

2011-10-01

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

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

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

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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.

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

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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.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

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Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

2017-12-01

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

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Jakobsen Maria

2011-02-01

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

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

Novel approaches to models of Alzheimer's disease pathology for drug screening and development.

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Shaughnessy, Laura; Chamblin, Beth; McMahon, Lori; Nair, Ayyappan; Thomas, Mary Beth; Wakefield, John; Koentgen, Frank; Ramabhadran, Ram

2004-01-01

Development of therapeutics for Alzheimer's disease (AD) requires appropriate cell culture models that reflect the errant biochemical pathways and animal models that reflect the pathological hallmarks of the disease as well as the clinical manifestations. In the past two decades AD research has benefited significantly from the use of genetically engineered cell lines expressing components of the amyloid-generating pathway, as well as from the study of transgenic mice that develop the pathological hallmarks of the disease, mainly neuritic plaques. The choice of certain cell types and the choice of mouse as the model organism have been mandated by the feasibility of introduction and expression of foreign genes into these model systems. We describe a universal and efficient gene-delivery system, using lentiviral vectors, that permits the development of relevant cell biological systems using neuronal cells, including primary neurons and animal models in mammalian species best suited for the study of AD. In addition, lentiviral gene delivery provides avenues for creation of novel models by direct and prolonged expression of genes in the brain in any vertebrate animal. TranzVector is a lentiviral vector optimized for efficiency and safety that delivers genes to cells in culture, in tissue explants, and in live animals regardless of the dividing or differentiated status of the cells. Genes can also be delivered efficiently to fertilized single-cell-stage embryos of a wide range of mammalian species, broadening the range of the model organism (from rats to nonhuman primates) for the study of disease mechanism as well as for development of therapeutics. Copyright 2004 Humana Press Inc.

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

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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.

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.

Intravenous delivery of HIV-based lentiviral vectors preferentially transduces F4/80+ and Ly-6C+ cells in spleen, important target cells in autoimmune arthritis.

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Ben T van den Brand

Full Text Available Antigen presenting cells (APCs play an important role in arthritis and APC specific gene therapeutic targeting will enable intracellular modulation of cell activity. Viral mediated overexpression is a potent approach to achieve adequate transgene expression levels and lentivirus (LV is useful for sustained expression in target cells. Therefore, we studied the feasibility of lentiviral mediated targeting of APCs in experimental arthritis. Third generation VSV-G pseudotyped self-inactivating (SIN-LV were injected intravenously and spleen cells were analyzed with flow cytometry for green fluorescent protein (GFP transgene expression and cell surface markers. Collagen-induced arthritis (CIA was induced by immunization with bovine collagen type II in complete Freund's adjuvant. Effect on inflammation was monitored macroscopically and T-cell subsets in spleen were analyzed by flow cytometry. Synovium from arthritic knee joints were analyzed for proinflammatory cytokine expression. Lentiviruses injected via the tail vein preferentially infected the spleen and transduction peaks at day 10. A dose escalating study showed that 8% of all spleen cells were targeted and further analysis showed that predominantly Ly6C+ and F4/80+ cells in spleen were targeted by the LV. To study the feasibility of blocking TAK1-dependent pathways by this approach, a catalytically inactive mutant of TAK1 (TAK1-K63W was overexpressed during CIA. LV-TAK1-K63W significantly reduced incidence and arthritis severity macroscopically. Further histological analysis showed a significant decrease in bone erosion in LV-TAK1-K63W treated animals. Moreover, systemic Th17 levels were decreased by LV-TAK1-K63W treatment in addition to diminished IL-6 and KC production in inflamed synovium. In conclusion, systemically delivered LV efficiently targets monocytes and macrophages in spleen that are involved in autoimmune arthritis. Moreover, this study confirms efficacy of TAK1 targeting in

High-throughput screening using pseudotyped lentiviral particles: a strategy for the identification of HIV-1 inhibitors in a cell-based assay.

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Garcia, Jean-Michel; Gao, Anhui; He, Pei-Lan; Choi, Joyce; Tang, Wei; Bruzzone, Roberto; Schwartz, Olivier; Naya, Hugo; Nan, Fa-Jun; Li, Jia; Altmeyer, Ralf; Zuo, Jian-Ping

2009-03-01

Two decades after its discovery the human immunodeficiency virus (HIV) is still spreading worldwide and killing millions. There are 25 drugs formally approved for HIV currently on the market, but side effects as well as the emergence of HIV strains showing single or multiple resistances to current drug-therapy are causes for concern. Furthermore, these drugs target only 4 steps of the viral cycle, hence the urgent need for new drugs and also new targets. In order to tackle this problem, we have devised a cell-based assay using lentiviral particles to look for post-entry inhibitors of HIV-1. We report here the assay development, validation as well as confirmation of the hits using both wild-type and drug-resistant HIV-1 viruses. The screening was performed on an original library, rich in natural compounds and pure molecules from Traditional Chinese Medicine pharmacopoeia, which had never been screened for anti-HIV activity. The identified hits belong to four chemical sub-families that appear to be all non-nucleoside reverse transcriptase inhibitors (NNRTIs). Secondary tests with live viruses showed that there was good agreement with pseudotyped particles, confirming the validity of this approach for high-throughput drug screens. This assay will be a useful tool that can be easily adapted to screen for inhibitors of viral entry.

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

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Andreas Holmgaard

2017-12-01

Full Text Available Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp Cas9, delivered by lentiviral vectors (LVs, can be used in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs induce in vitro indel formation at high frequencies (44%–93%. A single unilateral subretinal injection facilitates RPE-specific localization of the vector and disruption of Vegfa in isolated eGFP+ RPE cells obtained from mice five weeks after LV administration. Notably, sgRNA delivery results in the disruption of Vegfa with an in vivo indel formation efficacy of up to 84%. Sequencing of Vegfa-specific amplicons reveals formation of indels, including 4-bp deletions and 2-bp insertions. Taken together, our data demonstrate the capacity of lentivirus-delivered SpCas9 and sgRNAs as a developing therapeutic path in the treatment of ocular diseases, including age-related macular degeneration.

Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

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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.

Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.

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Kuroda, Hitoshi; Kutner, Robert H; Bazan, Nicolas G; Reiser, Jakob

2009-05-01

During the past 12 years, lentiviral 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. Despite significant progress, the production of high-titer high-quality lentiviral vectors is cumbersome and costly. The most commonly used method to produce lentiviral vectors involves transient transfection using calcium phosphate (CaP)-mediated precipitation of plasmid DNAs. However, inconsistencies in pH can cause significant batch-to-batch variations in lentiviral vector titers, making this method unreliable. This study describes optimized protocols for lentiviral vector production based on polyethylenimine (PEI)-mediated transfection, resulting in more consistent lentiviral vector stocks. To achieve this goal, simple production methods for high-titer lentiviral vector production involving transfection of HEK 293T cells immediately after plating were developed. Importantly, high titers were obtained with cell culture media lacking serum or other protein additives altogether. As a consequence, large-scale lentiviral vector stocks can now be generated with fewer batch-to-batch variations and at reduced costs and with less labor compared to the standard protocols.

In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

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Amari, Afshin; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Soleimani, Masoud; Mohammadi Amirabad, Leila; Tahoori, Mohammad Taher; Massumi, Mohammad

2015-08-01

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

Effect of specific silencing of EMMPRIN on the growth and cell cycle distribution of MCF-7 breast cancer cells.

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Yang, X Q; Yang, J; Wang, R; Zhang, S; Tan, Q W; Lv, Q; Meng, W T; Mo, X M; Li, H J

2015-12-02

The extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin family and shows increased expression in tumor cells. We examined the effect of RNAi-mediated EMMPRIN gene silencing induced by lentiviral on the growth and cycle distribution of MCF-7 breast cancer cells. Lentiviral expressing EMMPRIN-short hairpin RNA were packaged to infect MCF-7 cells. The inhibition efficiency of EMMPRIN was validated by real-time fluorescent quantitation polymerase chain reaction and western blotting. The effect of EMMPRIN on cell proliferation ability was detected using the MTT assay and clone formation experiments. Changes in cell cycle were detected by flow cytometry. EMMPRIN-short hairpin RNA-packaged lentiviral significantly down-regulated EMMPRIN mRNA and protein expression, significantly inhibited cell proliferation and in vitro tumorigenicity, and induced cell cycle abnormalities. Cells in the G0/G1 and G2/M phases were increased, while cells in the S phase were decreased after infection of MCF-7 cells for 3 days. The EMMPRIN gene facilitates breast cancer cell malignant proliferation by regulating cell cycle distribution and may be a molecular target for breast cancer gene therapy.

Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency.

Science.gov (United States)

Tran, Reginald; Myers, David R; Denning, Gabriela; Shields, Jordan E; Lytle, Allison M; Alrowais, Hommood; Qiu, Yongzhi; Sakurai, Yumiko; Li, William C; Brand, Oliver; Le Doux, Joseph M; Spencer, H Trent; Doering, Christopher B; Lam, Wilbur A

2017-10-04

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1 + ) and human (CD34 + ) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Antibacterial activity of silver-killed bacteria: the "zombies" effect

Science.gov (United States)

Wakshlak, Racheli Ben-Knaz; Pedahzur, Rami; Avnir, David

2015-04-01

We report a previously unrecognized mechanism for the prolonged action of biocidal agents, which we denote as the zombies effect: biocidally-killed bacteria are capable of killing living bacteria. The concept is demonstrated by first killing Pseudomonas aeruginosa PAO1 with silver nitrate and then challenging, with the dead bacteria, a viable culture of the same bacterium: Efficient antibacterial activity of the killed bacteria is observed. A mechanism is suggested in terms of the action of the dead bacteria as a reservoir of silver, which, due to Le-Chatelier's principle, is re-targeted to the living bacteria. Langmuirian behavior, as well as deviations from it, support the proposed mechanism.

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

Science.gov (United States)

Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

2013-02-28

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells

NARCIS (Netherlands)

Lamers, C.H.; Willemsen, R.; Elzakker, P. van; Steenbergen-Langeveld, S. van; Broertjes, M.; Oosterwijk-Wakka, J.C.; Oosterwijk, E.; Sleijfer, S.; Debets, R.; Gratama, J.W.

2011-01-01

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted

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.

Declarative language design for interactive visualization.

Science.gov (United States)

Heer, Jeffrey; Bostock, Michael

2010-01-01

We investigate the design of declarative, domain-specific languages for constructing interactive visualizations. By separating specification from execution, declarative languages can simplify development, enable unobtrusive optimization, and support retargeting across platforms. We describe the design of the Protovis specification language and its implementation within an object-oriented, statically-typed programming language (Java). We demonstrate how to support rich visualizations without requiring a toolkit-specific data model and extend Protovis to enable declarative specification of animated transitions. To support cross-platform deployment, we introduce rendering and event-handling infrastructures decoupled from the runtime platform, letting designers retarget visualization specifications (e.g., from desktop to mobile phone) with reduced effort. We also explore optimizations such as runtime compilation of visualization specifications, parallelized execution, and hardware-accelerated rendering. We present benchmark studies measuring the performance gains provided by these optimizations and compare performance to existing Java-based visualization tools, demonstrating scalability improvements exceeding an order of magnitude.

[Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

Science.gov (United States)

Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

2010-02-01

This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

Assessment of Integration-defective HIV-1 and EIAV Vectors In Vitro and In Vivo

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Scott Ellis

2012-01-01

Full Text Available The interest in integrase-defective lentiviral vectors (IDLVs stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. Here, we directly compared the transducing potential of IDLVs based on the equine infectious anemia virus (EIAV to the more commonly described HIV-1 IDLVs. IDLVs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. We show that both the single and the triple mutant HIV-1 IDLVs transduce the PC12 cells, but not the C2C12 cells, with similar efficiency to their parental HIV-1 vector. In contrast, the single and triple EIAV IDLVs did not efficiently transduce either differentiated cell line. Moreover, this HIV-1 IDLV-mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. Four weeks following stereotactic administration into adult rat brains, only the single HIV-1 IDLV mutant displayed a comparable transduction profile to the parental HIV-1 vector. In contrast, neither EIAV IDLV mutants showed significant reporter gene expression. This work indicates that the transducing potential of IDLVs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector.

Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

Science.gov (United States)

Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S.; Mok, Pooi Ling

2017-01-01

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours. PMID:28885562

The FORCE: A portable parallel programming language supporting computational structural mechanics

Science.gov (United States)

Jordan, Harry F.; Benten, Muhammad S.; Brehm, Juergen; Ramanan, Aruna

1989-01-01

This project supports the conversion of codes in Computational Structural Mechanics (CSM) to a parallel form which will efficiently exploit the computational power available from multiprocessors. The work is a part of a comprehensive, FORTRAN-based system to form a basis for a parallel version of the NICE/SPAR combination which will form the CSM Testbed. The software is macro-based and rests on the force methodology developed by the principal investigator in connection with an early scientific multiprocessor. Machine independence is an important characteristic of the system so that retargeting it to the Flex/32, or any other multiprocessor on which NICE/SPAR might be imnplemented, is well supported. The principal investigator has experience in producing parallel software for both full and sparse systems of linear equations using the force macros. Other researchers have used the Force in finite element programs. It has been possible to rapidly develop software which performs at maximum efficiency on a multiprocessor. The inherent machine independence of the system also means that the parallelization will not be limited to a specific multiprocessor.

Implementation of KRoC on Analog Devices' "SHARC" DSP

NARCIS (Netherlands)

Otten, G.W.; Schwirtz, M.H.; Schwirtz, Marcellinus H.; Bruis, R.; Bruis, R.; Broenink, Johannes F.; Bakkers, André; O'Neill, Brian C.

1996-01-01

This paper summarises the experiences gained at the Control Laboratory of the University of Twente in porting the Kent Retargetable occam Compiler -KroC -to the Analog Devices' ADSP21060 SHARC Digital Signal Processor. The choice of porting the KRoC to the DSP processor was in our view both a

Optimization of methods for the genetic modification of human T cells.

Science.gov (United States)

Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

2015-11-01

CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

CD47 is a Potential Target for the Treatment of Laryngeal Squamous Cell Carcinoma

OpenAIRE

ChunPing Yang; ShuFeng Gao; HaiZhen Zhang; Lian Xu; JianGuo Liu; Meiqun Wang; ShaoRong Zhang

2016-01-01

Background/Aims: This study aims to investigate the effect of CD47 on the development of laryngeal squamous cell carcinoma (LSCC) and the therapeutic potential of monoclonal antibody against CD47 and its ligand SIRPα in the treatment of LSCC. Methods: We firstly detected the expressions of CD47 mRNA and protein in LSCC and para-carcinoma tissues, introduced the most efficient CD47siRNA sequence into LSCC cells by lentiviral transfection and employed three monoclonal antibodies to evaluate the...

A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

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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

Lentiviral gene transfer into the dorsal root ganglion of adult rats

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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.

Re-targeting the Graze performance debugging tool for Java threads and analyzing the re-targeting to automatically parallelized (FORTRAN) code

OpenAIRE

Tsai, Pedro T. H.

2000-01-01

Approved for public release; distribution is unlimited This research focuses on the design of a language-independent concept, Glimpse, for performance debugging of multi-threaded programs. This research extends previous work on Graze, a tool designed and implemented for performance debugging of C++ programs. Not only is Glimpse easily portable among different programming languages, (i) it is useful in many different paradigms ranging from few long-lived threads to many short-lived...

Design of a novel integration-deficient lentivector technology that incorporates genetic and posttranslational elements to target human dendritic cells.

Science.gov (United States)

Tareen, Semih U; Kelley-Clarke, Brenna; Nicolai, Christopher J; Cassiano, Linda A; Nelson, Lisa T; Slough, Megan M; Vin, Chintan D; Odegard, Jared M; Sloan, Derek D; Van Hoeven, Neal; Allen, James M; Dubensky, Thomas W; Robbins, Scott H

2014-03-01

As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

Constraint driven operation assignment for retargetable VLIW compilers

NARCIS (Netherlands)

Bekooij, M.J.G.

2004-01-01

In veel consumenten elektronica producten worden processoren toegepast voor het bewerken van gedigitaliseerde signalen. Deze processoren zijn gewoonlijk ingebed in een systeem en moeten wat rekenkracht, vermogensverbruik en fabricage kosten aan stringente eisen voldoen. Door het optimaliseren van

Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity.

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Laura M J Ylinen

2010-08-01

Full Text Available TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5alpha but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.

Genetically engineering adenoviral vectors for gene therapy.

Science.gov (United States)

Coughlan, Lynda

2014-01-01

Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

Electroporation and use of hepatitis B virus envelope L proteins as bionanocapsules.

Science.gov (United States)

Yamada, Tadanori; Jung, Joohee; Seno, Masaharu; Kondo, Akihiko; Ueda, Masakazu; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

2012-06-01

Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.

Autonomous spacecraft landing through human pre-attentive vision

International Nuclear Information System (INIS)

Schiavone, Giuseppina; Izzo, Dario; Simões, Luís F; De Croon, Guido C H E

2012-01-01

In this work, we exploit a computational model of human pre-attentive vision to guide the descent of a spacecraft on extraterrestrial bodies. Providing the spacecraft with high degrees of autonomy is a challenge for future space missions. Up to present, major effort in this research field has been concentrated in hazard avoidance algorithms and landmark detection, often by reference to a priori maps, ranked by scientists according to specific scientific criteria. Here, we present a bio-inspired approach based on the human ability to quickly select intrinsically salient targets in the visual scene; this ability is fundamental for fast decision-making processes in unpredictable and unknown circumstances. The proposed system integrates a simple model of the spacecraft and optimality principles which guarantee minimum fuel consumption during the landing procedure; detected salient sites are used for retargeting the spacecraft trajectory, under safety and reachability conditions. We compare the decisions taken by the proposed algorithm with that of a number of human subjects tested under the same conditions. Our results show how the developed algorithm is indistinguishable from the human subjects with respect to areas, occurrence and timing of the retargeting. (paper)

Prospects for Foamy Viral Vector Anti-HIV Gene Therapy

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Arun K. Nalla

2016-03-01

Full Text Available Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC. Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

Delivering the Goods for Genome Engineering and Editing.

Science.gov (United States)

Skipper, Kristian Alsbjerg; Mikkelsen, Jacob Giehm

2015-08-01

A basic understanding of genome evolution and the life and impact of microorganisms, like viruses and bacteria, has been fundamental in the quest for efficient genetic therapies. The expanding tool box for genetic engineering now contains transposases, recombinases, and nucleases, all created from naturally occurring genome-modifying proteins. Whereas conventional gene therapies have sought to establish sustained expression of therapeutic genes, genomic tools are needed only in a short time window and should be delivered to cells ideally in a balanced "hit-and-run" fashion. Current state-of-the-art delivery strategies are based on intracellular production of protein from transfected plasmid DNA or in vitro-transcribed RNA, or from transduced viral templates. Here, we discuss advantages and challenges of intracellular production strategies and describe emerging approaches based on the direct delivery of protein either by transfer of recombinant protein or by lentiviral protein transduction. With focus on adapting viruses for protein delivery, we describe the concept of "all-in-one" lentiviral particles engineered to codeliver effector proteins and donor sequences for DNA transposition or homologous recombination. With optimized delivery methods-based on transferring DNA, RNA, or protein-it is no longer far-fetched that researchers in the field will indeed deliver the goods for somatic gene therapies.

MicroRNA-143 Downregulates Interleukin-13 Receptor Alpha1 in Human Mast Cells

Directory of Open Access Journals (Sweden)

Jianqiu Cheng

2013-08-01

Full Text Available MicroRNA-143 (miR-143 was found to be downregulated in allergic rhinitis, and bioinformatics analysis predicted that IL-13Rα1 was a target gene of miR-143. To understand the molecular mechanisms of miR-143 involved in the pathogenesis of allergic inflammation, recombinant miR-143 plasmid vectors were constructed, and human mast cell-1(HMC-1 cells which play a central role in the allergic response were used for study. The plasmids were transfected into HMC-1 cells using a lentiviral vector. Expression of IL-13Rα1 mRNA was then detected by reverse transcriptase polymerase chain reaction (RT-PCR and Western Blotting. The miR-143 lentiviral vector was successfully stably transfected in HMC-1 cells for target gene expression. Compared to the control, the target gene IL-13Rα1 was less expressed in HMC-1 transfected with miR-143 as determined by RT-PCR and Western Blotting (p < 0.05; this difference in expression was statistically significant and the inhibition efficiency was 71%. It indicates that miR-143 directly targets IL-13Rα1 and suppresses IL-13Rα1 expression in HMC-1 cells. Therefore, miR-143 may be associated with allergic reaction in human mast cells.

A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

Directory of Open Access Journals (Sweden)

Huiling Qiu

Full Text Available DNA vector-encoded Tough Decoy (TuD miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer, which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD vector in which only two sets of shorter oligonucleotides (< 60 mer were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324 were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.

Rapid In Vivo Validation of Tumor Suppressor Gene Function in Prostate Cancer Progression

Science.gov (United States)

2016-07-01

identification of the best sgRNA sequences and accelerated our ability to move to the in vivo studies proposed in Aim2. Our goal was to use CRISPR / Cas ...and to initiate prostate cancer in the mouse after injection of lentiviral particles expressing CRISPR / Cas components and Cre recombinase. Our initial...in vivo Our goal was to use CRISPR / Cas lentiviral transduction of the adult prostate to inactivate p53 or Rb. We aimed to recapitulate the effects of

Lentiviral transgenic microRNA-based shRNA suppressed mouse cytochromosome P450 3A (CYP3A expression in a dose-dependent and inheritable manner.

Directory of Open Access Journals (Sweden)

Yong Wang

Full Text Available Cytochomosome P450 enzymes (CYP are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44, and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P<0.01. This work laid down a foundation to further knock

Gene Therapy Approaches to Hemoglobinopathies.

Science.gov (United States)

Ferrari, Giuliana; Cavazzana, Marina; Mavilio, Fulvio

2017-10-01

Gene therapy for hemoglobinopathies is currently based on transplantation of autologous hematopoietic stem cells genetically modified with a lentiviral vector expressing a globin gene under the control of globin transcriptional regulatory elements. Preclinical and early clinical studies showed the safety and potential efficacy of this therapeutic approach as well as the hurdles still limiting its general application. In addition, for both beta-thalassemia and sickle cell disease, an altered bone marrow microenvironment reduces the efficiency of stem cell harvesting as well as engraftment. These hurdles need be addressed for gene therapy for hemoglobinopathies to become a clinical reality. Copyright © 2017 Elsevier Inc. All rights reserved.

Therapeutic hemoglobin levels after gene transfer in β-thalassemia mice and in hematopoietic cells of β-thalassemia and sickle cells disease patients.

Directory of Open Access Journals (Sweden)

Laura Breda

Full Text Available Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A and concurrently reducing the sickling tetramer (Hb S.Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these

Stromal interaction molecule 1 (STIM1) silencing inhibits tumor growth and promotes cell cycle arrest and apoptosis in hypopharyngeal carcinoma.

Science.gov (United States)

Sun, Yuanhao; Cui, Xiaobo; Wang, Jun; Wu, Shuai; Bai, Yunfei; Wang, Yaping; Wang, Boqian; Fang, Jugao

2015-05-01

As an important pathway maintaining the balance of intracellular calcium (Ca(2+)), store-operated Ca(2+) entry (SOCE) is critical for cellular functions. Stromal interaction molecule 1 (STIM1), a key component of SOCE, plays a dual role as an endoplasmic reticulum Ca(2+) receptor and an SOCE exciter. Aberrant expression of STIM1 could be discovered in several human cancer cells. However, the role of STIM1 in regulating human hypopharyngeal carcinoma still remains unclear. Real-time polymerase chain reaction (PCR) was used to detect expression of STIM1 in human hypopharyngeal carcinoma cell line FaDu. STIM1 on FaDu cells was knocked down by lentiviral transduction method. The biological impacts after knocking down of STIM1 on FaDu cells were investigated in vitro and in vivo. The result of real-time PCR showed that STIM1 was expressed in FaDu cells. Lentiviral transduction efficiently downregulated the expression of STIM1 in FaDu cells at both mRNA and protein levels. Significant downregulation of STIM1 on FaDu cells inhibited cell proliferation, induced cell cycle arrest in G0/G1 phase, promoted cell apoptosis, and restrained cell growth rate. The antigrowth effect of STIM1 silencing was also discovered in FaDu hypopharyngeal tumor model. Our findings indicate that STIM1 is likely to become a new therapeutic target for hypopharyngeal carcinoma treatment.

Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

Science.gov (United States)

Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

2015-05-01

Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy. © 2015 AlphaMed Press.

Evaluation of railway transportation efficiency based on super-cross efficiency

Science.gov (United States)

Kuang, Xiuyuan

2018-01-01

The efficiency of railway transportation is an important index. It can measure the development of railway transportation enterprises, and the efficiency of railway transportation has become a hot issue in the study of railway development. Data envelopment analysis (DEA) has been widely applied to railway efficiency analysis. In this paper, BBC model and super-cross efficiency model are constructed by using DEA theory, taking the 18 Railway Bureau as the research object, with the mileage, the number of employees, locomotive number, average daily loading number as input indicators, the passenger turnover, freight turnover and transport income as output indicators, then calculated and evaluated comprehensive efficiency, pure technical efficiency and scale efficiency. We get that the super-cross efficiency is more in line with the actual situation. Getting the super-cross efficiency is more in line with the actual situation.

