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Sample records for lentiviral vector systems

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

    Science.gov (United States)

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

    2005-02-18

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

  2. 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.

  3. 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.

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

    Science.gov (United States)

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

    2017-03-03

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

  5. Lentiviral Vector Gene Transfer to Porcine Airways

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    Patrick L Sinn

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

  8. 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 ...

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

    DEFF Research Database (Denmark)

    Aagaard, Lars; Amarzguioui, Mohammed; Sun, Guihua

    2007-01-01

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

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

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    Shen-shen ZHI

    2011-02-01

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

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

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

    2004-09-01

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

  12. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

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    Benjamin Geiling

    Full Text Available The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG and retroviral (pREG vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs. In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

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

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

    2010-03-01

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

  14. Targeting lentiviral vectors to antigen-specific immunoglobulins.

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

    2008-09-01

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

  15. 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.

  16. Neuron-specific RNA interference using lentiviral vectors

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  17. Packaging of HCV-RNA into lentiviral vector

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-04

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

  18. 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.

  19. 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.

  20. 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

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

    Science.gov (United States)

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

    2010-06-22

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

  2. Efficient transgenesis in farm animals by lentiviral vectors

    Science.gov (United States)

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

    2003-01-01

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

  3. Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing

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    Karine Cambon

    2017-06-01

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

  4. 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 ...

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

    Science.gov (United States)

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

    2011-07-01

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

  6. Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing.

    Science.gov (United States)

    Heider, Susanne; Muzard, Julien; Zaruba, Marianne; Metzner, Christoph

    2017-07-01

    Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.

  7. Lentiviral vectors for the treatment of primary immunodeficiencies.

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

    Semple-Rowland, Susan L; Berry, Jonathan

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Prokofjeva Maria M

    2013-01-01

    Full Text Available Abstract Background Despite progress in the development of combined antiretroviral therapies (cART, HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. Methods We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN LeGO vector technology. Results We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. Conclusions The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2017-03-06

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

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

    Science.gov (United States)

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

    2009-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Elizabeth M Everson

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Fernandez Soledad

    2006-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Sheng Zhou

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

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

    Directory of Open Access Journals (Sweden)

    Akiya Watakabe

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

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

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Daniel C Farley

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

  1. 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

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

    Science.gov (United States)

    Cetin, Ali; Callaway, Edward M

    2014-05-01

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

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

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    Li Ou

    2016-09-01

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

  4. [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.

  5. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-01

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

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

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    Jiasi Bai

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

  8. 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...

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

    OpenAIRE

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

    2010-01-01

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

  10. 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.

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

    Science.gov (United States)

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

    2008-09-01

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

  12. 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.

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

    Science.gov (United States)

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

    2012-06-01

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

  14. 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.

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

    Science.gov (United States)

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

    2012-10-01

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

  16. 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.

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

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    Zifu Zhang

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

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

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    Alessandra Gallinaro

    2018-02-01

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

  19. 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.

  20. Lentiviral vector delivery of human interleukin-7 (hIL-7 to human immune system (HIS mice expands T lymphocyte populations.

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    Ryan M O'Connell

    2010-08-01

    Full Text Available Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7 in Rag2-/-gammac-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-gammac-/- Human Immune System (HIS mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases.

  1. 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.

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

    Science.gov (United States)

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

    2009-12-01

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

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

    Science.gov (United States)

    Roy, I

    2001-11-01

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

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

    Science.gov (United States)

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

    2011-03-01

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

  5. 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.

  6. 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.

  7. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-07-01

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

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

    Science.gov (United States)

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

    2017-08-02

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

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  11. 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.

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

    LENUS (Irish Health Repository)

    McGinley, Lisa

    2012-01-31

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

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

    LENUS (Irish Health Repository)

    McGinley, Lisa

    2011-03-07

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

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

    Science.gov (United States)

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

    2016-09-01

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

  15. 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.

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

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

    2013-09-01

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

  17. 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.