Reversal of pathology in CHMP2B-mediated frontotemporal dementia patient cells using RNA interference

DEFF Research Database (Denmark)

Nielsen, Troels Tolstrup; Mizielinska, Sarah; Hasholt, Lis

2012-01-01

role in the pathogenesis of the disease. METHODS: In the present study, we used lentiviral vectors to efficiently knockdown CHMP2B by delivering microRNA embedded small hairpin RNAs. RESULTS: We show that CHMP2B can be efficiently knocked down in patient fibroblasts using an RNA interference approach......BACKGROUND: Frontotemporal dementia is the second most common form of young-onset dementia after Alzheimer's disease, and several genetic forms of frontotemporal dementia are known. A rare genetic variant is caused by a point mutation in the CHMP2B gene. CHMP2B is a component of the ESCRT......-III complex, which is involved in endosomal trafficking of proteins targeted for degradation in lysosomes. Mutations in CHMP2B result in abnormal endosomal structures in patient fibroblasts and patient brains, probably through a gain-of-function mechanism, suggesting that the endosomal pathway plays a central...

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

DEFF Research Database (Denmark)

Stenderup, Karin; Jakobsen, Maria; Rosada, Cecilia

2008-01-01

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

Menhir: An Environment for High Performance Matlab

Directory of Open Access Journals (Sweden)

Stéphane Chauveau

1999-01-01

Full Text Available In this paper we present Menhir a compiler for generating sequential or parallel code from the Matlab language. The compiler has been designed in the context of using Matlab as a specification language. One of the major features of Menhir is its retargetability to generate parallel and sequential C or Fortran code. We present the compilation process and the target system description for Menhir. Preliminary performances are given and compared with MCC, the MathWorks Matlab compiler.

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

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.

Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

International Nuclear Information System (INIS)

Campos, Samuel K.; Barry, Michael A.

2006-01-01

The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon

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...

Targeting GPR110 in HER2-Overexpressing Breast Cancers

Science.gov (United States)

2015-10-01

34assay"using"the"transwell" inserts"with" (A)"or"without" (B)"matrigel" for"SKBR3" cells "transfected"with"nonAtarge1ng"( NT1 )"orŗ"independent"GPR110"siRNAs...HER2 drug resistant cells and in tumorigenic population using a broad panel of cell line models. We have successfully generated inducible lentiviral...plasmids with GPR110 cDNA and cell lines that inducibly overexpress GPR110. Generation of cell lines with lentiviral plasmids containing GPR110

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

Directory of Open Access Journals (Sweden)

Motoharu Ono

Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.

Science.gov (United States)

Annunziato, Stefano; Kas, Sjors M; Nethe, Micha; Yücel, Hatice; Del Bravo, Jessica; Pritchard, Colin; Bin Ali, Rahmen; van Gerwen, Bas; Siteur, Bjørn; Drenth, Anne Paulien; Schut, Eva; van de Ven, Marieke; Boelens, Mirjam C; Klarenbeek, Sjoerd; Huijbers, Ivo J; van Miltenburg, Martine H; Jonkers, Jos

2016-06-15

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice. © 2016 Annunziato et al.; Published by Cold Spring Harbor Laboratory Press.

[Experimental study on human periodontal ligament cells transfected with human amelogenin gene].

Science.gov (United States)

Yu, Guang; Shu, Rong; Sun, Ying; Cheng, Lan; Song, Zhong-Chen; Zhang, Xiu-Li

2008-02-01

To construct the recombinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recombinant lentivirus, and evaluate the feasibility of applying modified PDLCs as seeds for a further periodontal reconstruction. The mature peptide of hAm cDNA was cloned and linked into the vector plasmid, the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein (GFP) with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR). The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR. The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering. Supported by National Natural Science Foundation of China (Grant No. 30672315).

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

Science.gov (United States)

Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

2015-01-01

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

Directory of Open Access Journals (Sweden)

Kate E. Hawkins

2016-03-01

Full Text Available The potential of induced pluripotent stem cells (iPSCs in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.

A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

Directory of Open Access Journals (Sweden)

Yu Takahashi

2018-01-01

Full Text Available Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC-derived intestinal organoids involving four methodological advances. (1 We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2 We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3 Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4 We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

Viral/Host interaction in viral infections

International Nuclear Information System (INIS)

Le Grand, R.

2006-01-01

The major objectives of the Neuro-virology Department (SNV for 'Service de Neurovirologie') are related to the study of host/pathogen interactions, particularly during primate lentiviral infections. Various experimental models have been developed such as non-human primates infected with the HIV-related simian immunodeficiency viruses (SIV), as an animal model of human AIDS. The current research programs of the SNV following four main directions: 1) Study of the pathogenesis of primate lentiviral infection, including mucosal transmission of HIV/SIV, primary infection, dissemination to various reservoirs, neuro-pathogenesis and hematopoietic disorders; 2) Prevention of HIV transmission, particularly through vaccination but also by means of microbicides applied to genital mucosa and post-exposure treatment with antiviral drugs; 3) Cellular and molecular pharmacology of new antiviral compounds; 4) Development of new primate models of human hematological disorders like chronic myeloid leukemia cells and development on new gene transfer in hematopoietic cells based on the use of lentiviral vectors Main programs of the SNV will be presented as well as the perspective focused on the use of non invasive in vivo imaging approaches for the exploration of immune and hematopoietic cells

Viral/Host interaction in viral infections

Energy Technology Data Exchange (ETDEWEB)

Le Grand, R. [CEA Fontenay-aux-Roses, Service de Neurovirologie, 92 (France)

2006-07-01

The major objectives of the Neuro-virology Department (SNV for 'Service de Neurovirologie') are related to the study of host/pathogen interactions, particularly during primate lentiviral infections. Various experimental models have been developed such as non-human primates infected with the HIV-related simian immunodeficiency viruses (SIV), as an animal model of human AIDS. The current research programs of the SNV following four main directions: 1) Study of the pathogenesis of primate lentiviral infection, including mucosal transmission of HIV/SIV, primary infection, dissemination to various reservoirs, neuro-pathogenesis and hematopoietic disorders; 2) Prevention of HIV transmission, particularly through vaccination but also by means of microbicides applied to genital mucosa and post-exposure treatment with antiviral drugs; 3) Cellular and molecular pharmacology of new antiviral compounds; 4) Development of new primate models of human hematological disorders like chronic myeloid leukemia cells and development on new gene transfer in hematopoietic cells based on the use of lentiviral vectors Main programs of the SNV will be presented as well as the perspective focused on the use of non invasive in vivo imaging approaches for the exploration of immune and hematopoietic cells.

Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron.

Science.gov (United States)

Johnson, Cayla M; Fisher, Derek J

2013-01-01

Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron's substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA(-) mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.

Juggling Efficiency

DEFF Research Database (Denmark)

Andersen, Rikke Sand; Vedsted, Peter

2015-01-01

on institutional logics, we illustrate how a logic of efficiency organise and give shape to healthcare seeking practices as they manifest in local clinical settings. Overall, patient concerns are reconfigured to fit the local clinical setting and healthcare professionals and patients are required to juggle...... efficiency in order to deal with uncertainties and meet more complex or unpredictable needs. Lastly, building on the empirical case of cancer diagnostics, we discuss the implications of the pervasiveness of the logic of efficiency in the clinical setting and argue that provision of medical care in today......'s primary care settings requires careful balancing of increasing demands of efficiency, greater complexity of biomedical knowledge and consideration for individual patient needs....

Chimeric TALE recombinases with programmable DNA sequence specificity.

Science.gov (United States)

Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

2012-11-01

Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

DEA-Risk Efficiency and Stochastic Dominance Efficiency of Stock Indices

OpenAIRE

Martin Branda; Miloš Kopa

2012-01-01

In this article, the authors deal with the efficiency of world stock indices. Basically, they compare three approaches: mean-risk, data envelopment analysis (DEA), and stochastic dominance (SD) efficiency. In the DEA methodology, efficiency is defined as a weighted sum of outputs compared to a weighted sum of inputs when optimal weights are used. In DEA-risk efficiency, several risk measures and functionals which quantify the risk of the indices (var, VaR, CVaR, etc.) as DEA inputs are used. ...

On-Board Rendezvous Targeting for Orion

Science.gov (United States)

Weeks, Michael W.; DSouza, Christopher N.

2010-01-01

The Orion On-board GNC system is among the most complex ever developed for a space mission. It is designed to operate autonomously (independent of the ground). The rendezvous system in particular was designed to operate on the far side of the moon, and in the case of loss-of-communications with the ground. The vehicle GNC system is designed to retarget the rendezvous maneuvers, given a mission plan. As such, all the maneuvers which will be performed by Orion, have been designed and are being incorporated into the flight code.

Predicting Moves-on-Stills for Comic Art Using Viewer Gaze Data.

Science.gov (United States)

Jain, Eakta; Sheikh, Yaser; Hodgins, Jessica

2016-01-01

Comic art consists of a sequence of panels of different shapes and sizes that visually communicate the narrative to the reader. The move-on-stills technique allows such still images to be retargeted for digital displays via camera moves. Today, moves-on-stills can be created by software applications given user-provided parameters for each desired camera move. The proposed algorithm uses viewer gaze as input to computationally predict camera move parameters. The authors demonstrate their algorithm on various comic book panels and evaluate its performance by comparing their results with a professional DVD.

Multi-directional program efficiency

DEFF Research Database (Denmark)

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

2016-01-01

The present paper analyses both managerial and program efficiencies of Lithuanian family farms, in the tradition of Charnes et al. (Manag Sci 27(6):668–697, 1981) but with the important difference that multi-directional efficiency analysis rather than the traditional data envelopment analysis...... 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)....

Energy Efficiency Plan 2009-2012; Energie Efficiency Plan 2009-2012

Energy Technology Data Exchange (ETDEWEB)

Meulen, M.M.W. (ed.)

2009-02-15

The aim of the Energy Efficiency Plan is to give an overview of the energy conservation plans of the Eindhoven University of Technology in Eindhoven, Netherlands, which must result in efficient use of energy conform the long-range agreements between businesses, industry and organizations and the Dutch government to improve energy efficiency (MJA3) [Dutch] Het doel van het EEP (Energie Efficiency Plan) is het in beeld brengen van de energiebesparingsplannen die leiden tot een efficienter gebruik van energie conform de MJA-3 afspraak (de derde Meerjaren Afspraak)

From energy efficiency towards resource efficiency within the Ecodesign Directive

DEFF Research Database (Denmark)

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

2017-01-01

on the most significant environmental impact has often resulted in a focus on energy efficiency in the use phase. Therefore, the Ecodesign Directive should continue to target resource efficiency aspects but also consider environ- mental aspects with a large improvement potential in addition to the most...... significant environmental impact. For the introduction of resource efficiency requirements into the Ecodesign Directive, these requirements have to be included in the preparatory study. It is therefore recommended to broaden the scope of the Methodology for the Ecodesign of Energy-related products and the Eco......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...

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

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.

Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

Science.gov (United States)

Zonari, Erika; Desantis, Giacomo; Petrillo, Carolina; Boccalatte, Francesco E; Lidonnici, Maria Rosa; Kajaste-Rudnitski, Anna; Aiuti, Alessandro; Ferrari, Giuliana; Naldini, Luigi; Gentner, Bernhard

2017-04-11

Ex vivo gene therapy based on CD34 + hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34 + cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E 2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34 + CD38 - cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Energy Efficiency Roadmap for Uganda, Making Energy Efficiency Count. Executive Summary

Energy Technology Data Exchange (ETDEWEB)

de la Rue du Can, Stephane; Pudleiner, David; Jones, David; Khan, Aleisha

2017-06-15

Like many countries in Sub-Saharan Africa, Uganda has focused its energy sector investments largely on increasing energy access by increasing energy supply. The links between energy efficiency and energy access, the importance of energy efficiency in new energy supply, and the multiple benefits of energy efficiency for the level and quality of energy available, have been largely overlooked. Implementing energy efficiency in parallel with expanding both the electricity grid and new clean energy generation reduces electricity demand and helps optimize the power supply so that it can serve more customers reliably at minimum cost. Ensuring efficient appliances are incorporated into energy access efforts provides improved energy services to customers. Energy efficiency is an important contributor to access to modern energy. This Energy Efficiency Roadmap for Uganda (Roadmap) is a response to the important role that electrical energy efficiency can play in meeting Uganda’s energy goals. Power Africa and the United Nations Sustainable Energy for All (SEforALL) initiatives collaborated with more than 24 stakeholders in Uganda to develop this document. The document estimates that if the most efficient technologies on the market were adopted, 2,224 gigawatt hours could be saved in 2030 across all sectors, representing 31% of the projected load. This translates into 341 megawatts of peak demand reductions, energy access to an additional 6 million rural customers and reduction of carbon dioxide emissions by 10.6 million tonnes in 2030. The Roadmap also finds that 91% of this technical potential is cost-effective, and 47% is achievable under conservative assumptions. The Roadmap prioritizes recommendations for implementing energy efficiency and maximizing benefits to meet the goals and priorities established in Uganda’s 2015 SEforALL Action Agenda. One important step is to create and increase demand for efficiency through long-term enabling policies and financial incentives

Killing machines: three pore-forming proteins of the immune system

Science.gov (United States)

McCormack, Ryan; de Armas, Lesley; Shiratsuchi, Motoaki

2014-01-01

The evolution of early multicellular eukaryotes 400–500 million years ago required a defensive strategy against microbial invasion. Pore-forming proteins containing the membrane-attack-complex-perforin (MACPF) domain were selected as the most efficient means to destroy bacteria or virally infected cells. The mechanism of pore formation by the MACPF domain is distinctive in that pore formation is purely physical and unspecific. The MACPF domain polymerizes, refolds, and inserts itself into bilayer membranes or bacterial outer cell walls. The displacement of surface lipid/carbohydrate molecules by the polymerizing MACPF domain creates clusters of large, water-filled holes that destabilize the barrier function and provide access for additional anti-bacterial or anti-viral effectors to sensitive sites that complete the destruction of the invader via enzymatic or chemical attack. The highly efficient mechanism of anti-microbial defense by a combined physical and chemical strategy using pore-forming MACPF-proteins has been retargeted during evolution of vertebrates and mammals for three purposes: (1) to kill extracellular bacteria C9/polyC9 evolved in conjunction with complement, (2) to kill virus infected and cancer cells perforin-1/polyperforin-1 CTL evolved targeted by NK and CTL, and (3) to kill intracellular bacteria transmembrane perforin-2/putative polyperforin-2 evolved targeted by phagocytic and nonphagocytic cells. Our laboratory has been involved in the discovery and description of each of the three pore-formers that will be reviewed here. PMID:24293008

Photosynthesis efficiency for different wavelengths; Fotosynthese-efficiency bij verschillende golflengten

Energy Technology Data Exchange (ETDEWEB)

Snel, J.F.H.; Meinen, E.; Bruins, M.A.; Van Ieperen, W.; Hogewoning, S.W.; Marcelis, L.F.M. [Wageningen UR Glastuinbouw, Wageningen (Netherlands)

2012-04-15

LED lighting has recently been introduced into Dutch horticulture. LED development so far indicates that in the near future LED's will be more energy efficient than high pressure sodium lamps. Crop light interception and photosynthesis efficiency are wavelength dependent. Therefore, LED colours for maximum crop photosynthesis, growth and development should be identified. Wageningen UR has investigated light interception and photosynthesis at different wavelengths for tomato, cucumber and rose. Measuring protocols and equipment were developed for leaf photosynthesis measurements in the laboratory and in greenhouses. A crop simulation model was used for up-scaling the leaf level results to crop level photosynthesis. For the vegetable crops the photosynthesis spectra are very similar to the generalised photosynthesis spectrum. Red light is most efficient for leaf photosynthesis. Light from red (ca. 645nm) LED's was maximally 13% more efficient than High Pressure Sodium light. For reddish leaves of the rose cultivar Prestige, red LED light was up to 35% more efficient. These figures apply to the momentary efficiency of leaf photosynthesis at 100 {mu}mol.m{sup -2}.s{sup -1} (PAR) and suggest that use of red light can lead to higher photosynthesis, especially for certain rose cultivars [Dutch] LED verlichting heeft zijn intrede gedaan in de Nederlandse glastuinbouw. De LED ontwikkeling laat zien dat in de nabije toekomst LED's efficiënter zijn dan SON-T verlichting. Lichtonderschepping en fotosynthese efficiëntie zijn afhankelijk van de kleur van het licht. Voor optimale fotosynthese, groei en ontwikkeling zouden de beste LED kleuren uitgezocht moeten worden. Wageningen UR heeft lichtonderschepping en fotosynthese bij verschillende lichtkleuren onderzocht bij tomaat, komkommer en roos. Protocollen en apparatuur werden ontwikkeld voor meting van bladfotosynthese en lichtonderschepping in het laboratorium en in de kas. Met een gewassimulatiemodel werd de

The SEAD global efficiency medal competition: accelerating market transformation for efficient televisions

Energy Technology Data Exchange (ETDEWEB)

Ravi, Kavita [US Department of Energy, Washington, DC (United States); Bennich, Peter [Swedish Energy Agency (Sweden); Cockburn, John [Natural Resources Canada, Ottawa (Canada); Doi, Naoko [Institute of Energy Economics (Japan); Garg, Sandeep [United Nations Development Programme, New York, NY (United States); Garnaik, S.P. [ICF International (India); Holt, Shane [Energy and Tourism, Canberra (Australia); Walker, Mike [Food and Rural Affairs (United Kingdom); Westbrook-Trenholm, Elizabeth [Natural Resources, Canada, Ottawa (Canada). Office of Energy Efficiency; Lising, Anna [Collaborative Labeling and Appliance Standards Program (United States); Pantano, Steve [Collaborative Labeling and Appliance Standards Program (United States); Khare, Amit [Collaborative Labeling and Appliance Standards Program (United States); Park, Won Young [Lawrence Berkeley National Lab., CA (United States)

2013-10-15

The Global Efficiency Medal competition, a cornerstone activity of the Super-efficient Equipment and Appliance Deployment (SEAD) Initiative, is an awards program that encourages the production and sale of super-efficient products. SEAD is a voluntary multinational government collaboration of the Clean Energy Ministerial (CEM). This winner-takes-all competition recognizes products with the best energy efficiency, guides early adopter purchasers towards the most efficient product choices and demonstrates the levels of energy efficiency achievable by commercially available and emerging technologies. The first Global Efficiency Medals were awarded to the most energy-efficient flat panel televisions; an iconic consumer purchase. SEAD Global Efficiency Medals were awarded to televisions that have proven to be substantially more energy efficient than comparable models available at the time of the competition (applications closed in the end of May 2012). The award-winning TVs consume between 33 to 44 percent less energy per 2 unit of screen area than comparable LED-backlit LCD televisions sold in each regional market and 50 to 60 percent less energy than CCFL-backlit LCD TVs. Prior to the launch of this competition, SEAD conducted an unprecedented international round-robin test (RRT) to qualify TV test laboratories to support verification testing for SEAD awards. The RRT resulted in increased test laboratory capacity and expertise around the world and ensured that the test results from participating regional test laboratories could be compared in a fair and transparent fashion. This paper highlights a range of benefits resulting from this first SEAD awards competition and encourages further investigation of the awards concept as a means to promote energy efficiency in other equipment types.

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

Transformation by stealth: the retargeting of home care services in Finland.

Science.gov (United States)

Kröger, Teppo; Leinonen, Anu

2012-05-01

This paper analyses the trends and changes that home care services for older people have undergone during the last two decades in Finland. The data used come from national social care statistics, covering the time period from 1990-2010. The results show that, in contrast to many other European nations that have expanded their home care provisions, the coverage levels in Finland have dropped dramatically during this period. Those with the highest needs do receive increased amounts of support, but others have become excluded from publicly funded home care provisions and often need to rely on family members. In most localities, public service provision is focused on personal care, and no longer covers household tasks. This major change of the character of the service is connected to three other recent trends that structure current provisions: the amalgamation of home-based social and healthcare services, the marketisation and emerging privatisation of care and the integration of informal family care into the formal care system. Overall, the changes represent weakening defamilisation, that is, decreasing public responsibility for the needs of many older people and, correspondingly, an increasing reliance on family carers. This full-scale transformation of home care has taken place without any real policy debate or major modification of legislation. No actual decision was ever made to thoroughly alter the character of home care in Finland: the transformation happened by stealth. © 2011 Blackwell Publishing Ltd.

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...

Comparison Between Several Integrase-defective Lentiviral Vectors Reveals Increased Integration of an HIV Vector Bearing a D167H Mutant

Directory of Open Access Journals (Sweden)

Muhammad Qamar Saeed

2014-01-01

Full Text Available HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the development of streamlined nuclear DNA circles functional for transgene expression. Few integrase mutant vectors have been successfully tested so far for gene transfer. They are cleared with time in mitotic cells, but stable within nondividing retina cells or neurons. Here, we compared six HIV vectors carrying different integrases, either wild type or with different mutations (D64V, D167H, Q168A, K186Q+Q214L+Q216L, and RRK262-264AAH shown to modify integrase enzymatic activity, oligomerization, or interaction with key cellular cofactor of HIV DNA integration as LEDGF/p75 or TNPO3. We show that these mutations differently affect the transduction efficiency as well as rates and patterns of integration of HIV-derived vectors suggesting their different processing in the nucleus. Surprisingly and most interestingly, we report that an integrase carrying the D167H substitution improves vector transduction efficiency and integration in both HEK-293T and primary CD34+ cells.

Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

Directory of Open Access Journals (Sweden)

Nicolas Grandchamp

Full Text Available Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.

Market Efficiency and Real Efficiency: The Connect and Disconnect via Feedback Effects

OpenAIRE

Liyan Yang; Itay Goldstein

2014-01-01

We study a model to explore the (dis)connect between market efficiency and real efficiency when real decision makers learn information from the market to guide their actions. We emphasize two channels that determine whether the two efficiency concepts are aligned. The "externality channel" says that individual learning outcomes may not always map into real efficiency because the presence of externality causes real decision makers to overuse the price information. The "(mis)match channel" emph...

DEA-Risk Efficiency and Stochastic Dominance Efficiency of Stock Indices

Czech Academy of Sciences Publication Activity Database

Branda, M.; Kopa, Miloš

2012-01-01

Roč. 62, č. 2 (2012), s. 106-124 ISSN 0015-1920 R&D Projects: GA ČR GAP402/10/1610 Grant - others:GA ČR(CZ) GAP402/12/0558 Program:GA Institutional research plan: CEZ:AV0Z10750506 Keywords : Data Envelopment Analysis * Risk measures * Index efficiency * Stochastic dominance Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 0.340, year: 2012 http://library.utia.cas.cz/separaty/2012/E/branda-dea-risk efficiency and stochastic dominance efficiency of stock indices.pdf

Contracting for Efficiency. A Best Practices Guide for Energy-Efficient Product Procurement

Energy Technology Data Exchange (ETDEWEB)

Bunch, Saralyn [Office of Energy Efficiency and Renewable Energy (EERE), Washington, DC (United States); Payne, Christopher [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

2016-04-01

The requirement to buy energy- and water-efficient products applies to federal purchases made through any procurement pathway (e.g., purchase cards, e-retailers, and solicitations) and to a wide variety of federal projects. The Federal Energy Management Program’s (FEMP's) Buy Energy-Efficient Products buyer overview fact sheet and Contracting for Efficiency best practices guide for product procurement are designed to support federal buyers in the purchase of energy- and water-efficient products.

Contracting for Efficiency: A Best Practices Guide for Energy Efficient Product Procurement

Energy Technology Data Exchange (ETDEWEB)

Bunch, Saralyn [Office of Energy Efficiency and Renewable Energy (EERE), Washington, DC (United States); Payne, Christopher [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

2017-11-01

The requirement to buy energy- and water-efficient products applies to federal purchases made through any procurement pathway (e.g., purchase cards, e-retailers, and solicitations) and to a wide variety of federal projects. The Federal Energy Management Program’s (FEMP's) Buy Energy-Efficient Products buyer overview fact sheet and Contracting for Efficiency best practices guide for product procurement are designed to support federal buyers in the purchase of energy- and water-efficient products.

Voyager 1 Saturn targeting strategy

Science.gov (United States)

Cesarone, R. J.

1980-01-01

A trajectory targeting strategy for the Voyager 1 Saturn encounter has been designed to accomodate predicted uncertainties in Titan's ephemeris while maximizing spacecraft safety and science return. The encounter is characterized by a close Titan flyby 18 hours prior to Saturn periapse. Retargeting of the nominal trajectory to account for late updates in Titan's estimated position can disperse the ascending node location, which is nominally situated at a radius of low expected particle density in Saturn's ring plane. The strategy utilizes a floating Titan impact vector magnitude to minimize this dispersion. Encounter trajectory characteristics and optimal tradeoffs are presented.

USE Efficiency: an innovative educational programme for energy efficiency in buildings

Science.gov (United States)

Papadopoulos, Theofilos A.; Christoforidis, Georgios C.; Papagiannis, Grigoris K.

2017-10-01

Power engineers are expected to play a pivotal role in transforming buildings into smart and energy-efficient structures, which is necessary since buildings are responsible for a considerable amount of the total energy consumption. To fulfil this role, a holistic approach in education is required, tackling subjects traditionally related to other engineering disciplines. In this context, USE Efficiency is an inter-institutional and interdisciplinary educational programme implemented in nine European Universities targeting energy efficiency in buildings. The educational programme effectively links professors, students, engineers and industry experts, creating a unique learning environment. The scope of the paper is to present the methodology and the general framework followed in the USE Efficiency programme. The proposed methodology can be adopted for the design and implementation of educational programmes on energy efficiency and sustainable development in higher education. End-of-course survey results showed positive feedback from the participating students, indicating the success of the programme.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein.

Science.gov (United States)

Zhu, Jie; Miao, Qiuhong; Tan, Yonggui; Guo, Huimin; Li, Chuanfeng; Chen, Zongyan; Liu, Guangqing

2016-11-01

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. Copyright © 2016 Elsevier B.V. All rights reserved.

An Evolutionary Efficiency Alternative to the Notion of Pareto Efficiency

NARCIS (Netherlands)

I.P. van Staveren (Irene)

2012-01-01

textabstractThe paper argues that the notion of Pareto efficiency builds on two normative assumptions: the more general consequentialist norm of any efficiency criterion, and the strong no-harm principle of the prohibition of any redistribution during the economic process that hurts at least one

Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer

Directory of Open Access Journals (Sweden)

Ouassila Habi

2010-01-01

Full Text Available The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM failure; thus correction of hematopoietic stem cells (HSCs through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs of FancC−/− mice. Using this approach, we show that FancC−/− HSCs were efficiently corrected. Intrafemoral gene transfer of the FancC gene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

Science.gov (United States)

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

Direct lentiviral-cyclooxygenase 2 application to the tendon-bone interface promotes osteointegration and enhances return of the pull-out tensile strength of the tendon graft in a rat model of biceps tenodesis.

Directory of Open Access Journals (Sweden)

Charles H Rundle

Full Text Available This study sought to determine if direct application of the lentiviral (LV-cyclooxygenase 2 (COX2 vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or βgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an

Efficient boiler operations sourcebook

Energy Technology Data Exchange (ETDEWEB)

Payne, F.W. (comp.)

1985-01-01

This book emphasizes the practical aspects of industrial and commercial boiler operations. It starts with a comprehensive review of general combustion and boiler fundamentals and then deals with specific efficiency improvement methods, and the cost savings which result. The book has the following chapter headings: boiler combustion fundamentals; boiler efficiency goals; major factors controlling boiler efficiency; boiler efficiency calculations; heat loss; graphical solutions; preparation for boiler testing; boiler test procedures; efficiency-related boiler maintenance procedures; boiler tune-up; boiler operational modifications; effect of water side and gas side scale deposits; load management; auxillary equipment to increase boiler efficiency; air preheaters and economizers; other types of auxillary equipment; combustion control systems and instrumentation; boiler O/sub 2/ trim controls; should you purchase a new boiler.; financial evaluation procedures; case studies. The last chapter includes a case study of a boiler burning pulverized coal and a case study of stoker-fired coal.

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......, our results indicate that pitfall traps are the most efficient for capturing shrews: not only do they have a higher efficiency (yield), but the taxonomic diversity of shrews is also higher when pitfall traps are used....

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.)

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.

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.

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

Science.gov (United States)

Masiuk, Katelyn E; Brown, Devin; Laborada, Jennifer; Hollis, Roger P; Urbinati, Fabrizia; Kohn, Donald B

2017-09-06

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 - cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 - cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 - cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 - cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

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.

Feedback and efficient behavior.

Directory of Open Access Journals (Sweden)

Sandro Casal

Full Text Available Feedback is an effective tool for promoting efficient behavior: it enhances individuals' awareness of choice consequences in complex settings. Our study aims to isolate the mechanisms underlying the effects of feedback on achieving efficient behavior in a controlled environment. We design a laboratory experiment in which individuals are not aware of the consequences of different alternatives and, thus, cannot easily identify the efficient ones. We introduce feedback as a mechanism to enhance the awareness of consequences and to stimulate exploration and search for efficient alternatives. We assess the efficacy of three different types of intervention: provision of social information, manipulation of the frequency, and framing of feedback. We find that feedback is most effective when it is framed in terms of losses, that it reduces efficiency when it includes information about inefficient peers' behavior, and that a lower frequency of feedback does not disrupt efficiency. By quantifying the effect of different types of feedback, our study suggests useful insights for policymakers.

Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

Science.gov (United States)

El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M; Rommelaere, Jean; Marchini, Antonio

2012-10-01

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

Tumor Targeting and Drug Delivery by Anthrax Toxin

Directory of Open Access Journals (Sweden)

Christopher Bachran

2016-07-01

Full Text Available Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

Tumor Targeting and Drug Delivery by Anthrax Toxin.

Science.gov (United States)

Bachran, Christopher; Leppla, Stephen H

2016-07-01

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

A rapid generation of adenovirus vector with a genetic modification in hexon protein.

Science.gov (United States)

Di, Bingyan; Mao, Qinwen; Zhao, Junli; Li, Xing; Wang, Dongyang; Xia, Haibin

2012-02-10

The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Lentivector Integration Sites in Ependymal Cells From a Model of Metachromatic Leukodystrophy: Non-B DNA as a New Factor Influencing Integration

Science.gov (United States)

McAllister, Robert G; Liu, Jiahui; Woods, Matthew W; Tom, Sean K; Rupar, C Anthony; Barr, Stephen D

2014-01-01

The blood–brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells. PMID:25158091

Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

Directory of Open Access Journals (Sweden)

Lindsey M. Skrdlant

2018-03-01

Full Text Available Lentiviral vectors are a common tool used to introduce new and corrected genes into cell therapy products for treatment of human diseases. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication competent lentivirus (RCL capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL prior to treatment of the patient. Current Food and Drug Administration (FDA guidelines recommend use of cell-based assays to this end, which can take up to 6 weeks for results. However, qPCR-based assays are a quick alternative for rapid assessment of RCL in products intended for fresh infusion. We describe here the development and qualification of a qPCR assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G] for RCL in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Our results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations.

Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.

Directory of Open Access Journals (Sweden)

Ran Taube

Full Text Available BACKGROUND: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv antibody fragments on human cells and lentivirus particles. METHODOLOGY/PRINCIPAL FINDINGS: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. CONCLUSIONS/SIGNIFICANCE: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.

Direct induction of hepatocyte-like cells from immortalized human bone marrow mesenchymal stem cells by overexpression of HNF4α

International Nuclear Information System (INIS)

Hu, Xiaojun; Xie, Peiyi; Li, Weiqiang; Li, Zhengran; Shan, Hong

2016-01-01

Hepatocytes from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are expected to be a useful source for cell transplantation. However, relatively low efficiency and repeatability of hepatic differentiation of human BM-MSCs remains an obstacle for clinical translation. Hepatocyte nuclear factor 4 alpha (HNF4α), a critical transcription factor, plays an essential role in the entire process of liver development. In this study, immortalized hBM-MSCs, UE7T-13 cells were transduced with a lentiviral vector containing HNF4α. The typical fibroblast-like morphology of the MSCs changed, and polygonal, epithelioid cells grew out after HNF4α transduction. In hepatocyte culture medium, HNF4α-transduced MSCs (E7-hHNF4α cells) strongly expressed the albumin (ALB), CYP2B6, alpha-1 antitrypsin (AAT), and FOXA2 mRNA and exhibited morphology markedly similar to that of mature hepatocytes. The E7-hHNF4α cells showed hepatic functions such as Indocyanine green (ICG) uptake and release, glycogen storage, urea production and ALB secretion. Approximately 28% of E7-hHNF4α cells expressed both ALB and AAT. Furthermore, these E7-hHNF4α cells via superior mesenteric vein (SMV) injection expressed human ALB in mouse chronic injured liver. In conclusion, this study represents a novel strategy by directly inducing hepatocyte-like cells from MSCs. - Highlights: • We overexpressed HNF4α in immortalized BM-MSCs by lentiviral transduction. • HNF4α-transduced MSCs transdifferentiated into hepatocytes with mature hepatic metabolic functions. • Our study represents a novel strategy by direct induction of hepatocyte-like cells from MSCs.

Current strides in AAV-derived vectors and SIN channels further ...

African Journals Online (AJOL)

A.S. Odiba

restored, hematopoietic stem cells has been used to terminate incurable blood ... gene and cell therapy approach are founded on either ex vivo gene incorporation into ..... generating integration-deficient lentiviral vectors (IDLV), which on.

Efficiency model of Russian banks

OpenAIRE

Pavlyuk, Dmitry

2006-01-01

The article deals with problems related to the stochastic frontier model of bank efficiency measurement. The model is used to study the efficiency of the banking sector of The Russian Federation. It is based on the stochastic approach both to the efficiency frontier location and to individual bank efficiency values. The model allows estimating bank efficiency values, finding relations with different macro- and microeconomic factors and testing some economic hypotheses.

How to subsidize energy efficiency under duopoly efficiently?

International Nuclear Information System (INIS)

Nie, Pu-yan; Yang, Yong-cong; Chen, You-hua; Wang, Zhao-hui

2016-01-01

Highlights: • This article captures the effects of output subsidy. • Firms without subsidy are not willing to improve energy efficiency. • Subsidy stimulates the subsidized firms’ outputs and deters the others’ outputs. • The subsidy intensity depends on firms’ position. • Overdue subsidy cannot reach the environmental object. - Abstract: Establishing a game theory model, this paper captures the effects of output subsidy on energy efficiency under Cournot competition and Stackelberg competition. Three types of subsidies are considered in the model, namely without subsidy, unilateral subsidy and bilateral subsidy. The findings indicate that firms without subsidy are not willing to improve energy efficiency. Also, subsidy stimulates the subsidized firms’ outputs while deters the outputs of other firms. Meanwhile, the equilibrium subsidy intensity depends on firms’ position. Furthermore, the minimal subsidy budgets under different situations are presented. Especially, given the fixed subsidy budget, the output of the subsidized firm is the highest if this firm plays the leading position. In addition, certain subsidy can reduce the total emission, while overdue subsidy cannot reach the environmental object.

Energy efficient policy impact in India: case study of investment in industrial energy efficiency

International Nuclear Information System (INIS)

Yang, M.

2006-01-01

The objective of this paper is to identify the effectiveness of energy policy and capital investment in energy efficiency technologies in the industrial sector in India. Indian energy policies relating to industrial energy efficiency over the past 25 years are briefly reviewed, and a comparison study of these energy efficiency policies and strategies in India and China has been carried out. Interviews were conducted with a number of government policy-making institutions and a national industrial development bank. The accounts of 26 industrial enterprises which applied and used a loan of the Asian Development Bank were audited for data collection. Field-visits to seven industrial entrepreneurs were undertaken in a case study. Methodologies used in this study include documentation, cross-country reviews on energy policies, questionnaire design and distribution in the industrial sector, and on-site auditing of energy efficiency technologies. This paper concludes that current energy policies and strategies in India need further improvement to promote energy efficiency investment and energy efficiency technology development in the industrial sector. This paper will interest those policy makers and industrial entrepreneurs who are willing to finance energy efficiency projects and improve energy efficiency in the industrial sector. (author)

Energy efficiency policy impact in India: case study of investment in industrial energy efficiency

International Nuclear Information System (INIS)

Yang Ming

2006-01-01

The objective of this paper is to identify the effectiveness of energy policy and capital investment in energy efficiency technologies in the industrial sector in India. Indian energy policies relating to industrial energy efficiency over the past 25 years are briefly reviewed, and a comparison study of these energy efficiency policies and strategies in India and China has been carried out. Interviews were conducted with a number of government policy-making institutions and a national industrial development bank. The accounts of 26 industrial enterprises which applied and used a loan of the Asian Development Bank were audited for data collection. Field-visits to seven industrial entrepreneurs were undertaken in a case study. Methodologies used in this study include documentation, cross-country reviews on energy policies, questionnaire design and distribution in the industrial sector, and on-site auditing of energy efficiency technologies. This paper concludes that current energy policies and strategies in India need further improvement to promote energy efficiency investment and energy efficiency technology development in the industrial sector. This paper will interest those policy makers and industrial entrepreneurs who are willing to finance energy efficiency projects and improve energy efficiency in the industrial sector

Energy efficiency networks; Energieeffizienz-Netzwerke

Energy Technology Data Exchange (ETDEWEB)

Gruber, Anna [Forschungsgesellschaft fuer Energiewirtschaft mbH (FfE GmbH), Muenchen (Germany)

2011-07-01

Energy efficiency networks are an attractive method to increase the energy efficiency and to reduce the costs and CO{sub 2} emissions of the companies operating in this network. A special feature of the energy efficiency networks is the exchange of experiences and training of the energy managers. Energy efficiency networks consist of about ten to fifteen locally domiciled companies. During the project period of three to four years, there are two main phases. In the first phase, the initial consultation phase, the actual state of a company is captured, and measures to increase the efficiency and energy conservation are identified. Parallel to this, in the second phase every three months a meeting with the participating companies takes place. Experience exchange and implementation of energy efficiency measures are the focus of these meetings. Initial studies show that the increase of the energy efficiency during participating in the energy efficiency network almost can be doubled in comparison to the average of the industry.

Energy efficiency: 2004 world overview

International Nuclear Information System (INIS)

2004-01-01

Since 1992 the World Energy Council (WEC) has been collaborating with ADEME (Agency for Environment and Energy Efficiency, France) on a joint project 'Energy Efficiency Policies and Indicators'. APERC (Asia Pacific Energy Research Centre) and OLADE (Latin American Energy Organisation) have also participated in the study, which has been monitoring and evaluating energy efficiency policies and their impacts around the world. WEC Member Committees have been providing data and information and ENERDATA (France) has provided technical assistance. This report, published in August 2004, presents and evaluates energy efficiency policies in 63 countries, with a specific focus on five policy measures, for which in-depth case studies were prepared by selected experts: - Minimum energy efficiency standards for household electrical appliances; - Innovative energy efficiency funds; - Voluntary/negotiated agreements on energy efficiency/ CO 2 ; - Local energy information centres; - Packages of measures. In particular, the report identifies the policy measures, which have proven to be the most effective, and can be recommended to countries which have recently embarked on the development and implementation of energy demand management policies. During the past ten years, the Kyoto Protocol and, more recently, emerging concerns about security of supply have raised, both the public and the political profile of energy efficiency. Almost all OECD countries and an increasing number of other countries are implementing energy efficiency policies adapted to their national circumstances. In addition to the market instruments (voluntary agreements, labels, information, etc.), regulatory measures are widely introduced where the market fails to give the right signals (buildings, appliances). In developing countries, energy efficiency is equally important, even if the drivers are different compared to industrialized countries. Reduction of greenhouse gas emissions and local pollution often have a

transgenesis techniques and its application in poultry production

African Journals Online (AJOL)

2015-05-27

May 27, 2015 ... reported another method - Lentiviral transgenesis which was reported to ... embryonic stem cells to differentiate in vitro to form embryoid ... induced differentiation. Thus ... are pluripotent and can thus be transmitted from one.

efficience technico-economique

African Journals Online (AJOL)

USER

ABSTRACT. TECHNICO-ECONOMIC EFFICIENCY : CASE OF THE PRODUCERS OF ONION AND POTATO IN KNOW IN MOROCCO. In the new context ... Key words : Technico-economic efficiency, stochastic frontier, potato production, onion production, Morocco ..... sont le labour, le cover-cropage et le traçage. 82 % des ...

Batch efficiency

Energy Technology Data Exchange (ETDEWEB)

Schwickerath, Ulrich; Silva, Ricardo; Uria, Christian, E-mail: Ulrich.Schwickerath@cern.c, E-mail: Ricardo.Silva@cern.c [CERN IT, 1211 Geneve 23 (Switzerland)

2010-04-01

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.

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

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.

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

Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

Directory of Open Access Journals (Sweden)

José L. Affranchino

2014-01-01

Full Text Available The lentiviral envelope glycoproteins (Env mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

African Journal of Biotechnology - Vol 9, No 13 (2010)

African Journals Online (AJOL)

Inheritance of fresh seed dormancy in Spanish-type peanut (Arachis ... Construction of a novel lentiviral vector carrying human B-domain-deleted factor VIII gene ... Studies on hemorrhagic pneumonia in Moschus sifanicus · EMAIL FREE FULL ...

Energy efficiency and behaviour

DEFF Research Database (Denmark)

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

separate key aspects hinders strategic energy efficiency planning. For this reason, the PLEEC project – “Planning for Energy Efficient Cities” – funded by the EU Seventh Framework Programme uses an integrative approach to achieve the sus‐ tainable, energy– efficient, smart city. By coordinating strategies...... to conduct behavioural interventions, to be presented in Deliverable 5.5., the final report. This report will also provide valuable information for the WP6 general model for an Energy-Smart City. Altogether 38 behavioural interventions are analysed in this report. Each collected and analysed case study...... of the European Union’s 20‐20‐20 plan is to improve energy efficiency by 20% in 2020. However, holistic knowledge about energy efficiency potentials in cities is far from complete. Currently, a WP4 location in PLEEC project page 3 variety of individual strategies and approaches by different stakeholders tackling...

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 ...

Energy efficiency in Swedish industry

International Nuclear Information System (INIS)

Zhang, Shanshan; Lundgren, Tommy; Zhou, Wenchao

2016-01-01

This paper assesses energy efficiency in Swedish industry. Using unique firm-level panel data covering the years 2001–2008, the efficiency estimates are obtained for firms in 14 industrial sectors by using data envelopment analysis (DEA). The analysis accounts for multi-output technologies where undesirable outputs are produced alongside with the desirable output. The results show that there was potential to improve energy efficiency in all the sectors and relatively large energy inefficiencies existed in small energy-use industries in the sample period. Also, we assess how the EU ETS, the carbon dioxide (CO_2) tax and the energy tax affect energy efficiency by conducting a second-stage regression analysis. To obtain consistent estimates for the regression model, we apply a modified, input-oriented version of the double bootstrap procedure of Simar and Wilson (2007). The results of the regression analysis reveal that the EU ETS and the CO_2 tax did not have significant influences on energy efficiency in the sample period. However, the energy tax had a positive relation with the energy efficiency. - Highlights: • We use DEA to estimate firm-level energy efficiency in Swedish industry. • We examine impacts of climate and energy policies on energy efficiency. • The analyzed policies are Swedish carbon and energy taxes and the EU ETS. • Carbon tax and EU ETS did not have significant influences on energy efficiency. • The energy tax had a positive relation with energy efficiency.

Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

Directory of Open Access Journals (Sweden)

Mayumi Oakland

2013-01-01

Full Text Available Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.

Gene transfer in rodents and primates as a new tool for modeling diseases in animals and assessing functions by in vivo imaging

Energy Technology Data Exchange (ETDEWEB)

Deglon, N. [Atomic Energy Commission (CEA), Dept. of Medical Research and MIRCen Program, 91 - Orsay (France)

2006-07-01

The identification of disease-causing genes in familial forms of neuro-degenerative disorders and the development of genetic models closely replicating human CNS pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions efficient delivery systems taking into account the specificity of the central nervous system are required to administer therapeutic candidates. In addition, there is a need to develop 1) genetic models in large animals that replicate late stages of the diseases and 2) imaging techniques suitable for longitudinal, quantitative and non-invasive evaluation of disease progression and the evaluation of new therapeutic strategies. Over the last few years, we have investigated the potential of lentiviral vectors as tool to model and treat CNS disorders. The use of lentiviral vectors to create animal model of these pathologies holds various advantages compared to classical transgenic approaches. Viral vectors are versatile, highly flexible tools to perform in vivo studies. Multiple genetic models can be created in a short period of time. High transduction efficiencies as well as robust and sustained trans-gene expression lead to the rapid appearance of functional and behavioral abnormalities and severe neuro-degeneration. Targeted injections in different brain areas can be used to investigate the regional specificity of the neuro-pathology and eliminate potential side effects associated with a widespread over-expression of the trans-gene. Finally, models can be established in different mammalian species including non-human primates, thereby providing an opportunity to assess complex behavioral changes and perform longitudinal follow-up of neuro-pathological alterations by imaging. We have demonstrated the proof of principle of this approach for Huntington's disease. We have shown that the intratriatal injection of lentiviral

Gene transfer in rodents and primates as a new tool for modeling diseases in animals and assessing functions by in vivo imaging

International Nuclear Information System (INIS)

Deglon, N.

2006-01-01

The identification of disease-causing genes in familial forms of neuro-degenerative disorders and the development of genetic models closely replicating human CNS pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions efficient delivery systems taking into account the specificity of the central nervous system are required to administer therapeutic candidates. In addition, there is a need to develop 1) genetic models in large animals that replicate late stages of the diseases and 2) imaging techniques suitable for longitudinal, quantitative and non-invasive evaluation of disease progression and the evaluation of new therapeutic strategies. Over the last few years, we have investigated the potential of lentiviral vectors as tool to model and treat CNS disorders. The use of lentiviral vectors to create animal model of these pathologies holds various advantages compared to classical transgenic approaches. Viral vectors are versatile, highly flexible tools to perform in vivo studies. Multiple genetic models can be created in a short period of time. High transduction efficiencies as well as robust and sustained trans-gene expression lead to the rapid appearance of functional and behavioral abnormalities and severe neuro-degeneration. Targeted injections in different brain areas can be used to investigate the regional specificity of the neuro-pathology and eliminate potential side effects associated with a widespread over-expression of the trans-gene. Finally, models can be established in different mammalian species including non-human primates, thereby providing an opportunity to assess complex behavioral changes and perform longitudinal follow-up of neuro-pathological alterations by imaging. We have demonstrated the proof of principle of this approach for Huntington's disease. We have shown that the intratriatal injection of lentiviral vector

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)

Efficiency profile method to study the hit efficiency of drift chambers

International Nuclear Information System (INIS)

Abyzov, A.; Bel'kov, A.; Lanev, A.; Spiridonov, A.; Walter, M.; Hulsbergen, W.

2002-01-01

A method based on the usage of efficiency profile is proposed to estimate the hit efficiency of drift chambers with a large number of channels. The performance of the method under real conditions of the detector operation has been tested analysing the experimental data from the HERA-B drift chambers

Energy efficiency in pumps

International Nuclear Information System (INIS)

Kaya, Durmus; Yagmur, E. Alptekin; Yigit, K. Suleyman; Kilic, Fatma Canka; Eren, A. Salih; Celik, Cenk

2008-01-01

In this paper, 'energy efficiency' studies, done in a big industrial facility's pumps, are reported. For this purpose; the flow rate, pressure and temperature have been measured for each pump in different operating conditions and at maximum load. In addition, the electrical power drawn by the electric motor has been measured. The efficiencies of the existing pumps and electric motor have been calculated by using the measured data. Potential energy saving opportunities have been studied by taking into account the results of the calculations for each pump and electric motor. As a conclusion, improvements should be made each system. The required investment costs for these improvements have been determined, and simple payback periods have been calculated. The main energy saving opportunities result from: replacements of the existing low efficiency pumps, maintenance of the pumps whose efficiencies start to decline at certain range, replacements of high power electric motors with electric motors that have suitable power, usage of high efficiency electric motors and elimination of cavitation problems

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......, a proportionator sampling and a systematic, uniform random sampling were simulated. We found that the proportionator was 50% to 90% more time efficient than systematic, uniform random sampling. The time efficiency of the autodisector on virtual slides was 60% to 100% better than the disector on tissue slides. We...... conclude that both the proportionator and the autodisector on virtual slides may improve efficiency of cell counting in stereology....

Energy efficient design

International Nuclear Information System (INIS)

1991-01-01

Solar Applications and Energy Efficiency in Building Design and Town Planning (RER/87/006) is a United Nations Development Programme (UNDP) project of the Governments of Albania, Bulgaria, Cyprus, The Czech and Slovak Federal Republic, France, Hungary, Malta, Poland, Turkey, United Kingdom and Yugoslavia. The project began in 1988 and comes to a conclusion at the end of 1991. It is to enhance the professional skills of practicing architects, engineers and town planners in European countries to design energy efficient buildings which reduce energy consumption and make greater use of passive solar heating and natural cooling techniques. The United Nations Economic Commission for Europe (ECE) is the Executing Agency of the project which is implemented under the auspices of the Committee on Energy, General Energy Programme of Work for 1990-1994, sub-programme 5 Energy Conservation and Efficiency (ECE/ENERGY/15). The project has five main outputs or results: an international network of institutions for low energy building design; a state-of-the-art survey of energy use in the built environment of European IPF countries; a simple computer program for energy efficient building design; a design guide and computer program operators' manual; and a series of international training courses in participating European IPF countries. Energy Efficient Design is the fourth output of the project. It comprises the design guide for practicing architects and engineers, for use mainly in mid-career training courses, and the operators' manual for the project's computer program

Measuring sustainability by Energy Efficiency Analysis for Korean Power Companies: A Sequential Slacks-Based Efficiency Measure

Directory of Open Access Journals (Sweden)

Ning Zhang

2014-03-01

Full Text Available Improving energy efficiency has been widely regarded as one of the most cost-effective ways to improve sustainability and mitigate climate change. This paper presents a sequential slack-based efficiency measure (SSBM application to model total-factor energy efficiency with undesirable outputs. This approach simultaneously takes into account the sequential environmental technology, total input slacks, and undesirable outputs for energy efficiency analysis. We conduct an empirical analysis of energy efficiency incorporating greenhouse gas emissions of Korean power companies during 2007–2011. The results indicate that most of the power companies are not performing at high energy efficiency. Sequential technology has a significant effect on the energy efficiency measurements. Some policy suggestions based on the empirical results are also presented.

More efficient together

DEFF Research Database (Denmark)

Zhang, Tian

2015-01-01

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 app...... competition between biotechnology and the food industry and avoid the environmental perturbation caused by intensive agriculture (3)....