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

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

    2018-03-23

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

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

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    Judith M Fontana

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

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

    Science.gov (United States)

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

    2013-09-01

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

  1. 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.

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

    Science.gov (United States)

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

    2015-12-01

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

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

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

    2007-07-01

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

  6. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

    Science.gov (United States)

    Azzouz, Mimoun; Martin-Rendon, Enca; Barber, Robert D; Mitrophanous, Kyriacos A; Carter, Emma E; Rohll, Jonathan B; Kingsman, Susan M; Kingsman, Alan J; Mazarakis, Nicholas D

    2002-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.

  7. 3' self-inactivating long terminal repeat inserts for the modulation of transgene expression from lentiviral vectors.

    Science.gov (United States)

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

    2012-04-01

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

  8. 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.

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

    Science.gov (United States)

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

    2013-02-28

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

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

    Science.gov (United States)

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

    2002-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Marino Michael P

    2009-02-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Sytwu Huey-Kang

    2009-08-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-07-21

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

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

    Science.gov (United States)

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

    2013-07-01

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

  19. Raster images vectorization system

    OpenAIRE

    Genytė, Jurgita

    2006-01-01

    The problem of raster images vectorization was analyzed and researched in this work. Existing vectorization systems are quite expensive, the results are inaccurate, and the manual vectorization of a large number of drafts is impossible. That‘s why our goal was to design and develop a new raster images vectorization system using our suggested automatic vectorization algorithm and the way to record results in a new universal vectorial file format. The work consists of these main parts: analysis...

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

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2005-01-01

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

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

    Science.gov (United States)

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

    2018-03-14

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

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Yao Deng

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

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

    African Journals Online (AJOL)

    USER

    2010-03-29

    Mar 29, 2010 ... compared with the use of a 2-tailed paired student's t test. Statistical analysis was performed using the GraphPad Prism 3.0 software. RESULTS. Verification of transfer vector .... Medico E, Naldini L (2005). Promoter trapping reveals significant differences in integration site selection between MLV and HIV ...

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

    Science.gov (United States)

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

    2011-03-01

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

  6. 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

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

    Directory of Open Access Journals (Sweden)

    Sablitzky Fred

    2004-01-01

    Full Text Available Abstract Background Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV and lentiviral (LV vectors into discrete regions of the forebrain. Results Recombinant AAV-Cre, AAV-GFP (green fluorescent protein and LV-Cre-EGFP (enhanced GFP were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. Conclusion AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

  8. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  11. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    Science.gov (United States)

    Wang, Weiming; Ye, Chaobaihui; Liu, Jingjing; Zhang, Di; Kimata, Jason T; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

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

    Science.gov (United States)

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

    2014-04-01

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

  13. Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator.

    Science.gov (United States)

    Ramezani, Ali; Hawley, Teresa S; Hawley, Robert G

    2003-06-15

    Retroviral vectors are the most efficient means of stable gene delivery to hematopoietic stem cells (HSCs). However, transgene expression from retroviral vectors is frequently subject to the negative influence of chromosomal sequences flanking the site of integration. Toward the development of autonomous transgene expression cassettes, we inserted the human interferon-beta scaffold attachment region (IFN-SAR) and the chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator both separately and together into a series of self-inactivating (SIN) lentiviral vector backbones. Transduced cells of the human CD34+ hematopoietic progenitor line KG1a-pooled populations as well as individual clones harboring single integrants--were analyzed for reporter expression during culture periods of up to 4 months. Vectors carrying both the 5'HS4 insulator and the IFN-SAR consistently outperformed control vectors without inserts as well as vectors carrying either element alone. The performance of a set of vectors containing the murine stem cell virus long terminal repeat as an internal promoter was subsequently assessed during in vitro monocytic differentiation of transduced primary human CD34+ cord blood cells. Similar to what was observed in the KG1a hematopoietic progenitor cell model, optimal reporter expression in primary monocytes was obtained with the vector bearing both regulatory elements. These findings indicate that the 5'HS4/IFN-SAR combination is particularly effective at maintaining open chromatin domains permissive for high-level transgene expression at early and late stages of hematopoietic development, and thus could be of utility in HSC-directed retroviral vector-mediated gene transfer applications.