Current-flow efficiency of networks

Science.gov (United States)

Liu, Kai; Yan, Xiaoyong

2018-02-01

Many real-world networks, from infrastructure networks to social and communication networks, can be formulated as flow networks. How to realistically measure the transport efficiency of these networks is of fundamental importance. The shortest-path-based efficiency measurement has limitations, as it assumes that flow travels only along those shortest paths. Here, we propose a new metric named current-flow efficiency, in which we calculate the average reciprocal effective resistance between all pairs of nodes in the network. This metric takes the multipath effect into consideration and is more suitable for measuring the efficiency of many real-world flow equilibrium networks. Moreover, this metric can handle a disconnected graph and can thus be used to identify critical nodes and edges from the efficiency-loss perspective. We further analyze how the topological structure affects the current-flow efficiency of networks based on some model and real-world networks. Our results enable a better understanding of flow networks and shed light on the design and improvement of such networks with higher transport efficiency.

Bandwidth efficient coding

CERN Document Server

Anderson, John B

2017-01-01

Bandwidth Efficient Coding addresses the major challenge in communication engineering today: how to communicate more bits of information in the same radio spectrum. Energy and bandwidth are needed to transmit bits, and bandwidth affects capacity the most. Methods have been developed that are ten times as energy efficient at a given bandwidth consumption as simple methods. These employ signals with very complex patterns and are called "coding" solutions. The book begins with classical theory before introducing new techniques that combine older methods of error correction coding and radio transmission in order to create narrowband methods that are as efficient in both spectrum and energy as nature allows. Other topics covered include modulation techniques such as CPM, coded QAM and pulse design.

Where is the Efficient Frontier

OpenAIRE

Jing Chen

2010-01-01

Tremendous effort has been spent on the construction of reliable efficient frontiers. However, mean-variance efficient portfolios constructed using sample means and covariance often perform poorly out of sample. We prove that, the capital market line is the efficient frontier for the risky assets in a financial market with liquid fixed income trading. This unified understanding of riskless asset as the boundary of risky assets relieves the burden of constructing efficient frontiers in asset a...

Energy efficiency in pumps

Energy Technology Data Exchange (ETDEWEB)

Kaya, Durmus; Yagmur, E. Alptekin [TUBITAK-MRC, P.O. Box 21, 41470 Gebze, Kocaeli (Turkey); Yigit, K. Suleyman; Eren, A. Salih; Celik, Cenk [Engineering Faculty, Kocaeli University, Kocaeli (Turkey); Kilic, Fatma Canka [Department of Air Conditioning and Refrigeration, Kocaeli University, Kullar, Kocaeli (Turkey)

2008-06-15

In this paper, ''energy efficiency'' studies, done in a big industrial facility's pumps, are reported. For this purpose; the flow rate, pressure and temperature have been measured for each pump in different operating conditions and at maximum load. In addition, the electrical power drawn by the electric motor has been measured. The efficiencies of the existing pumps and electric motor have been calculated by using the measured data. Potential energy saving opportunities have been studied by taking into account the results of the calculations for each pump and electric motor. As a conclusion, improvements should be made each system. The required investment costs for these improvements have been determined, and simple payback periods have been calculated. The main energy saving opportunities result from: replacements of the existing low efficiency pumps, maintenance of the pumps whose efficiencies start to decline at certain range, replacements of high power electric motors with electric motors that have suitable power, usage of high efficiency electric motors and elimination of cavitation problems. (author)

Productivity and energy efficiency

Energy Technology Data Exchange (ETDEWEB)

Lovins, H. [Rocky Mountain Inst., Snowmass, CO (United States)

1995-12-31

Energy efficient building and office design offers the possibility of significantly increased worker productivity. By improving lighting, heating and cooling, workers can be made more comfortable and productive. An increase of 1 percent in productivity can provide savings to a company that exceed its entire energy bill. Efficient design practices are cost effective just from their energy savings. The resulting productivity gains make them indispensable. This paper documents eight cases in which efficient lighting, heating, and cooling have measurably increased worker productivity, decreased absenteeism, and/or improved the quality of work performed. They also show that efficient lighting can measurably increase work quality by removing errors and manufacturing defects. The case studies presented include retrofit of existing buildings and the design of new facilities, and cover a variety of commercial and industrial settings. Each case study identifies the design changes that were most responsible for increased productivity. As the eight case studies illustrate, energy efficient design may be one of the least expensive ways for a business to improve the productivity of its workers and the quality of its product. (author). 15 refs.

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)

In Vitro Generation of Vascular Wall-Resident Multipotent Stem Cells of Mesenchymal Nature from Murine Induced Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Jennifer Steens

2017-04-01

Full Text Available Summary: The vascular wall (VW serves as a niche for mesenchymal stem cells (MSCs. In general, tissue-specific stem cells differentiate mainly to the tissue type from which they derive, indicating that there is a certain code or priming within the cells as determined by the tissue of origin. Here we report the in vitro generation of VW-typical MSCs from induced pluripotent stem cells (iPSCs, based on a VW-MSC-specific gene code. Using a lentiviral vector expressing the so-called Yamanaka factors, we reprogrammed tail dermal fibroblasts from transgenic mice containing the GFP gene integrated into the Nestin-locus (NEST-iPSCs to facilitate lineage tracing after subsequent MSC differentiation. A lentiviral vector expressing a small set of recently identified human VW-MSC-specific HOX genes then induced MSC differentiation. This direct programming approach successfully mediated the generation of VW-typical MSCs with classical MSC characteristics, both in vitro and in vivo. : In this article, Klein and colleagues show that iPSCs generated from skin fibroblasts of transgenic mice carrying a GFP gene under the control of the endogenous Nestin promoter to facilitate lineage tracing (NEST-iPSCs can be directly programmed toward mouse vascular wall-typical multipotent mesenchymal stem cells (VW-MSC by ectopic lentiviral expression of a previously defined VW-MSC-specific HOX code. Keywords: vascular wall-derived mesenchymal stem cells, HOX gene, induced pluripotent stem cells, direct programming, nestin

[Knockdown of dopamine receptor D2 upregulates the expression of adiogenic genes in mouse primary mesencephalic neurons].

Science.gov (United States)

Ding, Jiaqi; Chen, Xiaoli; Lin, Jiaji; Zhu, Junling; Li, Zhuyi

2018-01-01

Objective To study the effects of dopamine receptor D2 (DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing (HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes. Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation, mainly including delta(14)-sterol reductase, acetyl-coenzyme A synthetase, insulin-induced gene 1 protein and especially stearoyl-coenzyme A desaturase 1 (SCD1, 4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2.6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover, the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.

Are large farms more efficient? Tenure security, farm size and farm efficiency: evidence from northeast China

Science.gov (United States)

Zhou, Yuepeng; Ma, Xianlei; Shi, Xiaoping

2017-04-01

How to increase production efficiency, guarantee grain security, and increase farmers' income using the limited farmland is a great challenge that China is facing. Although theory predicts that secure property rights and moderate scale management of farmland can increase land productivity, reduce farm-related costs, and raise farmer's income, empirical studies on the size and magnitude of these effects are scarce. A number of studies have examined the impacts of land tenure or farm size on productivity or efficiency, respectively. There are also a few studies linking farm size, land tenure and efficiency together. However, to our best knowledge, there are no studies considering tenure security and farm efficiency together for different farm scales in China. In addition, there is little study analyzing the profit frontier. In this study, we particularly focus on the impacts of land tenure security and farm size on farm profit efficiency, using farm level data collected from 23 villages, 811 households in Liaoning in 2015. 7 different farm scales have been identified to further represent small farms, median farms, moderate-scale farms, and large farms. Technical efficiency is analyzed with stochastic frontier production function. The profit efficiency is regressed on a set of explanatory variables which includes farm size dummies, land tenure security indexes, and household characteristics. We found that: 1) The technical efficiency scores for production efficiency (average score = 0.998) indicate that it is already very close to the production frontier, and thus there is little room to improve production efficiency. However, there is larger space to raise profit efficiency (average score = 0.768) by investing more on farm size expansion, seed, hired labor, pesticide, and irrigation. 2) Farms between 50-80 mu are most efficient from the viewpoint of profit efficiency. The so-called moderate-scale farms (100-150 mu) according to the governmental guideline show no

Efficiency in man-machine communication

NARCIS (Netherlands)

Haakma, R.; Engel, F.L.

1990-01-01

Expressed in terms of speed and accuracy, intention transfer in goal-oriented inter-human communication can be very efficient. One of the mechanisms that make for efficient communication is early detection and repair of communication errors. Another important efficiency mechanism prevents repeated

The rebound effect of resource efficiency; Het reboundeffect bij resource efficiency

Energy Technology Data Exchange (ETDEWEB)

Oosterhuis, F.; Bouma, J. [Instituut voor Milieuvraagstukken IVM, Vrije Universiteit, Amsterdam (Netherlands); Hanemaaijer, A. [Planbureau voor de Leefomgeving PBL, Den Haag (Netherlands)

2013-12-15

As a result of efficient use of natural resources production and use of goods and services can be made cheaper, which increases demand. However, that might partially offset savings in energy and other natural resources. This so-called 'rebound effect' can not be ignored. It is therefore worthwhile to keep that in formulating 'resource efficiency' policy [Dutch] Door efficienter om te gaan met natuurlijke hulpbronnen kunnen de productie en het gebruik van goederen en diensten goedkoper worden, waardoor de vraag ernaar toeneemt. Dat kan de besparingen op energie en andere natuurlijke hulpbronnen deels weer teniet doen. Dit 'rebound-effect' is niet verwaarloosbaar. Het is zinvol om hier bij beleid gericht op 'resource efficiency' rekening mee te houden.

Energy efficiency and renewables policies: Promoting efficiency or facilitating monopsony?

International Nuclear Information System (INIS)

Brennan, Timothy J.

2011-01-01

The cliche in the electricity sector, the 'cheapest power plant is the one we don't build,' neglects the benefits of the energy that plant would generate. That economy-wide perspective need not apply in considering benefits to only consumers if not building that plant was the exercise of monopsony power. A regulator maximizing consumer welfare may need to avoid rationing demand at monopsony prices. Subsidizing energy efficiency to reduce electricity demand at the margin can solve that problem, if energy efficiency and electricity use are substitutes. Renewable energy subsidies, percentage use standards, or feed in tariffs may also serve monopsony as well with sufficient inelasticity in fossil fuel electricity supply. We may not observe these effects if the regulator can set price as well as quantity, lacks buyer-side market power, or is legally precluded from denying generators a reasonable return on capital. Nevertheless, the possibility of monopsony remains significant in light of the debate as to whether antitrust enforcement should maximize consumer welfare or total welfare. - Research Highlights: → Subsidizing energy efficiency can promote monopsony, if efficiency and use are substitutes. → Renewable energy subsidies, portfolio standards, or feed-in tariffs may also promote monopsony. → Effects require buyer-side market power and ability to deny generators a reasonable return. → Monopsony is significant in light of whether antitrust should maximize consumer or total welfare.

Efficient Learning Design

DEFF Research Database (Denmark)

Godsk, Mikkel

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...... 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...... 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...

Gene editing in hematopoietic stem cells: a potential therapeutic approach for Fanconi anemia

International Nuclear Information System (INIS)

Diez Cabezas, B.

2015-01-01

Gene therapy nowadays constitutes a safe and efficient treatment for a number of monogenic diseases affecting the hematopoietic system. Risks of insertional mutagenesis derived from the use of integrative vectors cannot, however, be completely excluded. Therefore, gene targeting has been proposed as a safer alternative, since the insertion of the herapeutic gene is driven to a specific locus in the genome. Gene targeting approaches are based on the use of specific nucleases which generate double strand breaks (DSBs) in a specific site of the genome,markedly enhancing the efficacy of homologous recombination (HR) with donor constructs harboring the gene of interest flanked by the corresponding homology arms. In this study we have optimized the conditions to target human lymphoblastic cell lines (LCLs) and also hematopoietic stem cells (HSCs) from healthy donors, with the final aim of correcting by gene editing the hematopoietic progenitor cells from Fanconi anemia subtype A (FA-A) patients. In particular, we have established a robust method to target both LCLs and HSCs in a safe harbor site in the genome, the AAVS1 locus. Our approach is based on the transduction of these cells with integrase-defective lentiviral vectors carrying a donor with the gene of interest, followed by the nucleofection of these cells with zinc finger nucleases used as mRNA. Using a control donor vector carrying the GFP reporter gene we have obtained, on average, 9.43% gene targeting efficiency in cord blood CD34+ cells from healthy donors. Moreover, we confirmed that gene targeting was also efficient in HSCs with long term and multipotent repopulation capacity, as demonstrated by transplants into immunodeficient mice. To improve the gene targeting efficiency, we investigated the feasibility of using gold nanoparticles, which were shown to improve the transduction efficiency of integrase-defective and competent lentiviral vectors in HSCs. This increment, however, did not lead to a higher gene

Gene editing in hematopoietic stem cells: a potential therapeutic approach for Fanconi anemia

Energy Technology Data Exchange (ETDEWEB)

Diez Cabezas, B.

2015-07-01

Gene therapy nowadays constitutes a safe and efficient treatment for a number of monogenic diseases affecting the hematopoietic system. Risks of insertional mutagenesis derived from the use of integrative vectors cannot, however, be completely excluded. Therefore, gene targeting has been proposed as a safer alternative, since the insertion of the herapeutic gene is driven to a specific locus in the genome. Gene targeting approaches are based on the use of specific nucleases which generate double strand breaks (DSBs) in a specific site of the genome,markedly enhancing the efficacy of homologous recombination (HR) with donor constructs harboring the gene of interest flanked by the corresponding homology arms. In this study we have optimized the conditions to target human lymphoblastic cell lines (LCLs) and also hematopoietic stem cells (HSCs) from healthy donors, with the final aim of correcting by gene editing the hematopoietic progenitor cells from Fanconi anemia subtype A (FA-A) patients. In particular, we have established a robust method to target both LCLs and HSCs in a safe harbor site in the genome, the AAVS1 locus. Our approach is based on the transduction of these cells with integrase-defective lentiviral vectors carrying a donor with the gene of interest, followed by the nucleofection of these cells with zinc finger nucleases used as mRNA. Using a control donor vector carrying the GFP reporter gene we have obtained, on average, 9.43% gene targeting efficiency in cord blood CD34+ cells from healthy donors. Moreover, we confirmed that gene targeting was also efficient in HSCs with long term and multipotent repopulation capacity, as demonstrated by transplants into immunodeficient mice. To improve the gene targeting efficiency, we investigated the feasibility of using gold nanoparticles, which were shown to improve the transduction efficiency of integrase-defective and competent lentiviral vectors in HSCs. This increment, however, did not lead to a higher gene

Studying the Efficiency of Industrial Dairy Farms of Saqqez and Divandarreh Cities: Using Super-Efficiency Approach

Directory of Open Access Journals (Sweden)

S.J. Mohammadi

2016-03-01

Full Text Available Introduction: In the developed world, particularly in developing countries, livestock is the most important agricultural sub-sector.Livestock of primary and secondary industries has an especial place in the national economybecause of their greatvalue of products, creating job opportunities, providing health products for consumers, increasing export income of the economy throughaccessing global markets of livestock products and finally their undeniable role in acquiring food security.The demand for milk in Iran increased due to an increase in population and the amount of milk production was also increased. The great share of increased produced milk goes to the industrial dairy farms. One of the major methods to increase the amount of milk production continually is to make its production efficient and improve economic conditions. The current study attempts to determine the efficiency and ranking of industrial dairy farms in Saqqez and Divandarreh cities using super-efficiency model. Materials and Methods: The statistical populations of the study are all active industrial dairy farms of Saqqez and Divandarreh cities which are about 19 farms. The required data for calculating the efficiency were gathered by surveying and completing questionnaires for the year 2013. In this study first, for each farm Data Envelopment Analysis (DEA method and GAMS software package were used to estimate super efficiency. Super efficiency is a form of modified DEA model in which each farm can get an efficiency greater than one. Then in order to make sure about being unbiased the obtained super-efficiency scores, the modified model of Banker and Gifford, was re-estimated and the conventional efficiency scores of farms were compared by normalizing and removing some of the scores of outlier farm based on pre-selected screens. The model has suggested conditions for which some of the estimates for dairy farms might have been contaminated with error.As a t result, it has been

Efficiency and Loading Evaluation of High Efficiency Mist Eliminators (HEME) - 12003

Energy Technology Data Exchange (ETDEWEB)

Giffin, Paxton K.; Parsons, Michael S.; Waggoner, Charles A. [Institute for Clean Energy Technology, Mississippi State University, 205 Research Blvd Starkville, MS 39759 (United States)

2012-07-01

High efficiency mist eliminators (HEME) are filters primarily used to remove moisture and/or liquid aerosols from an air stream. HEME elements are designed to reduce aerosol and particulate load on primary High Efficiency Particulate Air (HEPA) filters and to have a liquid particle removal efficiency of approximately 99.5% for aerosols down to sub-micron size particulates. The investigation presented here evaluates the loading capacity of the element in the absence of a water spray cleaning system. The theory is that without the cleaning system, the HEME element will suffer rapid buildup of solid aerosols, greatly reducing the particle loading capacity. Evaluation consists of challenging the element with a waste surrogate dry aerosol and di-octyl phthalate (DOP) at varying intervals of differential pressure to examine the filtering efficiency of three different element designs at three different media velocities. Also, the elements are challenged with a liquid waste surrogate using Laskin nozzles and large dispersion nozzles. These tests allow the loading capacity of the unit to be determined and the effectiveness of washing down the interior of the elements to be evaluated. (authors)

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.

Traveling-Wave Tube Efficiency Enhancement

Science.gov (United States)

Dayton, James A., Jr.

2011-01-01

Traveling-wave tubes (TWT's) are used to amplify microwave communication signals on virtually all NASA and commercial spacecraft. Because TWT's are a primary power user, increasing their power efficiency is important for reducing spacecraft weight and cost. NASA Glenn Research Center has played a major role in increasing TWT efficiency over the last thirty years. In particular, two types of efficiency optimization algorithms have been developed for coupled-cavity TWT's. The first is the phase-adjusted taper which was used to increase the RF power from 420 to 1000 watts and the RF efficiency from 9.6% to 22.6% for a Ka-band (29.5 GHz) TWT. This was a record efficiency at this frequency level. The second is an optimization algorithm based on simulated annealing. This improved algorithm is more general and can be used to optimize efficiency over a frequency bandwidth and to provide a robust design for very high frequency TWT's in which dimensional tolerance variations are significant.

Energy efficiency: from principles to reality

International Nuclear Information System (INIS)

Baudry, Paul; Ballot-Miguel, Benedicte; Binet, Guillaume; Bordigoni, Mathieu; Decellas, Fabrice; Hauser, Chantal; Hita, Laurent; Laurent, Marie-Helene; Osso, Dominique; Peureux, Jean-Louis; Pham Van Cang, Christian

2015-01-01

This collective publication proposes a comprehensive overview of issues related to energy efficiency: associated stakes, methods of assessment of energy savings and of their costs, methods of action for energy efficiency policies, application in the housing, office building and industry sectors based on energy consumption modes in these different sectors, and main technologies aimed at improving energy efficiency. The first chapter proposes an historical perspective on energy, outlines the crucial role of energy efficiency in today's and tomorrow's contexts, and discusses which are the different levers of action to increase this efficiency. The next chapters address methods of assessment of energy efficiency, identify and discuss the use of different potential sources of energy saving, propose an overview of the various objectives and instruments of policies for energy efficiency, and address the issue of energy efficiency in the housing sector, in the office building sector, and in the industry sector by indicating the current levels of energy consumption, by identifying the various potential sources of energy saving, and by indicating available technologies aimed at improving energy efficiency

Tolerance analysis on diffraction efficiency and polychromatic integral diffraction efficiency for harmonic diffractive optics

Science.gov (United States)

Shan, Mao

2016-10-01

In this dissertation, the mathematical model of effect of manufacturing errors including microstructure relative height error and relative width error on diffraction efficiency for the harmonic diffractive optical elements (HDEs) is set up. According to the expression of the phase delay and diffraction efficiency of the HDEs, the expression of diffraction efficiency of refraction and diffractive optical element with the microstructure height and periodic width errors in fabrication process is presented in this paper. Furthermore, the effect of manufacturing errors on diffraction efficiency for the harmonic diffractive optical elements is studied, and diffraction efficiency change is analyzed as the relative microstructure height-error in the same and in the opposite sign as well as relative width-error in the same and in the opposite sign. Example including infrared wavelength with materials GE has been discussed in this paper. Two kinds of manufacturing errors applied in 3.7 4.3um middle infrared and 8.7-11.5um far infrared optical system which results in diffraction efficiency and PIDE of HDEs are studied. The analysis results can be used for manufacturing error control in micro-structure height and periodic width. Results can be used for HDEs processing.

Energy efficiency in Norway (1997). Cross Country Comparison on Energy Efficiency Indicators - Phase 5

Energy Technology Data Exchange (ETDEWEB)

Alm, Leif Kristian

2000-02-01

This is the national report for Norway in phase 5 of the SAVE project 'Cross country comparison of energy efficiency indicators'. The report deals with energy use and energy efficiency in Norway the last 20 years, with a special emphasis on the period after 1990. A detailed sector analysis has been done, applying Laspeyres indices to attribute changes in energy use to either activity, structure or intensity (efficiency). Aggregating sectors, we have found a total efficiency improvement of maximum 7-8 TWH from 1990 to 1997. This corresponds to a saving of 0.5% per year. In the same period, final energy use per Gross Domestic Product (GDP) was reduced by approx 2.4% per year. Thereby most of the reduction in final energy intensity can not be attributed to increased energy efficiency. Almost all data are taken from official Norwegian statistics (Statistics Norway). (author)

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.

Efficiency of emergency exercises

International Nuclear Information System (INIS)

Zander, N.; Sogalla, M.

2011-01-01

In order to cope with accidents beyond the design basis within German nuclear power plants which possibly lead to relevant radiological consequences, the utilities as well as the competent authorities exist emergency organisations. The efficiency, capacity for teamwork and preparedness of such organisations should be tested by regular, efficient exercise activities. Such activities can suitably be based on scenarios which provide challenging tasks for all units of the respective emergency organisation. Thus, the demonstration and further development of the efficiency of the respective organisational structures, including their ability to collaborate, is promoted. (orig.)

Learning efficient correlated equilibria

KAUST Repository

Borowski, Holly P.; Marden, Jason R.; Shamma, Jeff S.

2014-01-01

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.

PV inverter test setup for European efficiency, static and dynamic MPPT efficiency evaluation

DEFF Research Database (Denmark)

Sera, Dezso; Teodorescu, Remus; Valentini, Massimo

2008-01-01

This paper concerns the evaluation of performance of grid-connected PV inverters in terms of conversion efficiency, European efficiency, static and dynamic MPP efficiency. Semi-automated tests were performed in the PV laboratory of the Institute of Energy Technology at the Aalborg University...... (Denmark) on a commercial transformerless PV inverter. Thanks to the available experimental test setups, that provide the required high measuring accuracy, and the developed PV simulator, which is required for MPPT performance evaluation, PV Inverters can be pretested before being tested by accredited...

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.

Energy efficiency of mobile soft robots.

Science.gov (United States)

Shui, Langquan; Zhu, Liangliang; Yang, Zhe; Liu, Yilun; Chen, Xi

2017-11-15

The performance of mobile soft robots is usually characterized by their locomotion/velocity efficiency, whereas the energy efficiency is a more intrinsic and fundamental criterion for the performance evaluation of independent or integrated soft robots. In this work, a general framework is established to evaluate the energy efficiency of mobile soft robots by considering the efficiency of the energy source, actuator and locomotion, and some insights for improving the efficiency of soft robotic systems are presented. Proposed as the ratio of the desired locomotion kinetic energy to the input mechanical energy, the energy efficiency of locomotion is found to play a critical role in determining the overall energy efficiency of soft robots. Four key factors related to the locomotion energy efficiency are identified, that is, the locomotion modes, material properties, geometric sizes, and actuation states. It is found that the energy efficiency of most mobile soft robots reported in the literature is surprisingly low (mostly below 0.1%), due to the inefficient mechanical energy that essentially does not contribute to the desired locomotion. A comparison of the locomotion energy efficiency for several representative locomotion modes in the literature is presented, showing a descending ranking as: jumping ≫ fish-like swimming > snake-like slithering > rolling > rising/turning over > inchworm-like inching > quadruped gait > earthworm-like squirming. Besides, considering the same locomotion mode, soft robots with lower stiffness, higher density and larger size tend to have higher locomotion energy efficiency. Moreover, a periodic pulse actuation instead of a continuous actuation mode may significantly reduce the input mechanical energy, thus improving the locomotion energy efficiency, especially when the pulse actuation matches the resonant states of the soft robots. The results presented herein indicate a large and necessary space for improving the locomotion energy

Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Guangming Wu

2011-07-01

Full Text Available Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH⁻/⁻ mice as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH⁻/⁻-induced pluripotent stem cells (iPS cells as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH⁻/⁻ iPS cell lines, we aggregated FAH⁻/⁻-iPS cells with tetraploid embryos and obtained entirely FAH⁻/⁻-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAH⁻/⁻ mice. Then, we transduced FAH cDNA into the FAH⁻/⁻-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl-1,3-cyclohexanedione. Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.

Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells

Directory of Open Access Journals (Sweden)

Lin Tong

2011-09-01

Full Text Available Abstract Background shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF and cell enzyme linked immunosorbent assays (cell ELISA, and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells. Results Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV. Conclusions Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.

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.

Monitoring changes in economy-wide energy efficiency: From energy-GDP ratio to composite efficiency index

International Nuclear Information System (INIS)

Ang, B.W.