  14. 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

  15. Targeting both viral and host determinants of human immunodeficiency virus entry, using a new lentiviral vector coexpressing the T20 fusion inhibitor and a selective CCL5 intrakine.

    Science.gov (United States)

    Petit, Nicolas; Dorgham, Karim; Levacher, Béatrice; Burlion, Aude; Gorochov, Guy; Marodon, Gilles

    2014-08-01

    Numerous strategies targeting early and late steps of the HIV life cycle have been proposed for gene therapy. However, targeting viral and host determinants of HIV entry is the only strategy that would prevent viral DNA-mediated CD4(+) cell death while diminishing the possibility for the virus to escape. To this end, we devised a bicistronic lentiviral vector expressing the membrane-bound form of the T20 fusion inhibitor, referred to as the C46 peptide, and a CCR5 superagonist, modified to sequester CCR5 away from the cell surface, referred to as the P2-CCL5 intrakine. We tested the effects of the vector on HIV infection and replication, using the human CEMR5 cell line expressing CD4 and CCR5, and primary human T cells. Transduced cells expressed the C46 peptide, detected with the 2F5 monoclonal antibody by flow cytometry. Expression of the P2-CCL5 intrakine correlates with lower levels of cell surface CCR5. Complete protection against HIV infection could be observed in cells expressing the protective transgenes. Importantly, we show that the combination of the transgenes was more potent than either transgene alone, showing the interest of expressing two entry inhibitors to inhibit HIV infection. Last, genetically modified cells possessed a selective advantage over nonmodified cells on HIV challenge in vitro, showing that modified cells were protected from HIV-induced cell death. Our results demonstrate that lentiviral vectors coexpressing the T20 fusion inhibitor and the P2-CCL5 intrakine represent promising tools for HIV gene therapy.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Takato Takenouchi

    2017-08-01

    Full Text Available The domestic pig is an important agricultural animal, and thus, infectious diseases that affect pigs can cause severe economic losses in the global swine industry. Various porcine pathogens target macrophages, which are classical innate immune cells. Although macrophages basically protect the host from pathogens, they also seem to contribute to infectious processes. Therefore, cultured macrophages can be used to develop in vitro models for studying not only genes associated with porcine innate immunity but also the infectious processes of porcine pathogens. However, the availability of porcine macrophage cell lines is limited. In this study, we describe a novel immortalized porcine kidney-derived macrophage (IPKM cell line, which was generated by transferring the SV40 large T antigen (SV40LT and porcine telomerase reverse transcriptase (pTERT genes into primary porcine kidney-derived macrophages using lentiviral vectors. The IPKM displayed a typical macrophage morphology and was routinely passaged (doubling time: about 4 days. These cells were immunostained for macrophage markers. In addition, they exhibited substantial phagocytosis of polystyrene microbeads and released inflammatory cytokines upon lipopolysaccharide (LPS stimulation. Furthermore, the maturation and secretion of interleukin-1β were observed after nigericin-induced inflammasome activation in LPS-primed IPKM. These findings suggest that IPKM exhibit the typical inflammatory characteristics of macrophages. By transferring the SV40LT and pTERT genes using lentiviral vectors, we also successfully immortalized macrophages derived from the peripheral blood of a low-density lipoprotein receptor-deficient pig. These results suggest that the co-expression of SV40LT and pTERT is an effective way of immortalizing porcine macrophages.

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

    Directory of Open Access Journals (Sweden)

    Hiroko Kita-Matsuo

    Full Text Available Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

  19. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

    Science.gov (United States)

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-04-19

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  20. Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4⁺ T-Cells.