2006-01-01

Since the 1973 world oil crisis, monitoring trends in energy efficiency at the economy-wide level has been an important component of energy strategy in many countries. To support this effort, various energy efficiency-related indicators have been developed. We examine some classical indicators which are often found in national and international energy studies in the 1970s and 1980s. We then describe the recent developments in using the index decomposition analysis to give an economy-wide composite energy efficiency index based on a bottom-up approach. This composite index is superior to the classical indicators as an economy-wide energy efficiency measure and has lately been adopted by a growing number of countries for national energy efficiency trend monitoring

Efficiency simulation of long neutron counter

International Nuclear Information System (INIS)

Hu Qingyuan; Li Bojun; Zhang De; Guo Hongsheng; Wang Dong; Yang Gaozhao; Si Fenni; Liu Jian

2008-01-01

In order to achieve the high efficiency and uniform sensitivity for neutrons with widely different energies, the efficiency of long boron trifluoride proportional counter imbedded in polyethylene moderator was simulated by MCNP code. The result shows that detective efficiency would increase with increasing moderator radius and response curve at higher energy would be ameliorated through adjusting the thickness of front moderator. Also we calculated the relative efficiencies for different energy of a detector whose efficiencies were calibrated on an accelerator. The simulated efficiency for D-D neutrons (2.4 MeV) is 75% of the efficiency for D-T neutrons (14.1 MeV), which is approximately agreed with experimental data, 61%. The validity of the simulated model was proved by the consistent results between calculation and experiment data. (authors)

Innovative-Simplified Nuclear Power Plant Efficiency Evaluation with High-Efficiency Steam Injector System

International Nuclear Information System (INIS)

Shoji, Goto; Shuichi, Ohmori; Michitsugu, Mori

2006-01-01

It is possible to establish simplified system with reduced space and total equipment weight using high-efficiency Steam Injectors (SI) instead of low-pressure feedwater heaters in Nuclear Power Plant (NPP). The SI works as a heat exchanger through direct contact between feedwater from condensers and extracted steam from turbines. It can get higher pressure than supplied steam pressure. The maintenance and reliability are still higher than the feedwater ones because SI has no movable parts. This paper describes the analysis of the heat balance, plant efficiency and the operation of this Innovative-Simplified NPP with high-efficiency SI. The plant efficiency and operation are compared with the electric power of 1100 MWe-class BWR system and the Innovative-Simplified BWR system with SI. The SI model is adapted into the heat balance simulator with a simplified model. The results show that plant efficiencies of the Innovated-Simplified BWR system are almost equal to original BWR ones. The present research is one of the projects that are carried out by Tokyo Electric Power Company, Toshiba Corporation, and six Universities in Japan, funded from the Institute of Applied Energy (IAE) of Japan as the national public research-funded program. (authors)

The International Database of Efficient Appliances (IDEA): A New Resource for Global Efficiency Policy

Energy Technology Data Exchange (ETDEWEB)

Gerke, Brian F; McNeil, Michael A; Tu, Thomas; Xu, Feiyang

2017-09-06

A major barrier to effective appliance efficiency program design and evaluation is a lack of data for determination of market baselines and cost-effective energy savings potential. The data gap is particularly acute in developing countries, which may have the greatest savings potential per unit GDP. To address this need, we are developing the International Database of Efficient Appliances (IDEA), which automatically compiles data from a wide variety of online sources to create a unified repository of information on efficiency, price, and features for a wide range of energy-consuming products across global markets. This paper summarizes the database framework and demonstrates the power of IDEA as a resource for appliance efficiency research and policy development. Using IDEA data for refrigerators in China and India, we develop robust cost-effectiveness indicators that allow rapid determination of savings potential within each market, as well as comparison of that potential across markets and appliance types. We discuss implications for future energy efficiency policy development.

Energy efficiency in the world and Turkey and investigation of energy efficiency in Turkish Industry

International Nuclear Information System (INIS)

Kavak, K.

2005-09-01

The reserves of fossil fuels which currently respond to the major part of world energy requirements are being running out very fast. Because it is forecasted that reserves of some fossil fuels like oil and natural gas will come to an end in the second half of this century, exploiting all energy resources in an efficient manner has great importance. Throughout the world where the energy demand grows continuously but the resources decrease gradually, many types of programs are implemented to provide efficient energy use. In Turkey, although there have been some efforts in last two decades, the importance of the issue could not be undersood yet. Turkey'sgeneral energy policy still focuses on supply security and finding ways to meet the growing demand, rather than decreasing the demand by energy efficiency. In this study, the possible opportunities and benefits that Turkey would gain by energy efficiency is pointed out. The studies about energy efficiency which have been conducted in the world and Turkey are examined. The measurement that can be taken in the sectors such as industry, power plants, buildings, transportation and the utilities of these measures for energy economy are indicated. The successful practices of energy efficiency studies in various countries, the state of some countries which pioneer efficiency implementations. Turkey's situation in energy in the light of basic indicators such as energy consumption per capita and enrgy intensity, the energy efficiency studies that have been done and should be done in various sectors of Turkey are also discussed in this thesis. Turkish industry's energy comsumption is analyzed as a seperate chapter by taking into consideration energy efficiency, energy intensity and energy resources. The general energy consumption and energy intensity tendencies of main manufacturing industries between 1995 and 2002 are explored and resource utilization ratios are investigated. This chapter provides to find out what kind of

Evaluation of porcine stem cells competence for somatic cell nuclear transfer and production of cloned animals

DEFF Research Database (Denmark)

Secher, Jan; Liu, Ying; Petkov, Stoyan

2017-01-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than...... somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem...... cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl...

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)

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.

Targeted gene therapy and cell reprogramming in Fanconi anemia

Science.gov (United States)

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-01-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

Promotion of Efficient Use of Energy

Energy Technology Data Exchange (ETDEWEB)

Harry Misuriello; DOE Project Officer - Keith Bennett

2006-01-25

The Department of Energy funded the Alliance to Save Energy to promote the efficient use of energy under a multiyear cooperative agreement. This funding allowed the Alliance to be innovative and flexible in its program development, and to initiate and enhance projects it would otherwise not have been able to pursue. The program period was 1999 through 2004. The mission of the Alliance to Save Energy is to promote energy efficiency domestically and worldwide. The Alliance followed this mission by working closely with consumers, government, policy makers, and energy efficient product and service providers. The projects that were initiated by the Alliance included communication and consumer education, policy analysis and research, the promotion of interaction among the energy efficiency industry, and international energy efficiency programs. The funding from the Department of Energy allowed the Alliance to study new issues in energy efficiency, draw public attention to those issues, and create targeted programs, such as the Efficient Windows Collaborative or the Green Schools program, which now function on their own to promote energy efficiency in important areas.

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

State-level benefits of energy efficiency

International Nuclear Information System (INIS)

Tonn, Bruce; Peretz, Jean H.

2007-01-01

This paper describes benefits attributable to state-level energy efficiency programs. Nationwide, state-level energy efficiency programs have targeted all sectors of the economy and have employed a wide range of methods to promote energy efficiency. Standard residential and industrial programs typically identify between 20% and 30% energy savings in homes and plants, respectively. Over a 20-year period of time, an average state that aggressively pursues even a limited array of energy efficiency programs can potentially reduce total state energy use by as much as 20%. Well-designed energy efficiency programs can be expected to help overcome numerous barriers to the market penetration of energy efficient technologies and accelerate the market penetration of the technologies. Energy efficiency programs are cost-effective; typical benefit-cost ratios exceed 3:1 and are much higher when non-energy and macroeconomic benefits are included. Indeed, energy efficiency and associated programs and investments can create significant numbers of new jobs and enhance state tax revenues. Several states have incorporated energy efficiency into their economic development programs. It should also be noted that increasing amounts of venture capital are being invested in the energy sector in general and in specific technologies like solar power in particular. (author)

State-Level Benefits of Energy Efficiency

Energy Technology Data Exchange (ETDEWEB)

Tonn, Bruce Edward [ORNL

2007-02-01

This report describes benefits attributable to state-level energy efficiency programs. Nationwide, state-level energy efficiency programs have targeted all sectors of the economy and have employed a wide range of methods to promote energy efficiency. Standard residential and industrial programs typically identify between 20 to 30% energy savings in homes and plants, respectively. Over a 20 year period of time, an average state that aggressively pursues even a limited array of energy efficiency programs can potentially reduce total state energy use by as much as 20%. Benefit-cost ratios of effective energy efficiency programs typically exceed 3 to 1 and are much higher when non-energy and macroeconomic benefits are included. Indeed, energy efficiency and associated programs and investments can create significant numbers of new jobs and enhance state tax revenues. Several states have incorporated energy efficiency into their economic development programs. It should also be noted that increasing amounts of venture capital are being invested in the energy sector in general and in specific technologies like solar power in particular. Well-designed energy efficiency programs can be expected to help overcome numerous barriers to the market penetration of energy efficient technologies and accelerate the market penetration of the technologies.

Energy efficiency in the British housing stock: Energy demand and the Homes Energy Efficiency Database

International Nuclear Information System (INIS)

Hamilton, Ian G.; Steadman, Philip J.; Bruhns, Harry; Summerfield, Alex J.; Lowe, Robert

2013-01-01

The UK Government has unveiled an ambitious retrofit programme that seeks significant improvement to the energy efficiency of the housing stock. High quality data on the energy efficiency of buildings and their related energy demand is critical to supporting and targeting investment in energy efficiency. Using existing home improvement programmes over the past 15 years, the UK Government has brought together data on energy efficiency retrofits in approximately 13 million homes into the Homes Energy Efficiency Database (HEED), along with annual metered gas and electricity use for the period of 2004–2007. This paper describes the HEED sample and assesses its representativeness in terms of dwelling characteristics, the energy demand of different energy performance levels using linked gas and electricity meter data, along with an analysis of the impact retrofit measures has on energy demand. Energy savings are shown to be associated with the installation of loft and cavity insulation, and glazing and boiler replacement. The analysis illustrates this source of ‘in-action’ data can be used to provide empirical estimates of impacts of energy efficiency retrofit on energy demand and provides a source of empirical data from which to support the development of national housing energy efficiency retrofit policies. - Highlights: • The energy efficiency level for 50% of the British housing stock is described. • Energy demand is influenced by size and age and energy performance. • Housing retrofits (e.g. cavity insulation, glazing and boiler replacements) save energy. • Historic differences in energy performance show persistent long-term energy savings

Industrial energy efficiency: A policy perspective

International Nuclear Information System (INIS)

Chandler, W.U.

1990-01-01

Policies that promote energy efficiency can work; but potential energy savings are unlikely to be realized without effective policy leadership. This article discusses the opportunities in several countries for increasing energy efficiency. Both ''open'' and centrally planned economies could be much more energy efficient. In the United States, for example, the government needs to stimulate energy efficiency. This could be done by sponsoring research to develop new processes, creating favourable financial conditions for investment in efficiency, and making the advantages of energy efficiency technologies better known. International collaboration in sponsoring research and transfer technologies could be of the greatest importance in improving energy efficiency in countries with centrally planned economies, including the Soviet Union, as well as in developing countries. Favourable conditions for achieving both economic development and environmental protection can be created through cooperation on the international level. (author). 24 refs, 4 tabs

Interaction between output efficiency and environmental efficiency : evidence from the textile industry in Jiangsu Province, China

NARCIS (Netherlands)

Jiang, Lei; Folmer, Henk; Bu, Maoliang

2016-01-01

Environmental efficiency improvement has played a crucial role in the theory and practice of stimulating clean production. This paper analyzes the interaction between environmental efficiency and output efficiency, particularly whether they reinforce each other or compete with each other, on the

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.

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.

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

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells

DEFF Research Database (Denmark)

Secher, Jan Ole Bertelsen; Ceylan, Ahmet; Mazzoni, Gianluca

2017-01-01

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to sh...

Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury

NARCIS (Netherlands)

Bartus, Katalin; James, Nicholas D; Didangelos, Athanasios; Bosch, Karen D; Verhaagen, J.; Yáñez-Muñoz, Rafael J; Rogers, John H; Schneider, Bernard L; Muir, Elizabeth M; Bradbury, Elizabeth J

2014-01-01

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate

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)

State Energy Efficiency Benefits and Opportunities

Science.gov (United States)

Describes the benefits of energy efficiency and how to assess its potential for your state. Also, find details on energy efficiency policies, programs, and resources available for furthering energy efficiency goals.

Manufacture of Third-Generation Lentivirus for Preclinical Use, with Process Development Considerations for Translation to Good Manufacturing Practice.

Science.gov (United States)

Gándara, Carolina; Affleck, Valerie; Stoll, Elizabeth Ann

2018-02-01

Lentiviral vectors are used in laboratories around the world for in vivo and ex vivo delivery of gene therapies, and increasingly clinical investigation as well as preclinical applications. The third-generation lentiviral vector system has many advantages, including high packaging capacity, stable gene expression in both dividing and post-mitotic cells, and low immunogenicity in the recipient organism. Yet, the manufacture of these vectors is challenging, especially at high titers required for direct use in vivo, and further challenges are presented by the process of translating preclinical gene therapies toward manufacture of products for clinical investigation. The goals of this paper are to report the protocol for manufacturing high-titer third-generation lentivirus for preclinical testing and to provide detailed information on considerations for translating preclinical viral vector manufacture toward scaled-up platforms and processes in order to make gene therapies under Good Manufacturing Practice that are suitable for clinical trials.

Market conditions affecting energy efficiency investments

International Nuclear Information System (INIS)

Seabright, J.

1996-01-01

The global energy efficiency market is growing, due in part to energy sector and macroeconomic reforms and increased awareness of the environmental benefits of energy efficiency. Many countries have promoted open, competitive markets, thereby stimulating economic growth. They have reduced or removed subsidies on energy prices, and governments have initiated energy conservation programs that have spurred the wider adoption of energy efficiency technologies. The market outlook for energy efficiency is quite positive. The global market for end-use energy efficiency in the industrial, residential and commercial sectors is now estimated to total more than $34 billion per year. There is still enormous technical potential to implement energy conservation measures and to upgrade to the best available technologies for new investments. For many technologies, energy-efficient designs now represent less than 10--20% of new product sales. Thus, creating favorable market conditions should be a priority. There are a number of actions that can be taken to create favorable market conditions for investing in energy efficiency. Fostering a market-oriented energy sector will lead to energy prices that reflect the true cost of supply. Policy initiatives should address known market failures and should support energy efficiency initiatives. And market transformation for energy efficiency products and services can be facilitated by creating an institutional and legal structure that favors commercially-oriented entities

Efficiency bounds for nonequilibrium heat engines

International Nuclear Information System (INIS)

Mehta, Pankaj; Polkovnikov, Anatoli

2013-01-01

We analyze the efficiency of thermal engines (either quantum or classical) working with a single heat reservoir like an atmosphere. The engine first gets an energy intake, which can be done in an arbitrary nonequilibrium way e.g. combustion of fuel. Then the engine performs the work and returns to the initial state. We distinguish two general classes of engines where the working body first equilibrates within itself and then performs the work (ergodic engine) or when it performs the work before equilibrating (non-ergodic engine). We show that in both cases the second law of thermodynamics limits their efficiency. For ergodic engines we find a rigorous upper bound for the efficiency, which is strictly smaller than the equivalent Carnot efficiency. I.e. the Carnot efficiency can be never achieved in single reservoir heat engines. For non-ergodic engines the efficiency can be higher and can exceed the equilibrium Carnot bound. By extending the fundamental thermodynamic relation to nonequilibrium processes, we find a rigorous thermodynamic bound for the efficiency of both ergodic and non-ergodic engines and show that it is given by the relative entropy of the nonequilibrium and initial equilibrium distributions. These results suggest a new general strategy for designing more efficient engines. We illustrate our ideas by using simple examples. -- Highlights: ► Derived efficiency bounds for heat engines working with a single reservoir. ► Analyzed both ergodic and non-ergodic engines. ► Showed that non-ergodic engines can be more efficient. ► Extended fundamental thermodynamic relation to arbitrary nonequilibrium processes

Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies

Directory of Open Access Journals (Sweden)

Fa Yang

2016-12-01

Full Text Available With the development of molecular cloning technology and the deep understanding of antibody engineering, there are diverse bispecific antibody formats from which to choose to pursue the optimal biological activity and clinical purpose. The single-chain-based bispecific antibodies usually bridge tumor cells with immune cells and form an immunological synapse because of their relatively small size. Bispecific antibodies in the IgG format include asymmetric bispecific antibodies and homodimerized bispecific antibodies, all of which have an extended blood half-life and their own crystalline fragment (Fc-mediated functions. Besides retargeting effector cells to the site of cancer, new applications were established for bispecific antibodies. Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Bispecific antibodies that can inhibit two correlated signaling molecules at the same time can be developed to overcome inherent or acquired resistance and to be more efficient angiogenesis inhibitors. Bispecific antibodies can also be used to treat hemophilia A by mimicking the function of factor VIII. Bispecific antibodies also have broad application prospects in bone disorders and infections and diseases of the central nervous system. The latest developments of the formats and application of bispecific antibodies will be reviewed. Furthermore, the challenges and perspectives are summarized in this review.

Modulation of the functional association between the HIV-1 intasome and the nucleosome by histone amino-terminal tails.

Science.gov (United States)

Benleulmi, Mohamed S; Matysiak, Julien; Robert, Xavier; Miskey, Csaba; Mauro, Eric; Lapaillerie, Delphine; Lesbats, Paul; Chaignepain, Stéphane; Henriquez, Daniel R; Calmels, Christina; Oladosu, Oyindamola; Thierry, Eloïse; Leon, Oscar; Lavigne, Marc; Andreola, Marie-Line; Delelis, Olivier; Ivics, Zoltán; Ruff, Marc; Gouet, Patrice; Parissi, Vincent

2017-11-28

Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.

MX: A beamline control system toolkit

Science.gov (United States)

Lavender, William M.

2000-06-01

The development of experimental and beamline control systems for two Collaborative Access Teams at the Advanced Photon Source has resulted in the creation of a portable data acquisition and control toolkit called MX. MX consists of a set of servers, application programs and libraries that enable the creation of command line and graphical user interface applications that may be easily retargeted to new and different kinds of motor and device controllers. The source code for MX is written in ANSI C and Tcl/Tk with interprocess communication via TCP/IP. MX is available for several versions of Unix, Windows 95/98/NT and DOS. It may be downloaded from the web site http://www.imca.aps.anl.gov/mx/.

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.)

Total-factor energy efficiency in developing countries

International Nuclear Information System (INIS)

Zhang Xingping; Cheng Xiaomei; Yuan Jiahai; Gao Xiaojun

2011-01-01

This paper uses a total-factor framework to investigate energy efficiency in 23 developing countries during the period of 1980-2005. We explore the total-factor energy efficiency and change trends by applying data envelopment analysis (DEA) window, which is capable of measuring efficiency in cross-sectional and time-varying data. The empirical results indicate that Botswana, Mexico and Panama perform the best in terms of energy efficiency, whereas Kenya, Sri Lanka, Syria and the Philippines perform the worst during the entire research period. Seven countries show little change in energy efficiency over time. Eleven countries experienced continuous decreases in energy efficiency. Among five countries witnessing continuous increase in total-factor energy efficiency, China experienced the most rapid rise. Practice in China indicates that effective energy policies play a crucial role in improving energy efficiency. Tobit regression analysis indicates that a U-shaped relationship exists between total-factor energy efficiency and income per capita. - Research Highlights: → To measure the total-factor energy efficiency using DEA window analysis. → Focus on an application area of developing countries in the period of 1980-2005. → A U-shaped relationship was found between total-factor energy efficiency and income.

Difference in leaf water use efficiency/photosynthetic nitrogen use efficiency of Bt-cotton and its conventional peer.

Science.gov (United States)

Guo, Ruqing; Sun, Shucun; Liu, Biao

2016-09-15

This study is to test the effects of Bt gene introduction on the foliar water/nitrogen use efficiency in cotton. We measured leaf stomatal conductance, photosynthetic rate, and transpiration rate under light saturation condition at different stages of a conventional cultivar (zhongmian no. 16) and its counterpart Bt cultivar (zhongmian no. 30) that were cultured on three levels of fertilization, based on which leaf instantaneous water use efficiency was derived. Leaf nitrogen concentration was measured to calculate leaf photosynthetic nitrogen use efficiency, and leaf δ(13)C was used to characterize long term water use efficiency. Bt cultivar was found to have lower stomatal conductance, net photosynthetic rates and transpiration rates, but higher instantaneous and long time water use efficiency. In addition, foliar nitrogen concentration was found to be higher but net photosynthetic rate was lower in the mature leaves of Bt cultivar, which led to lower photosynthetic nitrogen use efficiency. This might result from the significant decrease of photosynthetic rate due to the decrease of stomatal conductance. In conclusion, our findings show that the introduction of Bt gene should significantly increase foliar water use efficiency but decrease leaf nitrogen use efficiency in cotton under no selective pressure.

Orphan nuclear receptor TLX activates Wnt/β-catenin signalling to stimulate neural stem cell proliferation and self-renewal

Science.gov (United States)

Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T.; Gage, Fred H.; Evans, Ronald M.; Shi, Yanhong

2010-01-01

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/β-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/β-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active β-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a β-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active β-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active β-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active β-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/β-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode. PMID:20010817

Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.

Science.gov (United States)

Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T; Gage, Fred H; Evans, Ronald M; Shi, Yanhong

2010-01-01

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

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 in such set......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...... in such settings is proven under very general preference assumptions. The model is extended to include geographical location choice, a commodity space incorporating manufacturing imprecision and preferences for club-membership, schools and firms. Inefficiencies arising from household externalities or group...... 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...

ON THE ECONOMIC EFFICIENCY OF ORGANIZATIONS: TOWARD A SOLUTION OF THE EFFICIENT GOVERNMENT ENTERPRISE PARADOX

Directory of Open Access Journals (Sweden)

Robert E. Wright

2007-01-01

Full Text Available The modern widely-held joint-stock corporation appears to epitomize the economically efficient large-scale organization. Some scholars observe, however, that other types of organizations, including government-owned enterprises, have also achieved high degrees of efficiency and that some joint stock corporations have been inefficient. It is here argued that the economic efficiency of organizations is largely a function of two major variables—markets tructure and incentives within the organization—and not organizational form or ownership structure per se. Case studies of two business firms: a mutual life insurer and a family-controlled publisher, and two industries: higher education and custom construction, demonstrate the importance of internal incentives and market structures to organizational economic efficiency.

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.

An interdisciplinary perspective on industrial energy efficiency

International Nuclear Information System (INIS)

Palm, Jenny; Thollander, Patrik

2010-01-01

This paper combines engineering and social science approaches to enhance our understanding of industrial energy efficiency and broaden our perspective on policy making in Europe. Sustainable development demands new strategies, solutions, and policy-making approaches. Numerous studies of energy efficiency potential state that cost-effective energy efficiency technologies in industry are not always implemented for various reasons, such as lack of information, procedural impediments, and routines not favoring energy efficiency. Another reason for the efficiency gap is the existence of particular values, unsupportive of energy efficiency, in the dominant networks of a branch of trade. Analysis indicates that different sectors of rather closed communities have established their own tacit knowledge, perceived truths, and routines concerning energy efficiency measures. Actors in different industrial sectors highlight different barriers to energy efficiency and why cost-effective energy efficiency measures are not being implemented. The identified barriers can be problematized in relation to the social context to understand their existence and how to resolve them.

Consumer preferences for automobile energy-efficiency grades

International Nuclear Information System (INIS)

Koo, Yoonmo; Kim, Chang Seob; Hong, Junhee; Choi, Ie-Jung; Lee, Jongsu

2012-01-01

Recently, increases in energy prices have made energy conservation and efficiency improvements even more essential than in the past. However, consumers experience difficulty in obtaining reliable information regarding energy efficiency, so that many countries have implemented regulations to enforce energy-efficiency grade labeling. In this study, consumer preferences regarding energy efficiency grades are analyzed by the mixed logit and MDCEV model based on the revealed preference data of past automobile purchases. Findings show that consumers rationally apply information on energy efficiency grades when purchasing automobiles. However, they tend to show inefficiency in automobile usage patterns. This study discusses political implications of energy efficiency policies as they might impact consumer behaviors of automobile purchase and usage. - Highlights: ► We model discrete choice model to evaluate energy-efficiency grade regulation. ► Consumers apply information on energy efficiency grades when purchasing automobiles. ► However, they tend to show inefficiency in automobile usage patterns. ► The policies for efficient automobile usage are discussed.

Electrical efficiency and droop in MQW LEDs

Science.gov (United States)

Malyutenko, V. K.

2014-02-01

It is believed that low power conversion efficiency in commercial MQW LEDs occurs as a result of efficiency droop, current-induced dynamic degradation of the internal quantum efficiency, injection efficiency, and extraction efficiency. Broadly speaking, all these "quenching" mechanisms could be referred to as the optical losses. The vast advances of high-power InGaN and AlGaInP MQW LEDs have been achieved by addressing these losses. In contrast to these studies, in this paper we consider an alternative approach to make high-power LEDs more efficient. We identify current-induced electrical efficiency degradation (EED) as a strong limiting factor of power conversion efficiency. We found that EED is caused by current crowding followed by an increase in current-induced series resistance of a device. By decreasing the current spreading length, EED also causes the optical efficiency to degrade and stands for an important aspect of LED performance. This paper gives scientists the opportunity to look for different attributes of EED.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

Directory of Open Access Journals (Sweden)

Ciervo Alessandra

2007-10-01

Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

Science.gov (United States)

Savarino, Andrea; Pistello, Mauro; D'Ostilio, Daniela; Zabogli, Elisa; Taglia, Fabiana; Mancini, Fabiola; Ferro, Stefania; Matteucci, Donatella; De Luca, Laura; Barreca, Maria Letizia; Ciervo, Alessandra; Chimirri, Alba; Ciccozzi, Massimo; Bendinelli, Mauro

2007-01-01

Background Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in

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.