    Science.gov (United States)

    Urusov, Felix; Glazkova, Dina; Omelchenko, Denis; Bogoslovskaya, Elena; Tsyganova, Galina; Kersting, Katerina; Shipulin, German; Pokrovsky, Vadim

    2018-02-04

    C-C chemokine receptor type 5 (CCR5) is utilized by human immunodeficiency virus (HIV) as a co-receptor for cell entry. Suppression of the CCR5 gene by artificial microRNAs (amiRNAs) could confer cell resistance. In previous work, we created a lentivector that encoded the polycistron of two identical amiRNAs that could effectively suppress CCR5. However, tandem repeats in lentiviral vectors led to deletions of the repeated sequences during reverse transcription of the vector RNA. To solve this problem, we have created a new amiRNA against CCR5, mic1002, which has a different microRNA scaffold and targets a different sequence. Replacing one of the two identical tandem amiRNAs in the polycistron with the mic1002 amiRNA increased the accuracy of its lentiviral vector transfer while retaining its ability to effectively suppress CCR5. A lentiviral vector containing two heterogenic amiRNAs significantly inhibited HIV replication in a vector-transduced human CD4⁺ lymphocyte culture.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Hanieh Jalali

    2016-02-01

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

  3. Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD/SCIDγc-/-mice.

    Science.gov (United States)

    Levy, C; Fusil, F; Amirache, F; Costa, C; Girard-Gagnepain, A; Negre, D; Bernadin, O; Garaulet, G; Rodriguez, A; Nair, N; Vandendriessche, T; Chuah, M; Cosset, F-L; Verhoeyen, E

    2016-12-01

    Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc -/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc -/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein

  4. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach

    Directory of Open Access Journals (Sweden)

    Francesca Spanevello

    2016-01-01

    Full Text Available Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1 were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4+ T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  5. LV305, a dendritic cell-targeting integration-deficient ZVex(TM)-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response.

    Science.gov (United States)

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; Ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1.

  6. LV305, a dendritic cell-targeting integration-deficient ZVexTM-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response

    Science.gov (United States)

    Albershardt, Tina Chang; Campbell, David James; Parsons, Andrea Jean; Slough, Megan Merrill; ter Meulen, Jan; Berglund, Peter

    2016-01-01

    We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1. PMID:27626061

  7. Inhibiting PHD2 in bone marrow mesenchymal stem cells via lentiviral vector-mediated RNA interference facilitates the repair of periodontal tissue defects in SD rats

    Science.gov (United States)

    Chen, Changxing; Li, Houxuan; Jiang, Jun; Zhang, Qian; Yan, Fuhua

    2017-01-01

    Hypoxia-inducible factors (HIFs) play an important role in angiogenesis, and they can activate the expression of several downstream angiogenic factors. HIF-1 is a major transcriptor of HIFs, composed of α and β subunits. Prolyl hydroxylase domain-containing protein 2 (PHD2) is the main catabolic enzyme for HIF-1α, and it can accelerate its degradation under normoxic conditions. PHD2 expression in bone marrow mesenchymal stem cells (BMMSCs) of SD rats was down-regulated under normoxic conditions in this study by utilizing lentiviral vector-mediated RNA interference to promote HIF-1α accumulation, thus enhancing the expression of angiogenic factors. A tissue-engineered compound was constructed using the composite collagen membrane of BMMSCs after PHD2 gene silencing to repair periodontal fenestration defects in SD rats. The results of this study indicated that, after PHD2 gene silencing, the osteogenic differentiation of BMMSCs was enhanced in vitro, the resistance of cells to oxidative stress was also validated in vitro, thereby illustrating the promotion of the repair of artificially constructed periodontal tissue defects in rats. The results of this study provide a reference and guidance for future applications of RNA interference in periodontal tissue engineering and serve as a basis for improving the survival of seed cells in recipient tissues. PMID:29069818

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

    Science.gov (United States)

    Huston, Marshall W; Riegman, Adriaan R A; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P; Wagemaker, Gerard

    2014-10-01

    Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning.

  9. 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.

  10. 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

  11. In vitro incorporation of a cell-binding protein to a lentiviral vector using an engineered split intein enables targeted delivery of genetic cargo.

    Science.gov (United States)

    Chamoun-Emanuelli, Ana M; Wright, Gus; Roger, Smith; Münch, Robert C; Buchholz, Christian J; Chen, Zhilei

    2015-12-01

    Gene therapy represents a promising therapeutic paradigm for addressing many disorders, but the absence of a vector that can be robustly and reproducibly functionalized with cell-homing functionality to mediate the delivery of genetic cargo specifically to target cells following systemic administration has stood as a major impediment. In this study, a high-affinity protein-protein pair comprising a splicing-deficient naturally split intein was used as molecular Velcro to append a HER2/neu-binding protein (DARPin) onto the surface of a binding-deficient, fusion-competent lentivirus. HER2/neu-specific lentiviruses created using this in vitro pseudotyping approach were able to deliver their genetic reporter cargo specifically to cells that express the target receptor at high levels in a co-culture. We envision that the described technology could provide a powerful, broadly applicable platform for the incorporation of cell-targeting functionality onto viral vectors. © 2015 Wiley Periodicals, Inc.

  12. 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.

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

    Science.gov (United States)

    Pridans, Clare; Lillico, Simon; Whitelaw, Bruce; Hume, David A

    2014-01-01

    The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large “cargo” for applications in gene therapy and vaccine delivery. PMID:26015955

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

    Directory of Open Access Journals (Sweden)

    Natalia Vapniarsky

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

  15. 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 ...

  16. First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient.

    Science.gov (United States)

    Pollack, Seth M; Lu, Hailing; Gnjatic, Sacha; Somaiah, Neeta; O'Malley, Ryan B; Jones, Robin L; Hsu, Frank J; Ter Meulen, Jan

    2017-10-01

    Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.

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

    Directory of Open Access Journals (Sweden)

    Nisha G Sosale

    2016-01-01

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

  18. The evaporative vector: Homogeneous systems

    International Nuclear Information System (INIS)

    Klots, C.E.

    1987-05-01

    Molecular beams of van der Waals molecules are the subject of much current research. Among the methods used to form these beams, three-sputtering, laser ablation, and the sonic nozzle expansion of neat gases - yield what are now recognized to be ''warm clusters.'' They contain enough internal energy to undergo a number of first-order processes, in particular that of evaporation. Because of this evaporation and its attendant cooling, the properties of such clusters are time-dependent. The states of matter which can be arrived at via an evaporative vector on a typical laboratory time-scale are discussed. Topics include the (1) temperatures, (2) metastability, (3) phase transitions, (4) kinetic energies of fragmentation, and (5) the expression of magical properties, all for evaporating homogeneous clusters

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

    Science.gov (United States)

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

    2016-01-01

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

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

    African Journals Online (AJOL)

    Yomi

    2012-01-24

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

  1. 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

  2. Vector coherent states for nanoparticle systems

    Energy Technology Data Exchange (ETDEWEB)

    Aremua, Isiaka [Institut de Mathematiques et de Sciences Physiques (IMSP), University of Abomey-Calavi, 01 BP 613 Porto-Novo (Benin); Hounkonnou, Mahouton Norbert, E-mail: iaremua@imsp-uac.org, E-mail: norbert.hounkonnou@cipma.uac.bj [International Chair of Mathematical Physics and Applications (ICMPA-UNESCO Chair), University of Abomey-Calavi, 072 BP 50 Cotonou (Benin)

    2011-11-18

    The first part of this work deals with a formalism of vector coherent states construction for a system of M Fermi-type modes associated with N bosonic modes. Then follows a generalization to a Hamiltonian describing the translational motion of the center of mass of a nanoparticle. The latter gives rise to a new mechanism for the electronic energy relaxation in nanocrystals, intensively studied today in condensed matter physics. Finite degeneracies of the involved Hamiltonian systems are also investigated. The defined vector coherent states satisfy relevant mathematical properties of continuity, resolution of identity, temporal stability and action identity. (paper)

  3. High performance computing on vector systems

    CERN Document Server

    Roller, Sabine

    2008-01-01

    Presents the developments in high-performance computing and simulation on modern supercomputer architectures. This book covers trends in hardware and software development in general and specifically the vector-based systems and heterogeneous architectures. It presents innovative fields like coupled multi-physics or multi-scale simulations.