Framing car fuel efficiency : linearity heuristic for fuel consumption and fuel-efficiency ratings

NARCIS (Netherlands)

Schouten, T.M.; Bolderdijk, J.W.; Steg, L.

2014-01-01

People are sensitive to the way information on fuel efficiency is conveyed. When the fuel efficiency of cars is framed in terms of fuel per distance (FPD; e.g. l/100 km), instead of distance per units of fuel (DPF; e.g. km/l), people have a more accurate perception of potential fuel savings. People

Overview of Ecological Agriculture with High Efficiency

OpenAIRE

Huang, Guo-qin; Zhao, Qi-guo; Gong, Shao-lin; Shi, Qing-hua

2012-01-01

From the presentation, connotation, characteristics, principles, pattern, and technologies of ecological agriculture with high efficiency, we conduct comprehensive and systematic analysis and discussion of the theoretical and practical progress of ecological agriculture with high efficiency. (i) Ecological agriculture with high efficiency was first advanced in China in 1991. (ii) Ecological agriculture with high efficiency highlights "high efficiency", "ecology", and "combination". (iii) Ecol...

Energy Efficient Mobile Operating Systems

OpenAIRE

Muhammad Waseem

2013-01-01

Energy is an important resource in mobile computers now days. It is important to manage energy in efficient manner so that energy consumption will be reduced. Developers of operating system decided to increase the battery life time of mobile phones at operating system level. So, design of energy efficient mobile operating system is the best way to reduce the energy consumption in mobile devices. In this paper, currently used energy efficient mobile operating system is discussed and compared. ...

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.

Ecological efficiency in China and its influencing factors-a super-efficient SBM metafrontier-Malmquist-Tobit model study.

Science.gov (United States)

Ma, Xiaojun; Wang, Changxin; Yu, Yuanbo; Li, Yudong; Dong, Biying; Zhang, Xinyu; Niu, Xueqi; Yang, Qian; Chen, Ruimin; Li, Yifan; Gu, Yihan

2018-05-15

Ecological problem is one of the core issues that restrain China's economic development at present, and it is urgently needed to be solved properly and effectively. Based on panel data from 30 regions, this paper uses a super efficiency slack-based measure (SBM) model that introduces the undesirable output to calculate the ecological efficiency, and then uses traditional and metafrontier-Malmquist index method to study regional change trends and technology gap ratios (TGRs). Finally, the Tobit regression and principal component analysis methods are used to analysis the main factors affecting eco-efficiency and impact degree. The results show that about 60% of China's provinces have effective eco-efficiency, and the overall ecological efficiency of China is at the superior middling level, but there is a serious imbalance among different provinces and regions. Ecological efficiency has an obvious spatial cluster effect. There are differences among regional TGR values. Most regions show a downward trend and the phenomenon of focusing on economic development at the expense of ecological protection still exists. Expansion of opening to the outside, increases in R&D spending, and improvement of population urbanization rate have positive effects on eco-efficiency. Blind economic expansion, increases of industrial structure, and proportion of energy consumption have negative effects on eco-efficiency.

Design for energy efficiency: Energy efficient industrialized housing research program. Progress report

Energy Technology Data Exchange (ETDEWEB)

Kellett, R.; Berg, R.; Paz, A.; Brown, G.Z.

1991-03-01

Since 1989, the U.S. Department of Energy has sponsored the Energy Efficient Industrialized Housing research program (EEIH) to improve the energy efficiency of industrialized housing. Two research centers share responsibility for this program: The Center for Housing Innovation at the University of Oregon and the Florida Solar Energy Center, a research institute of the University of Central Florida. Additional funding is provided through the participation of private industry, state governments and utilities. The program is guided by a steering committee comprised of industry and government representatives. This report summarizes Fiscal Year (FY) 1990 activities and progress, and proposed activities for FY 1991 in Task 2.1 Design for Energy Efficiency. This task establishes a vision of energy conservation opportunities in critical regions, market segments, climate zones and manufacturing strategies significant to industrialized housing in the 21st Century. In early FY 1990, four problem statements were developed to define future housing demand scenarios inclusive of issues of energy efficiency, housing design and manufacturing. Literature surveys were completed to assess seven areas of influence for industrialized housing and energy conservation in the future. Fifty-five future trends were identified in computing and design process; manufacturing process; construction materials, components and systems; energy and environment; demographic context; economic context; and planning policy and regulatory context.

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

Efficiency of Compressed Air Energy Storage

DEFF Research Database (Denmark)

Elmegaard, Brian; Brix, Wiebke

2011-01-01

The simplest type of a Compressed Air Energy Storage (CAES) facility would be an adiabatic process consisting only of a compressor, a storage and a turbine, compressing air into a container when storing and expanding when producing. This type of CAES would be adiabatic and would if the machines...... were reversible have a storage efficiency of 100%. However, due to the specific capacity of the storage and the construction materials the air is cooled during and after compression in practice, making the CAES process diabatic. The cooling involves exergy losses and thus lowers the efficiency...... of the storage significantly. The efficiency of CAES as an electricity storage may be defined in several ways, we discuss these and find that the exergetic efficiency of compression, storage and production together determine the efficiency of CAES. In the paper we find that the efficiency of the practical CAES...

Resource allocation based on cost efficiency

DEFF Research Database (Denmark)

Dehnokhalaji, Akram; Ghiyasi, Mojtaba; Korhonen, Pekka

2017-01-01

-objective linear programming problem using two different strategies. First, we propose an RA model which keeps the cost efficiencies of units unchanged. This is done assuming fixed technical and allocative efficiencies. The approach is based on the assumption that the decision maker (DM) may not have big changes......In this paper, we consider a resource allocation (RA) problem and develop an approach based on cost (overall) efficiency. The aim is to allocate some inputs among decision making units (DMUs) in such way that their cost efficiencies improve or stay unchanged after RA. We formulate a multi...... in the structure of DMUs within a short term. The second strategy does not impose any restrictions on technical and allocative efficiencies. It guarantees that none of the cost efficiencies of DMUs get worse after RA, and the improvement for units is possible if it is feasible and beneficial. Two numerical...

Prediction and design of efficient exciplex emitters for high-efficiency, thermally activated delayed-fluorescence organic light-emitting diodes.

Science.gov (United States)

Liu, Xiao-Ke; Chen, Zhan; Zheng, Cai-Jun; Liu, Chuan-Lin; Lee, Chun-Sing; Li, Fan; Ou, Xue-Mei; Zhang, Xiao-Hong

2015-04-08

High-efficiency, thermally activated delayed-fluorescence organic light-emitting diodes based on exciplex emitters are demonstrated. The best device, based on a TAPC:DPTPCz emitter, shows a high external quantum efficiency of 15.4%. Strategies for predicting and designing efficient exciplex emitters are also provided. This approach allow prediction and design of efficient exciplex emitters for achieving high-efficiency organic light-emitting diodes, for future use in displays and lighting applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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 in such set...... in OLG, learning in OLG and in games, optimal pricing of derivative securities, the impact of heterogeneity...

Logistics, Management and Efficiency

OpenAIRE

Mircea UDRESCU; Sandu CUTURELA

2014-01-01

The problem related to the efficiency of the management for organization is general being the concern off all managers. In the present essay we consider that the efficacy of the organization begins from the structural systemization of the organizational management into general management, management of logistics and management of production which demands a new managerial process, more competitive based on economic efficiency.

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.

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.

Energy Efficiency Requirements in Building Codes, Energy Efficiency Policies for New Buildings. IEA Information Paper

Energy Technology Data Exchange (ETDEWEB)

Laustsen, Jens

2008-03-15

The aim of this paper is to describe and analyse current approaches to encourage energy efficiency in building codes for new buildings. Based on this analysis the paper enumerates policy recommendations for enhancing how energy efficiency is addressed in building codes and other policies for new buildings. This paper forms part of the IEA work for the G8 Gleneagles Plan of Action. These recommendations reflect the study of different policy options for increasing energy efficiency in new buildings and examination of other energy efficiency requirements in standards or building codes, such as energy efficiency requirements by major renovation or refurbishment. In many countries, energy efficiency of buildings falls under the jurisdiction of the federal states. Different standards cover different regions or climatic conditions and different types of buildings, such as residential or simple buildings, commercial buildings and more complicated high-rise buildings. There are many different building codes in the world and the intention of this paper is not to cover all codes on each level in all countries. Instead, the paper details different regions of the world and different ways of standards. In this paper we also evaluate good practices based on local traditions. This project does not seek to identify one best practice amongst the building codes and standards. Instead, different types of codes and different parts of the regulation have been illustrated together with examples on how they have been successfully addressed. To complement this discussion of efficiency standards, this study illustrates how energy efficiency can be improved through such initiatives as efficiency labelling or certification, very best practice buildings with extremely low- or no-energy consumption and other policies to raise buildings' energy efficiency beyond minimum requirements. When referring to the energy saving potentials for buildings, this study uses the analysis of recent IEA

On the Use of Student Data in Efficiency Analysis--Technical Efficiency in Swedish Upper Secondary School

Science.gov (United States)

Waldo, Staffan

2007-01-01

While individual data form the base for much empirical analysis in education, this is not the case for analysis of technical efficiency. In this paper, efficiency is estimated using individual data which is then aggregated to larger groups of students. Using an individual approach to technical efficiency makes it possible to carry out studies on a…

The International Database of Efficient Appliances (IDEA): A new tool to support appliance energy-efficiency deployment

International Nuclear Information System (INIS)

Gerke, Brian F.; McNeil, Michael A.; Tu, Thomas

2017-01-01

Highlights: •IDEA collects, organizes, and stores data on appliance features and efficiency. •Data can be gathered for any appliance type on any international market. •IDEA enables new approaches to efficiency deployment and monitoring. •Techniques are developed for cross-market comparison of energy-savings potential. •We find significant cost-effective energy-savings potential from efficiency for Indian and Chinese refrigerators. -- Abstract: Appliance energy-efficiency programs are a central component of many countries’ energy-policy portfolios. A major barrier to optimal implementation of these programs is lack of data to determine market baselines, assess the potential for cost-effective energy savings, and track markets over time to evaluate and verify program impacts. To address this gap, we have developed the International Database of Efficient Appliances (IDEA), a suite of software tools that automatically gathers data that is currently dispersed across various online sources and compiles it into a unified repository of information on efficiency, price, and features for a diversity of appliances and devices in markets around the world. In this article we describe the framework and functionality of IDEA, and we demonstrate its power as a resource for research and policy development related to appliance energy efficiency. Using IDEA data for refrigerators in China and India, we assess the potential for cost-effective energy savings within each market by computing robust indicators that can also be easily compared across different appliances and markets. We find that significant cost-effective savings are available on both markets. We discuss implications for the development of future energy-efficiency deployment programs.

Energy Efficiency Policy and Carbon Pricing

Energy Technology Data Exchange (ETDEWEB)

NONE

2011-07-01

The main message of this paper is that while carbon pricing is a prerequisite for least-cost carbon mitigation strategies, carbon pricing is not enough to overcome all the barriers to cost-effective energy efficiency actions. Energy efficiency policy should be designed carefully for each sector to ensure optimal outcomes for a combination of economic, social and climate change goals. This paper aims to examine the justification for specific energy efficiency policies in economies with carbon pricing in place. The paper begins with an inventory of existing market failures that attempt to explain the limited uptake of energy efficiency. These market failures are investigated to see which can be overcome by carbon pricing in two subsectors -- electricity use in residential appliances and heating energy use in buildings. This analysis finds that carbon pricing addresses energy efficiency market failures such as externalities and imperfect energy markets. However, several market and behavioural failures in the two subsectors are identified that appear not to be addressed by carbon pricing. These include: imperfect information; principal-agent problems; and behavioural failures. In this analysis, the policies that address these market failures are identified as complementary to carbon pricing and their level of interaction with carbon pricing policies is relatively positive. These policies should be implemented when they can improve energy efficiency effectively and efficiently (and achieve other national goals such as improving socio-economic efficiency).

Promotion of energy efficiency in enterprises

International Nuclear Information System (INIS)

Beltrani, G.; Schelske, O.; Peter, D.; Oettli, B.

2003-01-01

This comprehensive report for the Swiss Federal Office of Energy (SFOE) presents the results of a study made within the framework of the research programme on energy-economics fundamentals on how the energy efficiency of enterprises can be improved. The report first examines the present state of affairs in Swiss enterprises and looks into the interaction of energy efficiency and environmental management systems. ISO 14001 certification is discussed and examples are given of the responses of various enterprises to a survey concerning the role of energy efficiency in environmental management. Both hindrances and success factors for the embedding of energy-efficiency measures in environmental management activities are discussed and examples are given. Instruments available in Switzerland and from abroad that can be used to promote energy efficiency in enterprises are discussed. Four particular instruments are presented; guidelines and computer-based tools that help in the making of energy-relevant investment decisions, incentives to take part in an energy-benchmark system for small and medium-sized enterprises (SME), low-interest loans for investments in energy-efficiency for SMEs and the closer definition of 'continuous improvement' of energy efficiency within the framework of ISO 14001. The results of a survey amongst those involved are discussed. The report is concluded with recommendations for the implementation of the guidelines and for improvements in the integration of energy efficiency in environmental management systems

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.

Interactions between Energy Efficiency Programs funded under the Recovery Act and Utility Customer-Funded Energy Efficiency Programs

Energy Technology Data Exchange (ETDEWEB)

Goldman, Charles A.; Stuart, Elizabeth; Hoffman, Ian; Fuller, Merrian C.; Billingsley, Megan A.

2011-02-25

Since the spring of 2009, billions of federal dollars have been allocated to state and local governments as grants for energy efficiency and renewable energy projects and programs. The scale of this American Reinvestment and Recovery Act (ARRA) funding, focused on 'shovel-ready' projects to create and retain jobs, is unprecedented. Thousands of newly funded players - cities, counties, states, and tribes - and thousands of programs and projects are entering the existing landscape of energy efficiency programs for the first time or expanding their reach. The nation's experience base with energy efficiency is growing enormously, fed by federal dollars and driven by broader objectives than saving energy alone. State and local officials made countless choices in developing portfolios of ARRA-funded energy efficiency programs and deciding how their programs would relate to existing efficiency programs funded by utility customers. Those choices are worth examining as bellwethers of a future world where there may be multiple program administrators and funding sources in many states. What are the opportunities and challenges of this new environment? What short- and long-term impacts will this large, infusion of funds have on utility customer-funded programs; for example, on infrastructure for delivering energy efficiency services or on customer willingness to invest in energy efficiency? To what extent has the attribution of energy savings been a critical issue, especially where administrators of utility customer-funded energy efficiency programs have performance or shareholder incentives? Do the new ARRA-funded energy efficiency programs provide insights on roles or activities that are particularly well-suited to state and local program administrators vs. administrators or implementers of utility customer-funded programs? The answers could have important implications for the future of U.S. energy efficiency. This report focuses on a selected set of ARRA

Efficient Fingercode Classification

Science.gov (United States)

Sun, Hong-Wei; Law, Kwok-Yan; Gollmann, Dieter; Chung, Siu-Leung; Li, Jian-Bin; Sun, Jia-Guang

In this paper, we present an efficient fingerprint classification algorithm which is an essential component in many critical security application systems e. g. systems in the e-government and e-finance domains. Fingerprint identification is one of the most important security requirements in homeland security systems such as personnel screening and anti-money laundering. The problem of fingerprint identification involves searching (matching) the fingerprint of a person against each of the fingerprints of all registered persons. To enhance performance and reliability, a common approach is to reduce the search space by firstly classifying the fingerprints and then performing the search in the respective class. Jain et al. proposed a fingerprint classification algorithm based on a two-stage classifier, which uses a K-nearest neighbor classifier in its first stage. The fingerprint classification algorithm is based on the fingercode representation which is an encoding of fingerprints that has been demonstrated to be an effective fingerprint biometric scheme because of its ability to capture both local and global details in a fingerprint image. We enhance this approach by improving the efficiency of the K-nearest neighbor classifier for fingercode-based fingerprint classification. Our research firstly investigates the various fast search algorithms in vector quantization (VQ) and the potential application in fingerprint classification, and then proposes two efficient algorithms based on the pyramid-based search algorithms in VQ. Experimental results on DB1 of FVC 2004 demonstrate that our algorithms can outperform the full search algorithm and the original pyramid-based search algorithms in terms of computational efficiency without sacrificing accuracy.

Thrust Efficiency, Energy Efficiency, and the Role of VDF in Hall Thruster Performance Analysis (Preprint)

National Research Council Canada - National Science Library

Larson, C. W; Hargus, William A; Brown, Daniel L

2007-01-01

...) of the propellant jet on the conversion of anode electrical energy to jet kinetic energy. This enabled a mathematically rigorous distinction to be made between thrust efficiency and energy efficiency...

Thrust Efficiency, Energy Efficiency, and the Role of VDF in Hall Thruster Performance Analysis (Postprint)

National Research Council Canada - National Science Library

Larson, C. W; Hargus, William A; Brown, Daniel L

2007-01-01

...) of the propellant jet on the conversion of anode electrical energy to jet kinetic energy. This enabled a mathematically rigorous distinction to be made between thrust efficiency and energy efficiency...

[Characteristics of phosphorus uptake and use efficiency of rice with high yield and high phosphorus use efficiency].

Science.gov (United States)

Li, Li; Zhang, Xi-Zhou; Li, Tinx-Xuan; Yu, Hai-Ying; Ji, Lin; Chen, Guang-Deng

2014-07-01

A total of twenty seven middle maturing rice varieties as parent materials were divided into four types based on P use efficiency for grain yield in 2011 by field experiment with normal phosphorus (P) application. The rice variety with high yield and high P efficiency was identified by pot experiment with normal and low P applications, and the contribution rates of various P efficiencies to yield were investigated in 2012. There were significant genotype differences in yield and P efficiency of the test materials. GRLu17/AiTTP//Lu17_2 (QR20) was identified as a variety with high yield and high P efficiency, and its yields at the low and normal rates of P application were 1.96 and 1.92 times of that of Yuxiang B, respectively. The contribution rate of P accumulation to yield was greater than that of P grain production efficiency and P harvest index across field and pot experiments. The contribution rates of P accumulation and P grain production efficiency to yield were not significantly different under the normal P condition, whereas obvious differences were observed under the low P condition (66.5% and 26.6%). The minimal contribution to yield was P harvest index (11.8%). Under the normal P condition, the contribution rates of P accumulation to yield and P harvest index were the highest at the jointing-heading stage, which were 93.4% and 85.7%, respectively. In addition, the contribution rate of P accumulation to grain production efficiency was 41.8%. Under the low P condition, the maximal contribution rates of P accumulation to yield and grain production efficiency were observed at the tillering-jointing stage, which were 56.9% and 20.1% respectively. Furthermore, the contribution rate of P accumulation to P harvest index was 16.0%. The yield, P accumulation, and P harvest index of QR20 significantly increased under the normal P condition by 20.6%, 18.1% and 18.2% respectively compared with that in the low P condition. The rank of the contribution rates of P

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...

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.

Efficiency snakes and energy ladders: A (meta-)frontier demand analysis of electricity consumption efficiency in Chinese households

International Nuclear Information System (INIS)

Broadstock, David C.; Li, Jiajia; Zhang, Dayong

2016-01-01

Policy makers presently lack access to quantified estimates – and hence an explicit understanding – of energy consumption efficiency within households, creating a potential gap between true efficiency levels and the necessarily assumed efficiency levels that policy makers adopt in designing and implementing energy policy. This paper attempts to fill this information gap by empirically quantifying electricity consumption efficiency for a sample of more than 7,000 households. Adopting the recently introduced ‘frontier demand function’ due to Filippini and Hunt (2011) but extending it into the metafrontier context – to control for structural heterogeneity arising from location type – it is shown that consumption efficiency is little more than 60% on average. This implies huge potential for energy reduction via the expansion of schemes to promote energy efficiency. City households, which are the wealthiest in the sample, are shown to define the metafrontier demand function (and hence have the potential to be the most efficient households), but at the same time exhibit the largest inefficiencies. These facts together allow for a potential refinement on the household energy ladder concept, suggesting that wealth affords access to the best technologies thereby increasing potential energy efficiency (the ‘traditional view of the household energy ladder), but complementary to this these same households are most inefficient. This has implications for numerous areas of policy, including for example the design of energy assistance schemes, identification of energy education needs/priorities as well more refined setting of subsidies/tax-credit policies. - Highlights: •Frontier demand functions are estimated for a sample of 7102 Chinese households. •Metafrontier methods capture heterogeneity arising from urban form (e.g. cities, towns and villages). •Wealthier houses have higher efficiency potential, but are in fact less efficient in their consumption of

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

Investigation of the radiative efficiency and threshold in InGaN laser diodes under the influence of efficiency droop

International Nuclear Information System (INIS)

Ryu, Han-Youl

2012-01-01

Based on the rate equation model of semiconductor lasers, the radiative efficiency and threshold current density of InGaN-based blue laser diodes (LDs) are theoretically investigated, including the effect of efficiency droop in the InGaN quantum wells. The peak point of the radiative efficiency versus current density relation is used as the parameter of the rate equation analysis. The threshold current density of InGaN blue LDs is found to depend strongly on the maximum radiative efficiency at low current density, implying that improving the maximum efficiency is important to maintain a high radiative efficiency at a large current density and to achieve a low-threshold lasing action under the influence of efficiency droop.

SUSTAINABLE ECONOMIC GROWTH AND ECO-EFFICIENCY

Directory of Open Access Journals (Sweden)

Mariana\tLUPAN

2015-06-01

Full Text Available The current economic and social contexts have brought forth the issues regarding growth and sustainability. The concept of growth has always been linked to an increase in consumption levels, and this inevitably led to pressures on the environment and on the resources that support human activity. Given these circumstances, the question whether we can avoid an environmental disaster while maintaining economic growth, has become more stringent. We chose to approach this aspect by examining the concept of eco-efficiency, a concept that embodies aspects of both economic efficiency and environmental efficiency. Eco-efficiency can be regarded as the effectiveness with which resources are used in order to create products and services that satisfy human needs. Based on this idea, the last decade has produced an increasing number of studies on eco-efficiency and how it can be measured and implemented in the production of goods and services, but also in the field regarding demand patterns. An analysis regarding the aspects of eco-efficiency at the macro level of the Romanian economy is in line with the current environmental concerns, thus I have chosen to cover these questions, as well as the evolution of the locale economy towards a more sustainable development. The outcome of the examined aspects shows that, in spite of an increase in eco-efficiency levels, energy and material consumption and emissions have increased. This raises the question if measuring economic and environmental efficiency by reporting to the GDP value is becoming obsolete and if there is a need to revaluate eco-efficiency indicators in order to measure the transition to a greener and more sustainable development from different points of view.

Efficient secure two-party protocols

CERN Document Server

Hazay, Carmit

2010-01-01

The authors present a comprehensive study of efficient protocols and techniques for secure two-party computation -- both general constructions that can be used to securely compute any functionality, and protocols for specific problems of interest. The book focuses on techniques for constructing efficient protocols and proving them secure. In addition, the authors study different definitional paradigms and compare the efficiency of protocols achieved under these different definitions.The book opens with a general introduction to secure computation and then presents definitions of security for a

RAM-efficient external memory sorting

DEFF Research Database (Denmark)

Arge, Lars; Thorup, Mikkel

2013-01-01

In recent years a large number of problems have been considered in external memory models of computation, where the complexity measure is the number of blocks of data that are moved between slow external memory and fast internal memory (also called I/Os). In practice, however, internal memory time...... often dominates the total running time once I/O-efficiency has been obtained. In this paper we study algorithms for fundamental problems that are simultaneously I/O-efficient and internal memory efficient in the RAM model of computation....

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

Energy and Water Efficiency on Campus | NREL

Science.gov (United States)

Energy and Water Efficiency on Campus Energy and Water Efficiency on Campus NREL ensures the resiliency of our future energy and water systems through energy efficiency strategies and technologies , renewable energy, and water efficiency on the NREL campus. FY17 Energy Intensity. The South Table Mountain

Optimization analysis of propulsion motor control efficiency

Directory of Open Access Journals (Sweden)

CAI Qingnan

2017-12-01

Full Text Available [Objectives] This paper aims to strengthen the control effect of propulsion motors and decrease the energy used during actual control procedures.[Methods] Based on the traditional propulsion motor equivalence circuit, we increase the iron loss current component, introduce the definition of power matching ratio, calculate the highest efficiency of a motor at a given speed and discuss the flux corresponding to the power matching ratio with the highest efficiency. In the original motor vector efficiency optimization control module, an efficiency optimization control module is added so as to achieve motor efficiency optimization and energy conservation.[Results] MATLAB/Simulink simulation data shows that the efficiency optimization control method is suitable for most conditions. The operation efficiency of the improved motor model is significantly higher than that of the original motor model, and its dynamic performance is good.[Conclusions] Our motor efficiency optimization control method can be applied in engineering to achieve energy conservation.

Energy efficiency

International Nuclear Information System (INIS)

Marvillet, Ch.; Tochon, P.; Mercier, P.

2004-01-01

World energy demand is constantly rising. This is a legitimate trend, insofar as access to energy enables enhanced quality of life and sanitation levels for populations. On the other hand, such increased consumption generates effects that may be catastrophic for the future of the planet (climate change, environmental imbalance), should this growth conform to the patterns followed, up to recent times, by most industrialized countries. Reduction of greenhouse gas emissions, development of new energy sources and energy efficiency are seen as the major challenges to be taken up for the world of tomorrow. In France, the National Energy Debate indeed emphasized, in 2003, the requirement to control both demand for, and offer of, energy, through a strategic orientation law for energy. The French position corresponds to a slightly singular situation - and a privileged one, compared to other countries - owing to massive use of nuclear power for electricity generation. This option allows France to be responsible for a mere 2% of worldwide greenhouse gas emissions. Real advances can nonetheless still be achieved as regards improved energy efficiency, particularly in the transportation and residential-tertiary sectors, following the lead, in this respect, shown by industry. These two sectors indeed account for over half of the country CO 2 emissions (26% and 25% respectively). With respect to transportation, the work carried out by CEA on the hydrogen pathway, energy converters, and electricity storage has been covered by the preceding chapters. As regards housing, a topic addressed by one of the papers in this chapter, investigations at CEA concern integration of the various devices enabling value-added use of renewable energies. At the same time, the organization is carrying through its activity in the extensive area of heat exchangers, allowing industry to benefit from improved understanding in the modeling of flows. An activity evidenced by advances in energy efficiency for

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

40 CFR Appendix A to Subpart Mmmm... - Alternative Capture Efficiency and Destruction Efficiency Measurement and Monitoring Procedures...

Science.gov (United States)

2010-07-01

... alternative procedure for destruction efficiency determines the organic carbon content of the volatiles... portion of the coating and the efficiency of the capture system. The organic carbon content of the control... to the surrounding atmosphere. 3.0Control Device Efficiency. 3.1Determine the weight fraction carbon...

CD133-targeted Gene Transfer Into Long-term Repopulating Hematopoietic Stem Cells

NARCIS (Netherlands)

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwaeble, Joachim; Kaufmann, Kerstin B.; Mueller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J.; Grez, Manuel

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem

Energy Efficiency: Finding Leadership Opportunities

Directory of Open Access Journals (Sweden)

William Rosehart

2014-01-01

Full Text Available Between 1995 and 2011, the population of Alberta increased by roughly 40 per cent, but energy use in the province grew much faster, with a 62 per cent increase over the same period. In the industrial sector, the province’s largest energy consumer, demands grew 110 per cent. In mining and oil-and-gas extraction specifically, energy use over that period soared, growing by 355 per cent. That remarkable growth in energy consumption creates a particular challenge for Alberta Premier Alison Redford, who in 2011 ordered her ministers to develop a plan that “would make Alberta the national leader in energy efficiency and sustainability.” The province is still waiting. The incentives to become more energy efficient are not particularly strong in Alberta. The province’s terrain and size favour larger and less-efficient vehicles. Energy in the province is abundant, so there is little cause for concern over energy security. And energy is relatively affordable, particularly for a population that is more affluent than the Canadian average. There is little pressure on Albertans to radically alter their energy consumption behaviour. Yet, improved energy efficiency could position businesses in Alberta to become even more globally competitive, in addition to leading to improved air quality and public health. And for a province racing to keep up with growing energy demand, effective measures that promote conservation will prove much cheaper than adding yet more expensive infrastructure to the energy network. Many other jurisdictions have already provided examples of methods Alberta could employ to effectively promote energy conservation. First, Alberta must set hard targets for its goals to save energy, and then monitor that progress through transparent accounting, measuring and reporting. The provincial government can also nurture a culture of energy conservation, by formally and publicly recognizing leadership in efficiency improvements in industry and

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.

Role of executive agencies for energy efficiency with a view on activities of Serbian Energy Efficiency Agency

Directory of Open Access Journals (Sweden)

Kovačić Bojan J.

2012-01-01

Full Text Available Many countries, particularly in Europe, have executive energy efficiency agencies at national, regional and local levels that are organized in different ways. For all of them, it is common that there are existing strategic needs in their countries for enhancement of conditions and measures for rational use of energy and fuels. Serbian Energy Efficiency Agency was established in 2002 within the reform of the energy sector in Serbia and its current status was defined in 2004 by the Energy Law. It contributes to the improvement of social responsibility towards energy in all structures of the state and society, by proposing energy efficiency incentives, promoting importance of energy efficiency, as well as by managing energy efficiency and renewable energy programs and projects.

Adult-Brain-Derived Neural Stem Cells Grafting into a Vein Bridge Increases Postlesional Recovery and Regeneration in a Peripheral Nerve of Adult Pig

Directory of Open Access Journals (Sweden)

Olivier Liard

2012-01-01

Full Text Available We attempted transplantation of adult neural stem cells (ANSCs inside an autologous venous graft following surgical transsection of nervis cruralis with 30 mm long gap in adult pig. The transplanted cell suspension was a primary culture of neurospheres from adult pig subventricular zone (SVZ which had been labeled in vitro with BrdU or lentivirally transferred fluorescent protein. Lesion-induced loss of leg extension on the thigh became definitive in controls but was reversed by 45–90 days after neurosphere-filled vein grafting. Electromyography showed stimulodetection recovery in neurosphere-transplanted pigs but not in controls. Postmortem immunohistochemistry revealed neurosphere-derived cells that survived inside the venous graft from 10 to 240 post-lesion days and all displayed a neuronal phenotype. Newly formed neurons were distributed inside the venous graft along the severed nerve longitudinal axis. Moreover, ANSC transplantation increased CNPase expression, indicating activation of intrinsic Schwann cells. Thus ANSC transplantation inside an autologous venous graft provides an efficient repair strategy.

Tip, an Lck-interacting protein of Herpesvirus saimiri, causes Fas- and Lck-dependent apoptosis of T lymphocytes

International Nuclear Information System (INIS)

Hasham, Muneer G.; Tsygankov, Alexander Y.

2004-01-01

Saimiriine herpesvirus-2 (Herpesvirus saimiri) transforms T lymphocytes, including human, to continuous growth in vitro. H. saimiri-induced transformation is becoming an important tool of T-cell biology, including studies of HIV replication. Two proteins of H. saimiri subgroup C, Tip and StpC, are essential for T-cell transformation. In spite of the important role of these proteins, their biological functions and the molecular mechanisms of their action remain insufficiently understood. To further elucidate the effects of Tip on T cells, we transduced T lymphocytes, using an efficient lentiviral gene transfer system, to express Tip in the absence of other H. saimiri proteins. Our results indicate that Tip specifically inhibits IL-2 production by human T lymphocytes. Furthermore, Tip promotes T-cell apoptosis, which appears to be the reason for the observed decrease in IL-2 production. Finally, the apoptotic effect of Tip in T cells is mediated by Fas and requires the presence of active Lck in the cell

CD47 is a Potential Target for the Treatment of Laryngeal Squamous Cell Carcinoma

Directory of Open Access Journals (Sweden)

ChunPing Yang

2016-11-01

Full Text Available Background/Aims: This study aims to investigate the effect of CD47 on the development of laryngeal squamous cell carcinoma (LSCC and the therapeutic potential of monoclonal antibody against CD47 and its ligand SIRPα in the treatment of LSCC. Methods: We firstly detected the expressions of CD47 mRNA and protein in LSCC and para-carcinoma tissues, introduced the most efficient CD47siRNA sequence into LSCC cells by lentiviral transfection and employed three monoclonal antibodies to evaluate their anti-LSCC effects in vitro and in vivo. Results: We observed that the mRNA and protein expressions of CD47 in LSCC tissue had significant increase in LSCC tissues compared with those in para-carcinoma tissue (p Conclusion: The results suggested a critical role of CD47 in LSCC development and the promising treatment of antiCD47/SIRPα and/or CD47siRNA in LSCC.

Energy Efficiency in Norway 1990-2000

Energy Technology Data Exchange (ETDEWEB)

Rosenberg, Eva

2003-06-01

This is the national report for Norway in the EU/SAVE project ''Indicators for Energy Efficiency Monitoring and Target setting (ODYSSEE)''. The report deals with energy use and energy efficiency in Norway 1990-2000 (2001 for overall energy use). Final energy use per Gross Domestic Product (GDP) was reduced by approximately 1.9% pr year in the period 1990 to 2001. The energy efficiency improvement has been calculated to 0.6% pr year, while the role of structural changes has been 1.3% pr year. A detailed sector analysis has been done, applying Laspeyres indices to attribute changes in energy use to activity, structure or intensity (efficiency). Aggregating sectors, we have found a total efficiency improvement of approximately 11 TWh from 1990 to 2000. (author)

Separating environmental efficiency into production and abatement efficiency. A nonparametric model with application to U.S. power plants

Energy Technology Data Exchange (ETDEWEB)

Hampf, Benjamin

2011-08-15

In this paper we present a new approach to evaluate the environmental efficiency of decision making units. We propose a model that describes a two-stage process consisting of a production and an end-of-pipe abatement stage with the environmental efficiency being determined by the efficiency of both stages. Taking the dependencies between the two stages into account, we show how nonparametric methods can be used to measure environmental efficiency and to decompose it into production and abatement efficiency. For an empirical illustration we apply our model to an analysis of U.S. power plants.

Swimming efficiency in a shear-thinning fluid

Science.gov (United States)

Nganguia, Herve; Pietrzyk, Kyle; Pak, On Shun

2017-12-01

Micro-organisms expend energy moving through complex media. While propulsion speed is an important property of locomotion, efficiency is another factor that may determine the swimming gait adopted by a micro-organism in order to locomote in an energetically favorable manner. The efficiency of swimming in a Newtonian fluid is well characterized for different biological and artificial swimmers. However, these swimmers often encounter biological fluids displaying shear-thinning viscosities. Little is known about how this nonlinear rheology influences the efficiency of locomotion. Does the shear-thinning rheology render swimming more efficient or less? How does the swimming efficiency depend on the propulsion mechanism of a swimmer and rheological properties of the surrounding shear-thinning fluid? In this work, we address these fundamental questions on the efficiency of locomotion in a shear-thinning fluid by considering the squirmer model as a general locomotion model to represent different types of swimmers. Our analysis reveals how the choice of surface velocity distribution on a squirmer may reduce or enhance the swimming efficiency. We determine optimal shear rates at which the swimming efficiency can be substantially enhanced compared with the Newtonian case. The nontrivial variations of swimming efficiency prompt questions on how micro-organisms may tune their swimming gaits to exploit the shear-thinning rheology. The findings also provide insights into how artificial swimmers should be designed to move through complex media efficiently.

The efficiency of life insurance and family Takaful in Malaysia: Relative efficiency using the stochastic cost frontier analysis

Science.gov (United States)

Baharin, Roziana; Isa, Zaidi

2013-04-01

This paper focuses on the Stochastic cost Frontier Analysis (SFA) approach, in an attempt to measure the relationship between efficiency and organizational structure for Takaful and insurance operators in Malaysia's dual financial system. This study applied a flexible cost functional form i.e., Fourier Flexible Functional Form, for a sample consisting of 19 firms, chosen between 2002 and 2010, by employing the Battese and Coelli invariant efficiency model. The findings show that on average, there is a significant difference in cost efficiency between the Takaful industry and the insurance industry. It was found that Takaful has lower cost efficiency than conventional insurance, which shows that the organization form has an influence on efficiency. Overall, it was observed that the level of efficiency scores for both life insurance and family Takaful do not vary across time.

Agronomic efficiency and physiologic efficiency determination of Azolla-N and Urea-N applied to lowland rice

International Nuclear Information System (INIS)

Sisworo, Elsje L.; Rasjid, Havid; Sisworo, Widjang H.; Wemay, Johannis; Djawanas, Amrin

1994-01-01

Data from three field experiments using 15 N labelled Azolla and urea as an N source have been used to determine the agronomical and physiological efficiency of the N-Azoll and N-Urea applied tolowland rice. In general physiological efficiency of N from N-sources applied was lower than the agronomical efficiency. It seems that the increase of N-rates would enhance the agronomical and physiological efficiency of N applied to lowland rice but on the other hand the efficiency of N derived from the applied N-source decreased. Azolla+inorganic N-fertilizer when applied to lowland rice could increase rice yield expresses in dry weight of grain and straw above the control plants. Applying Azolla+inorganic N-fertilizer in the farmer's field located in West Java and West Sumatera often showed higher yields compared to when applied with inorganic N-fertilizer alone although the latter has higher total N-rates. (author). 9 refs, 8 tabs, 5 figs

Propulsive efficiency and non- expert swimmers performance

Directory of Open Access Journals (Sweden)

Tiago Barbosa

2009-12-01

Full Text Available Propulsive efficiency is one of the most interesting issues for competitive swimming researchers, has it presents significant relationships with the swimmer’s biophysical behavior and his/her performance. Although propulsive efficiency is a variable that has been quite studied in elite swimmers, there is no research on this issue in young and non-expert swimmers. Thus, the aim of this study was to: (i estimate the propulsive efficiency on non-expert swimmers; (ii identify biomechanical and anthropometrical parameters that are associated with propulsive efficiency; (iii identify the association between the propulsive efficiency and swim performance. Twenty-eight non-expert swimmers participated on this study. It was assessed the propulsive efficiency, biomechanical and anthropometrical parameters, as well as, the swim performance. The propulsive efficiency of non-expert swimmers is lower than data reported in the literature to higher competitive levels swimmers and there are no significant differences between boys and girls. It was also noted that several biomechanical and anthropometrical parameters, as well as, the swim performance are associated with the propulsive efficiency.

World's Most Efficient Solar Cell

Science.gov (United States)

World's Most Efficient Solar Cell National Renewable Energy Laboratory, Spectrolab Set Record For , 1999 - A solar cell that can convert sunlight to electricity at a record-setting 32 percent efficiency on Earth. Spectrolab of Sylmar, Calif., "grew" the record-setting solar cell. After

Eco-efficiency in industrial production

NARCIS (Netherlands)

von Raesfeld Meijer, Ariane M.; de Bakker, F.G.A.; Groen, Arend J.

2001-01-01

English AbstractThis report of the MATRIC project investigated 'Eco-efficiency in industrial production'. After a general introduction into the domain of eco-efficiency, the first part of this report further focusses on the organisation of Product-Oriented Environmental Management (POEM), which is

Magnetic blocking direct-recovery efficiency

International Nuclear Information System (INIS)

Whealton, J.H.; Wooten, J.H.; McGaffey, R.W.

1981-10-01

The ion recovery efficiency of a transverse magnetic field monochromatic direct recovery device intended for intense neutral beams is examined theoretically by solving a Poisson-Vlasov equation. An optimum in recovery efficiency is obtained for finite ion current density and excess initial speed

Efficient Environmental Policy with Imperfect Compliance

Energy Technology Data Exchange (ETDEWEB)

Sandmo, A. [Norwegian School of Economics and Business Administration, Bergen (Norway)

2002-09-01

Discussions of efficient environmental policy tend to recommend taxes rather than quotas on grounds of efficiency; a uniform tax will equalize marginal abatement cost between polluters. When polluters' actions are imperfectly observable, the distinction between taxes and quotas becomes less clear. Taxes may be evaded by underreporting of emissions, while quota violations will not always be discovered. This paper explores the conditions under which the efficiency properties of taxes continue to hold even when evasion is possible, and the extent to which the fine for quota violations plays the same role as a tax on emissions with similar efficiency properties.

Energy efficiency action plan. Policy action plan for promotion of energy efficiency in the Czech Republic to 2010

International Nuclear Information System (INIS)

1999-08-01

Energy efficiency and renewable energy production contribute to the three major goals of the national energy policy of the Czech Republic: overall competitiveness, security of supply and environmental protection. Therefore, the Czech government aims to promote these two sustainable options. The Energy Policy White Paper, which is being developed at the time of writing (June 1999), will provide the general framework for the future role of energy efficiency and renewable energy in the Czech Republic. In addition, it is necessary to develop specific policies. The National Energy Efficiency Study aimed to support the Czech government in the formulation of energy efficiency and renewable energy policy. The National Energy Efficiency Study has resulted in the following documents: (1) The Energy Efficiency Action Plan focuses on promotion of energy efficiency in end-use (this report); The Renewable Energy Action Plan (separate report; ECN-C--99-064) deals with policy on promotion of renewable energy production. These two Action Plans provide policy makers in the Czech government with essential information on potentials, targets, budgets and recommended policy instruments. The core of the Action Plans is the list of concrete policy actions, ready for implementation; and (2) The National Energy Efficiency Study NEES (separate report; ECN-C--99-063). This report is the background document to the two Action Plans. It contains detailed information on options and measures, potentials, barriers and policy instruments for energy efficiency and renewables. The main part is a detailed outline for a new energy efficiency and renewable policy. Also, it includes recommendations for financing schemes to overcome the investment constraints in the Czech Republic. Finally, a list of concrete projects is included to support project identification

Series-Tuned High Efficiency RF-Power Amplifiers

DEFF Research Database (Denmark)

Vidkjær, Jens

2008-01-01

An approach to high efficiency RF-power amplifier design is presented. It addresses simultaneously efficiency optimization and peak voltage limitations when transistors are pushed towards their power limits.......An approach to high efficiency RF-power amplifier design is presented. It addresses simultaneously efficiency optimization and peak voltage limitations when transistors are pushed towards their power limits....

Fourth Annual Report on Energy Efficiency

International Nuclear Information System (INIS)

Di Franco, Nino; Bertini, Ilaria; Federici, Alessandro; Moneta Roberto

2015-01-01

Here we present the main elements of the annual report on energy efficiency 2015. The results indicate that, thanks to national policies for energy efficiency, Italy saved over 7.5 million tons of oil equivalent per year in the period 2005-2013. Compared to the National Plan for Energy Efficiency 2014, the report shows that the 2020 objectives have already been achieved for more than 20%, with residential (35.7% of the target) and industry (26.6%) among the sectors that contributed most to this result. Substantial savings could result from the agribusiness sector through the dissemination of efficient technologies in the logistics and large retail chains. A key role lies with the banks: 86% of banks has developed products dedicated to efficiency, necessitating guidelines for replicability of projects, and audit and rating to assess their quality [it

Combustion phasing for maximum efficiency for conventional and high efficiency engines

International Nuclear Information System (INIS)

Caton, Jerald A.

2014-01-01

Highlights: • Combustion phasing for max efficiency is a function of engine parameters. • Combustion phasing is most affected by heat transfer, compression ratio, burn duration. • Combustion phasing is less affected by speed, load, equivalence ratio and EGR. • Combustion phasing for a high efficiency engine was more advanced. • Exergy destruction during combustion as functions of combustion phasing is reported. - Abstract: The importance of the phasing of the combustion event for internal-combustion engines is well appreciated, but quantitative details are sparse. The objective of the current work was to examine the optimum combustion phasing (based on maximum bmep) as functions of engine design and operating variables. A thermodynamic, engine cycle simulation was used to complete this assessment. As metrics for the combustion phasing, both the crank angle for 50% fuel mass burned (CA 50 ) and the crank angle for peak pressure (CA pp ) are reported as functions of the engine variables. In contrast to common statements in the literature, the optimum CA 50 and CA pp vary depending on the design and operating variables. Optimum, as used in this paper, refers to the combustion timing that provides the maximum bmep and brake thermal efficiency (MBT timing). For this work, the variables with the greatest influence on the optimum CA 50 and CA pp were the heat transfer level, the burn duration and the compression ratio. Other variables such as equivalence ratio, EGR level, engine speed and engine load had a much smaller impact on the optimum CA 50 and CA pp . For the conventional engine, for the conditions examined, the optimum CA 50 varied between about 5 and 11°aTDC, and the optimum CA pp varied between about 9 and 16°aTDC. For a high efficiency engine (high dilution, high compression ratio), the optimum CA 50 was 2.5°aTDC, and the optimum CA pp was 7.8°aTDC. These more advanced values for the optimum CA 50 and CA pp for the high efficiency engine were

Management of efficient use of energy and energy efficiency markets in Europe

International Nuclear Information System (INIS)

Lutz, Wolfang F.

1999-01-01

The present paper is based on the study S ystematization of European Legal, regulatory, and Institutional Frameworks for the Efficient Use of Energy , conducted in the framework of the project entitled Building up the Institutional and Regulatory Design to Consolidate Modernization of Energy Policies in the Countries of Latin America: Efficient Use of energy, implemented by the United Nations Economic Commission for Latin America and the Caribbean, in cooperation with the Synergy Programme of the European Commission of the Directorate General of Energy. (The author)

High-efficiency airfoil rudders applied to submarines

Directory of Open Access Journals (Sweden)

ZHOU Yimei

2017-03-01

Full Text Available Modern submarine design puts forward higher and higher requirements for control surfaces, and this creates a requirement for designers to constantly innovate new types of rudder so as to improve the efficiency of control surfaces. Adopting the high-efficiency airfoil rudder is one of the most effective measures for improving the efficiency of control surfaces. In this paper, we put forward an optimization method for a high-efficiency airfoil rudder on the basis of a comparative analysis of the various strengths and weaknesses of the airfoil, and the numerical calculation method is adopted to analyze the influence rule of the hydrodynamic characteristics and wake field by using the high-efficiency airfoil rudder and the conventional NACA rudder comparatively; at the same time, a model load test in a towing tank was carried out, and the test results and simulation calculation obtained good consistency:the error between them was less than 10%. The experimental results show that the steerage of a high-efficiency airfoil rudder is increased by more than 40% when compared with the conventional rudder, but the total resistance is close:the error is no more than 4%. Adopting a high-efficiency airfoil rudder brings much greater lifting efficiency than the total resistance of the boat. The results show that high-efficiency airfoil rudder has obvious advantages for improving the efficiency of control, giving it good application prospects.

Encouraging energy efficiency: Policies and programs

International Nuclear Information System (INIS)

Anon.

1992-01-01

Successfully overcoming the barriers to higher energy efficiency requires development of policies designed for specific users and locations. Reform of energy pricing, which entails removing subsidies and beginning internalization of externalities, is critical to give technology producers and users proper signals for investment and management decisions. But while a rise in energy prices increases the amount of energy-efficiency improvement that is cost-effective, it does not remove other barriers that deter investment. Minimum efficiency standards or agreements can raise the market floor, and are important because they affect the entire market in the near-term. But they may not raise the celining very much, and do little to push the efficiency frontier. To accomplish these goals, incentives and other market-development strategies are needed. Utility programs in particular can play a key role in pushing energy efficiency beyond the level where users are likely to invest on their own. Policies, programs, and pricing should complement one another. Pricing reform alone will not overcome the many entrenched barriers to higher energy efficiency, but trying to accelerate energy efficiency improvement without addressing energy pricing problems will lead to limited success. Whether tagerting new equipment or management of existing systems, policies must reflect a thorough understanding of the particular system and an awareness of the motivations of the actors. 25 refs

Energy efficiency in transport and mobility from an eco-efficiency viewpoint

International Nuclear Information System (INIS)

Uson, Alfonso Aranda; Capilla, Antonio Valero; Bribian, Ignacio Zabalza; Scarpellini, Sabina; Sastresa, Eva Llera

2011-01-01

European Union countries' current energy policies for the transport sector promote, amongst other initiatives; urban mobility plans, the renewal of fleets of cars and industrial vehicles and the introduction of biofuel. From the point of view of eco-efficiency and Life Cycle Assessment (LCA), energy policies must go further. The objective of this paper is to analyse the current transport model and the policies on energy efficiency being promoted in the EU from a LCA point of view. Special attention has been paid to private vehicles, in assessing the environmental impact of the various stages of manufacture, their use and disposal, and the consequences of plans to renew fleets. How old should a vehicle ideally be so that when it is changed, the embodied energy in the materials of the vehicle is less than the gain in energy efficiency due to changing the model for example? In addition the paper analyses the different means of transport in the energy consumption-time ratio from a LCA viewpoint. The fact that reducing transport times leads to greater energy consumption gives rise to the question: how long does nature take to repair the environmental damage caused?

Absorption Efficiency of Receiving Antennas

DEFF Research Database (Denmark)

Andersen, Jørgen Bach; Frandsen, Aksel

2005-01-01

A receiving antenna with a matched load will always scatter some power. This paper sets an upper and a lower bound on the absorption efficiency (absorbed power over sum of absorbed and scattered powers), which lies between 0 and 100% depending on the directivities of the antenna and scatter...... patterns. It can approach 100% as closely as desired, although in practice this may not be an attractive solution. An example with a small endfire array of dipoles shows an efficiency of 93%. Several examples of small conical horn antennas are also given, and they all have absorption efficiencies less than...

Energy efficiency standards and innovation

Science.gov (United States)

Morrison, Geoff

2015-01-01

Van Buskirk et al (2014 Environ. Res. Lett. 9 114010) demonstrate that the purchase price, lifecycle cost and price of improving efficiency (i.e. the incremental price of efficiency gain) decline at an accelerated rate following the adoption of the first energy efficiency standards for five consumer products. The authors show these trends using an experience curve framework (i.e. price/cost versus cumulative production). While the paper does not draw a causal link between standards and declining prices, they provide suggestive evidence using markets in the US and Europe. Below, I discuss the potential implications of the work.

Efficient Wideband Spectrum Sensing with Maximal Spectral Efficiency for LEO Mobile Satellite Systems

Directory of Open Access Journals (Sweden)

Feilong Li

2017-01-01

Full Text Available The usable satellite spectrum is becoming scarce due to static spectrum allocation policies. Cognitive radio approaches have already demonstrated their potential towards spectral efficiency for providing more spectrum access opportunities to secondary user (SU with sufficient protection to licensed primary user (PU. Hence, recent scientific literature has been focused on the tradeoff between spectrum reuse and PU protection within narrowband spectrum sensing (SS in terrestrial wireless sensing networks. However, those narrowband SS techniques investigated in the context of terrestrial CR may not be applicable for detecting wideband satellite signals. In this paper, we mainly investigate the problem of joint designing sensing time and hard fusion scheme to maximize SU spectral efficiency in the scenario of low earth orbit (LEO mobile satellite services based on wideband spectrum sensing. Compressed detection model is established to prove that there indeed exists one optimal sensing time achieving maximal spectral efficiency. Moreover, we propose novel wideband cooperative spectrum sensing (CSS framework where each SU reporting duration can be utilized for its following SU sensing. The sensing performance benefits from the novel CSS framework because the equivalent sensing time is extended by making full use of reporting slot. Furthermore, in respect of time-varying channel, the spatiotemporal CSS (ST-CSS is presented to attain space and time diversity gain simultaneously under hard decision fusion rule. Computer simulations show that the optimal sensing settings algorithm of joint optimization of sensing time, hard fusion rule and scheduling strategy achieves significant improvement in spectral efficiency. Additionally, the novel ST-CSS scheme performs much higher spectral efficiency than that of general CSS framework.

Incentives to improve energy efficiency in EU Grids

Energy Technology Data Exchange (ETDEWEB)

Papaefthymiou, G.; Beestermoeller, C.; Gardiner, A.

2013-04-15

The Energy Efficiency Directive (2012/27/EU) includes provisions related to network tariffs and regulation. It is timely therefore to revisit the potential options for energy efficiency in grids, the treatment of energy efficiency in network tariffs and alternative policies for improving energy efficiency. This project builds on work done previously for the European Copper Institute in this area. In this paper, we concentrate on energy efficiency in electricity network design and operation. Other articles in the Directive relate to the role of the network tariffs and regulations in enabling or incentivising the provision of energy efficiency to end users. In section 2, we describe technical efficiency measures to reduce losses (improve energy efficiency) in the grid. Section 3 reviews grid tariffs in three countries to identify whether they provide incentives or disincentives for energy efficiency in the grid. Section 4 discusses and evaluates alternative regulations for energy efficiency in grids. Section 5 concludes and discusses the main components of the optimal policy framework.

Compound Synthesis or Growth and Development of Roots/Stomata Regulate Plant Drought Tolerance or Water Use Efficiency/Water Uptake Efficiency.

Science.gov (United States)

Meng, Lai-Sheng

2018-04-11

Water is crucial to plant growth and development because it serves as a medium for all cellular functions. Thus, the improvement of plant drought tolerance or water use efficiency/water uptake efficiency is important in modern agriculture. In this review, we mainly focus on new genetic factors for ameliorating drought tolerance or water use efficiency/water uptake efficiency of plants and explore the involvement of these genetic factors in the regulation of improving plant drought tolerance or water use efficiency/water uptake efficiency, which is a result of altered stomata density and improving root systems (primary root length, hair root growth, and lateral root number) and enhanced production of osmotic protectants, which is caused by transcription factors, proteinases, and phosphatases and protein kinases. These results will help guide the synthesis of a model for predicting how the signals of genetic and environmental stress are integrated at a few genetic determinants to control the establishment of either water use efficiency or water uptake efficiency. Collectively, these insights into the molecular mechanism underpinning the control of plant drought tolerance or water use efficiency/water uptake efficiency may aid future breeding or design strategies to increase crop yield.

Multiresolution molecular mechanics: Implementation and efficiency

Energy Technology Data Exchange (ETDEWEB)

Biyikli, Emre; To, Albert C., E-mail: albertto@pitt.edu

2017-01-01

Atomistic/continuum coupling methods combine accurate atomistic methods and efficient continuum methods to simulate the behavior of highly ordered crystalline systems. Coupled methods utilize the advantages of both approaches to simulate systems at a lower computational cost, while retaining the accuracy associated with atomistic methods. Many concurrent atomistic/continuum coupling methods have been proposed in the past; however, their true computational efficiency has not been demonstrated. The present work presents an efficient implementation of a concurrent coupling method called the Multiresolution Molecular Mechanics (MMM) for serial, parallel, and adaptive analysis. First, we present the features of the software implemented along with the associated technologies. The scalability of the software implementation is demonstrated, and the competing effects of multiscale modeling and parallelization are discussed. Then, the algorithms contributing to the efficiency of the software are presented. These include algorithms for eliminating latent ghost atoms from calculations and measurement-based dynamic balancing of parallel workload. The efficiency improvements made by these algorithms are demonstrated by benchmark tests. The efficiency of the software is found to be on par with LAMMPS, a state-of-the-art Molecular Dynamics (MD) simulation code, when performing full atomistic simulations. Speed-up of the MMM method is shown to be directly proportional to the reduction of the number of the atoms visited in force computation. Finally, an adaptive MMM analysis on a nanoindentation problem, containing over a million atoms, is performed, yielding an improvement of 6.3–8.5 times in efficiency, over the full atomistic MD method. For the first time, the efficiency of a concurrent atomistic/continuum coupling method is comprehensively investigated and demonstrated.

Energy efficiency in Norway 1990-2002. Monitoring tools for energy efficiency in Europe: The Odyssee and MURE projects

Energy Technology Data Exchange (ETDEWEB)

Rosenberg, Eva; Espegren, Kari Aamodt

2004-08-01

This report presents an analysis of energy efficiency trends in Norway on the basis of energy efficiency indicators extracted from the Odyssee data base, maintained and updated in the framework of the SAVE programme. This analysis focuses on the period 1990-2001/2002. It also examines the policies and measures implemented in the field of energy efficiency with a focus on the years 2000-2003. Final energy use per Gross Domestic Product (GDP) was reduced by approximately 2% pr year in the period 1990 to 2002. The energy efficiency improvement has been calculated to 0.7% pr year, while the role of structural changes has been 1.3% pr year. A detailed sector analysis has been done, applying Laspeyres indices to attribute changes in energy use to activity, structure or intensity (efficiency). We have found in total efficiency improvement of approximately 15 TWh from 1990 to 2001. (Author)

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.

Energy efficiency initiatives: Indian experience

Energy Technology Data Exchange (ETDEWEB)

Dey, Dipankar [ICFAI Business School, Kolkata, (IBS-K) (India)

2007-07-01

India, with a population of over 1.10 billion is one of the fastest growing economies of the world. As domestic sources of different conventional commercial energy are drying up, dependence on foreign energy sources is increasing. There exists a huge potential for saving energy in India. After the first 'oil shock' (1973), the government of India realized the need for conservation of energy and a 'Petroleum Conservation Action Group' was formed in 1976. Since then many initiatives aiming at energy conservation and improving energy efficiency, have been undertaken (the establishment of Petroleum Conservation Research Association in 1978; the notification of Eco labelling scheme in 1991; the formation of Bureau of Energy Efficiency in 2002). But no such initiative was successful. In this paper an attempt has been made to analyze the changing importance of energy conservation/efficiency measures which have been initiated in India between 1970 and 2005.The present study tries to analyze the limitations and the reasons of failure of those initiatives. The probable reasons are: fuel pricing mechanism (including subsidies), political factors, corruption and unethical practices, influence of oil and related industry lobbies - both internal and external, the economic situation and the prolonged protection of domestic industries. Further, as India is opening its economy, the study explores the opportunities that the globally competitive market would offer to improve the overall energy efficiency of the economy. The study suggests that the Bureau of Energy Efficiency (BEE) - the newly formed nodal agency for improving energy efficiency of the economy may be made an autonomous institution where intervention from the politicians would be very low. For proper implementation of different initiatives to improve energy efficiency, BEE should involve more the civil societies (NGO) from the inception to the implementation stage of the programs. The paper also

Efficient convolutional sparse coding

Science.gov (United States)

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.

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

Energy efficiency of electrical infrared heating elements

International Nuclear Information System (INIS)

Brown, K.J.; Farrelly, R.; O’Shaughnessy, S.M.; Robinson, A.J.

2016-01-01

Highlights: • Characterization of the radiant energy efficiency of infrared heating elements. • Performed for a commercially available ceramic heater element for two cases. • Total radiant power and net radiant efficiency is computed. • Radiant efficiencies are strongly dependant on the input power to the element. • In-plane efficiencies depend on the distance from the heater. - Abstract: A measurement system has been designed to characterize the radiant energy efficiency of infrared heating elements. The system also allows for measurement of the radiant heat flux distribution emitted from radiant heater assemblies. To facilitate these, a 6-axis robotic arm is fitted with a Schmidt–Boelter radiant heat flux gauge. A LabVIEW interface operates the robot and positions the sensor in the desired location and subsequently acquires the desired radiant heat flux measurement. To illustrate the functionality of the measurement system and methodology, radiant heat flux distributions and efficiency calculations are performed for a commercially available ceramic heater element for two cases. In the first, a spherical surface is traced around the entire heater assembly and the total radiant power and net radiant efficiency is computed. In the second, 50 cm × 50 cm vertical planes are traced parallel to the front face of the heater assembly at distances between 10 cm and 50 cm and the in-plane power and efficiencies are computed. The results indicate that the radiant efficiencies are strongly dependant on the input power to the element and, for the in-plane efficiencies, depend on the distance from the heater.

Benchmarking urban energy efficiency in the UK

International Nuclear Information System (INIS)

Keirstead, James

2013-01-01

This study asks what is the ‘best’ way to measure urban energy efficiency. There has been recent interest in identifying efficient cities so that best practices can be shared, a process known as benchmarking. Previous studies have used relatively simple metrics that provide limited insight on the complexity of urban energy efficiency and arguably fail to provide a ‘fair’ measure of urban performance. Using a data set of 198 urban UK local administrative units, three methods are compared: ratio measures, regression residuals, and data envelopment analysis. The results show that each method has its own strengths and weaknesses regarding the ease of interpretation, ability to identify outliers and provide consistent rankings. Efficient areas are diverse but are notably found in low income areas of large conurbations such as London, whereas industrial areas are consistently ranked as inefficient. The results highlight the shortcomings of the underlying production-based energy accounts. Ideally urban energy efficiency benchmarks would be built on consumption-based accounts, but interim recommendations are made regarding the use of efficiency measures that improve upon current practice and facilitate wider conversations about what it means for a specific city to be energy-efficient within an interconnected economy. - Highlights: • Benchmarking is a potentially valuable method for improving urban energy performance. • Three different measures of urban energy efficiency are presented for UK cities. • Most efficient areas are diverse but include low-income areas of large conurbations. • Least efficient areas perform industrial activities of national importance. • Improve current practice with grouped per capita metrics or regression residuals

Energy efficiency practices among road freight hauliers

International Nuclear Information System (INIS)

Liimatainen, Heikki; Stenholm, Pekka; Tapio, Petri; McKinnon, Alan

2012-01-01

The reduction of greenhouse gases (GHG) is a highly prevalent public policy goal among European Union member countries. In the new White Paper on transport, the role of road freight transports in this is strongly emphasized. This far, however, the efficiency practices utilised in logistics firms are less studied. Drawing from policy goals and new survey data on 295 road transport firms our results show that hauliers are aware of the possible energy efficiency actions but lack the knowledge and resources to fully utilize them. Energy efficiency seems also to be unimportant for many shippers, so there are no incentives for hauliers to improve it. Examples from various countries show that clear energy efficiency improvements can be achieved with active cooperation between hauliers, shippers and policy makers. Such cooperation can be developed in Finland through the sectoral energy efficiency agreements. The novelty and the utility of these results allow scholars to answer important open questions in the national-level determinants of enhancing energy efficiency practices among road freight hauliers, and contribute to our understanding of how these can be fostered in public policies. - Highlights: ► Hauliers still monitor their fuel consumption with unsophisticated methods. ► Larger hauliers are more active in energy efficiency related issues than smaller ones. ► Hauliers are aware of energy efficiency actions, but lack knowledge of implementation. ► Finnish energy efficiency agreement provides a good framework for public policies. ► Companies that monitor and improve energy efficiency may gain competitive advantage.

Continuity and boundary conditions in thermodynamics: From Carnot's efficiency to efficiencies at maximum power

Science.gov (United States)

Ouerdane, H.; Apertet, Y.; Goupil, C.; Lecoeur, Ph.

2015-07-01

Classical equilibrium thermodynamics is a theory of principles, which was built from empirical knowledge and debates on the nature and the use of heat as a means to produce motive power. By the beginning of the 20th century, the principles of thermodynamics were summarized into the so-called four laws, which were, as it turns out, definitive negative answers to the doomed quests for perpetual motion machines. As a matter of fact, one result of Sadi Carnot's work was precisely that the heat-to-work conversion process is fundamentally limited; as such, it is considered as a first version of the second law of thermodynamics. Although it was derived from Carnot's unrealistic model, the upper bound on the thermodynamic conversion efficiency, known as the Carnot efficiency, became a paradigm as the next target after the failure of the perpetual motion ideal. In the 1950's, Jacques Yvon published a conference paper containing the necessary ingredients for a new class of models, and even a formula, not so different from that of Carnot's efficiency, which later would become the new efficiency reference. Yvon's first analysis of a model of engine producing power, connected to heat source and sink through heat exchangers, went fairly unnoticed for twenty years, until Frank Curzon and Boye Ahlborn published their pedagogical paper about the effect of finite heat transfer on output power limitation and their derivation of the efficiency at maximum power, now mostly known as the Curzon-Ahlborn (CA) efficiency. The notion of finite rate explicitly introduced time in thermodynamics, and its significance cannot be overlooked as shown by the wealth of works devoted to what is now known as finite-time thermodynamics since the end of the 1970's. The favorable comparison of the CA efficiency to actual values led many to consider it as a universal upper bound for real heat engines, but things are not so straightforward that a simple formula may account for a variety of situations. The

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.

Monitoring tools for energy efficiency in Europe

Energy Technology Data Exchange (ETDEWEB)

NONE

2002-07-01

This document brings together the different definitions of the indicators used in the European Odyssee project on energy efficiency indicators. This project was initiated in 1990. It benefits from the combined support of the SAVE programme of the European Commission, of Ademe and of 15 national Efficiency Agencies within the European network of energy efficiency agencies. The objective of the project is to develop and maintain indicators that enable to review progress in energy efficiency and CO{sub 2} emissions abatement, by sector, end-use, etc.. for each country and the EU as a whole. To reach this objective, all data and indicators are stored in a common database called ODYSSEE that is regularly updated. A common methodology is used to produce comparative energy efficiency indicators from the database. The definitions presented in this document concern: 1) the general points (energy intensity, consumption, savings, efficiency, the unit consumption effect and index, the technological effect or savings, the substitution effect and the behavioural/management effect); 2) the macro-indicators (primary and final energy intensities at constant structure, at purchasing power parities, at reference economic structure); 3) industry (energy intensity of industry/manufacturing, of industry at constant structure and at reference structure, unit consumption of steel, cement etc.., process effect); 4) transports (energy intensity, unit consumption of vehicles, average specific consumption, test specific consumption, unit consumption, specific consumption, behavioural energy savings; 5) households and services (unit consumption, specific consumption, energy intensity of households, appliances); 6) transformations (apparent efficiency of energy sector or transformations, efficiency at constant fuel mix, efficiency of electricity sector). The same work is made for the 'key energy efficiency indicators', for the 'aggregate energy efficiency indicators' for

High Efficiency of Two Efficient QSDC with Authentication Is at the Cost of Their Security

International Nuclear Information System (INIS)

Su-Juan, Qin; Qiao-Yan, Wen; Luo-Ming, Meng; Fu-Chen, Zhu

2009-01-01

Two efficient protocols of quantum secure direct communication with authentication [Chin. Phys. Lett. 25 (2008) 2354] were recently proposed by Liu et al. to improve the efficiency of two protocols presented in [Phys. Rev. A 75 (2007) 026301] by four Pauli operations. We show that the high efficiency of the two protocols is at the expense of their security. The authenticator Trent can reach half the secret by a particular attack strategy in the first protocol. In the second protocol, not only Trent but also an eavesdropper outside can elicit half-information about the secret from the public declaration

Premium Efficiency Motors And Market Penetration Policy

Energy Technology Data Exchange (ETDEWEB)

Benhaddadi, Mohamed; Olivier, Guy

2010-09-15

This paper illustrates the induced enormous energy saving potential, permitted by using high-efficiency motors. Furthermore, the most important barriers to larger high-efficiency motors utilization are identified, and some incentives recommendations are given to overcome identified impediments. The authors consider that there is a strong case to enhance incentives policies for larger market penetration. The US Energy Policy Act and the Canadian Energy Efficient Act have lead to North American leadership on motor efficiency implementation. North America is not on the leading edge for energy saving and conservation. Motor efficiency is an exception that should be at least maintained.

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…

Efficiency and abatement costs of energy-related CO2 emissions in China: A slacks-based efficiency measure

International Nuclear Information System (INIS)

Choi, Yongrok; Zhang, Ning; Zhou, P.

2012-01-01

Highlights: ► We employ a slacks-based DEA model to estimate the energy efficiency and shadow prices of CO 2 emissions in China. ► The empirical study shows that China was not performing CO 2 -efficiently. ► The average of estimated shadow prices of CO 2 emissions is about $7.2. -- Abstract: This paper uses nonparametric efficiency analysis technique to estimate the energy efficiency, potential emission reductions and marginal abatement costs of energy-related CO 2 emissions in China. We employ a non-radial slacks-based data envelopment analysis (DEA) model for estimating the potential reductions and efficiency of CO 2 emissions for China. The dual model of the slacks-based DEA model is then used to estimate the marginal abatement costs of CO 2 emissions. An empirical study based on China’s panel data (2001–2010) is carried out and some policy implications are also discussed.

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.

Deep-blue efficient OLED based on NPB with little efficiency roll-off under high current density

Science.gov (United States)

Liu, Jian

2017-03-01

NPB usually is used as a hole-transport layer in OLED. In fact, it is a standard pure blue-emission material. However, its light-emitting efficiency in OLED is low due to emissive nature of organic material. Herein, a deep-blue OLDE based on NPB was fabricated. The light-emitting efficiency of the device demonstrates a moderate value, and efficiency roll-off is little under high current density. The device demonstrates that the electroplex's emission decreases with increasing electric field intensity.

Learning Layouts for Single-Page Graphic Designs.

Science.gov (United States)

O'Donovan, Peter; Agarwala, Aseem; Hertzmann, Aaron

2014-08-01

This paper presents an approach for automatically creating graphic design layouts using a new energy-based model derived from design principles. The model includes several new algorithms for analyzing graphic designs, including the prediction of perceived importance, alignment detection, and hierarchical segmentation. Given the model, we use optimization to synthesize new layouts for a variety of single-page graphic designs. Model parameters are learned with Nonlinear Inverse Optimization (NIO) from a small number of example layouts. To demonstrate our approach, we show results for applications including generating design layouts in various styles, retargeting designs to new sizes, and improving existing designs. We also compare our automatic results with designs created using crowdsourcing and show that our approach performs slightly better than novice designers.

Modeling international trends in energy efficiency

International Nuclear Information System (INIS)

Stern, David I.

2012-01-01

I use a stochastic production frontier to model energy efficiency trends in 85 countries over a 37-year period. Differences in energy efficiency across countries are modeled as a stochastic function of explanatory variables and I estimate the model using the cross-section of time-averaged data, so that no structure is imposed on technological change over time. Energy efficiency is measured using a new energy distance function approach. The country using the least energy per unit output, given its mix of outputs and inputs, defines the global production frontier. A country's relative energy efficiency is given by its distance from the frontier—the ratio of its actual energy use to the minimum required energy use, ceteris paribus. Energy efficiency is higher in countries with, inter alia, higher total factor productivity, undervalued currencies, and smaller fossil fuel reserves and it converges over time across countries. Globally, technological change was the most important factor counteracting the energy-use and carbon-emissions increasing effects of economic growth.

Thermodynamically efficient solar concentrators

Science.gov (United States)

Winston, Roland

2012-10-01

Non-imaging Optics is the theory of thermodynamically efficient optics and as such depends more on thermodynamics than on optics. Hence in this paper a condition for the "best" design is proposed based on purely thermodynamic arguments, which we believe has profound consequences for design of thermal and even photovoltaic systems. This new way of looking at the problem of efficient concentration depends on probabilities, the ingredients of entropy and information theory while "optics" in the conventional sense recedes into the background.

Energy Efficiency in Norway 1996-1999. Monitoring tools for energy efficiency in Europe: the ODYSSEE and MURE projects

Energy Technology Data Exchange (ETDEWEB)

Rosenberg, Eva

2002-05-01

This is the national report for Norway in the EU/SAVE project ''Monitoring tools for energy efficiency in Europe: the ODYSSEE and MURE projects''. The report deals with energy use and energy efficiency in Norway 1990-1999. Final energy use per Gross Domestic Product (GDP) was reduced by approximately 1.6% per year from 1990 to 1999. The energy efficiency improvement has been calculated to 0.4% pr year, while the role of structural changes has been 1.2% pr year. A detailed sector analysis has been done, applying Laspeyres indices to attribute changes in energy use to activity, structure or intensity (efficiency). Aggregating sectors, we have found a total efficiency improvement of approximately 8 TWh from 1990 to 1999. (author)

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).

Metasurface holograms reaching 80% efficiency.

Science.gov (United States)

Zheng, Guoxing; Mühlenbernd, Holger; Kenney, Mitchell; Li, Guixin; Zentgraf, Thomas; Zhang, Shuang

2015-04-01

Surfaces covered by ultrathin plasmonic structures--so-called metasurfaces--have recently been shown to be capable of completely controlling the phase of light, representing a new paradigm for the design of innovative optical elements such as ultrathin flat lenses, directional couplers for surface plasmon polaritons and wave plate vortex beam generation. Among the various types of metasurfaces, geometric metasurfaces, which consist of an array of plasmonic nanorods with spatially varying orientations, have shown superior phase control due to the geometric nature of their phase profile. Metasurfaces have recently been used to make computer-generated holograms, but the hologram efficiency remained too low at visible wavelengths for practical purposes. Here, we report the design and realization of a geometric metasurface hologram reaching diffraction efficiencies of 80% at 825 nm and a broad bandwidth between 630 nm and 1,050 nm. The 16-level-phase computer-generated hologram demonstrated here combines the advantages of a geometric metasurface for the superior control of the phase profile and of reflectarrays for achieving high polarization conversion efficiency. Specifically, the design of the hologram integrates a ground metal plane with a geometric metasurface that enhances the conversion efficiency between the two circular polarization states, leading to high diffraction efficiency without complicating the fabrication process. Because of these advantages, our strategy could be viable for various practical holographic applications.

Energy efficiency rating of districts, case Finland

International Nuclear Information System (INIS)

Hedman, Åsa; Sepponen, Mari; Virtanen, Mikko

2014-01-01

There is an increasing political pressure on the city planning to create more energy efficient city plans. Not only do the city plans have to enable and promote energy efficient solutions, but it also needs to be clearly assessed how energy efficient the plans are. City planners often have no or poor know how about energy efficiency and building technologies which makes it difficult for them to answer to this need without new guidelines and tools. An easy to use tool for the assessment of the energy efficiency of detailed city plans was developed. The aim of the tool is for city planners to easily be able to assess the energy efficiency of the proposed detailed city plan and to be able to compare the impacts of changes in the plan. The tool is designed to be used with no in-depth knowledge about energy or building technology. With a wide use of the tool many missed opportunities for improving energy efficiency can be avoided. It will provide better opportunities for sustainable solutions leading to less harmful environmental impact and reduced emissions. - Highlights: • We have created a tool for assessing energy efficiency of detailed city plans. • The energy source is the most important factor for efficiency of districts in Finland. • Five case districts in Finland were analyzed. • In this paper one residential district has in-depth sensitivity analyses done

Relationship between efficiency and pedal rate in cycling

DEFF Research Database (Denmark)

Hansen, E A; Sjøgaard, G

2007-01-01

Cycling was performed to test the following two hypotheses: (1) muscular efficiency is unrelated to pedal rate (61, 88, and 115 r.p.m.) for a group of subjects with a wide range of slow twitch (ST) fibers in spite of decreasing whole-body efficiency and (2) muscular efficiency correlates positively...... with % ST muscle fibers, and this correlation is more pronounced at low pedal rates than at high pedal rates. Whole-body gross efficiency decreased from 20-22% at 61 r.p.m. to 15-18% at 115 r.p.m. Mean muscular efficiency for all subjects (n=16) was approximately 26%, with delta efficiency being constant...... and muscular efficiency (taking internal power into account) slightly increasing with pedal rate. Muscular efficiency correlated positively (R(2)=0.25) with % ST fibers (21-97% ST in m. vastus lateralis) at 115 r.p.m. while not at 61 and 88 r.p.m. In conclusion, the decrease in whole-body gross efficiency...

Assessing the efficiency of US electricity markets

International Nuclear Information System (INIS)

Arciniegas, I.; Barrett, C.; Marathe, A.

2003-01-01

The recent California's energy crisis has raised doubts about the benefits of energy deregulation. While it is true that the California electricity market is in turmoil, other electricity markets like the Pennsylvania-NewJersey-Maryland (PJM) are doing fine. This paper assesses the mark of efficiency reached by the electricity markets in California, New York, and PJM. It also compares the degree of efficiency across markets (forward vs. real time) and across time. No significant differences between the California and PJM electricity markets were discovered in the year of California's energy crisis (2000) using the co-integration tests. This research suggests that differences in price behavior between these two markets during 2000 did not arise from differences in efficiency. According to our analysis and measures of efficiency, PJM and California electricity markets are more efficient than the New York market. Also, as these markets become more mature over time, their efficiency level goes up. We also found evidence that a multi-settlement scheduling system leads to higher efficiency. (author)

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.