  4. Lentiviral delivery of short hairpin RNAs

    Science.gov (United States)

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

    2009-01-01

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

  5. Reversible Vector Ratchet Effect in Skyrmion Systems

    Science.gov (United States)

    Ma, Xiaoyu; Reichhardt, Charles; Reichhardt, Cynthia

    Magnetic skyrmions are topological non-trivial spin textures found in several magnetic materials. Since their motion can be controlled using ultralow current densities, skyrmions are appealing for potential applications in spintronics as information carriers and processing devices. In this work, we studied the collective transport properties of driven skyrmions based on a particle-like model with molecular dynamics (MD) simulation. Our results show that ac driven skyrmions interacting with an asymmetric substrate provide a realization of a new class of ratchet system, which we call a vector ratchet, that arises due to the effect of the Magnus term on the skyrmion dynamics. In a vector ratchet, the dc motion induced by the ac drive can be described as a vector that can be rotated up to 360 degrees relative to the substrate asymmetry direction. This could represent a new method for controlling skyrmion motion for spintronic applications.

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

    Science.gov (United States)

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

    2015-12-15

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

  7. System for Automated Calibration of Vector Modulators

    Science.gov (United States)

    Lux, James; Boas, Amy; Li, Samuel

    2009-01-01

    Vector modulators are used to impose baseband modulation on RF signals, but non-ideal behavior limits the overall performance. The non-ideal behavior of the vector modulator is compensated using data collected with the use of an automated test system driven by a LabVIEW program that systematically applies thousands of control-signal values to the device under test and collects RF measurement data. The technology innovation automates several steps in the process. First, an automated test system, using computer controlled digital-to-analog converters (DACs) and a computer-controlled vector network analyzer (VNA) systematically can apply different I and Q signals (which represent the complex number by which the RF signal is multiplied) to the vector modulator under test (VMUT), while measuring the RF performance specifically, gain and phase. The automated test system uses the LabVIEW software to control the test equipment, collect the data, and write it to a file. The input to the Lab - VIEW program is either user-input for systematic variation, or is provided in a file containing specific test values that should be fed to the VMUT. The output file contains both the control signals and the measured data. The second step is to post-process the file to determine the correction functions as needed. The result of the entire process is a tabular representation, which allows translation of a desired I/Q value to the required analog control signals to produce a particular RF behavior. In some applications, corrected performance is needed only for a limited range. If the vector modulator is being used as a phase shifter, there is only a need to correct I and Q values that represent points on a circle, not the entire plane. This innovation has been used to calibrate 2-GHz MMIC (monolithic microwave integrated circuit) vector modulators in the High EIRP Cluster Array project (EIRP is high effective isotropic radiated power). These calibrations were then used to create

  8. Lentiviral vectors for treatment of haemophilia

    OpenAIRE

    Ward, N. J.

    2010-01-01

    Haemophilia A and B are X‐linked recessive disorders caused by defects in coagulation factors (F) VIII and IX, respectively. Severe cases of haemophilia are characterised by episodes of spontaneous bleeding, predominantly into the joints and muscles, and can result in permanent disability and even mortality if left untreated. The haemophilias are compelling candidates for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagul...

  9. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  10. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

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

    DEFF Research Database (Denmark)

    GLUD, AN; Hedegaard, Claus; nielsen, MS

    2010-01-01

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

  12. Nitrous Oxide Liquid Injection Thrust Vector Control System Testing Project

    Data.gov (United States)

    National Aeronautics and Space Administration — A Nitrous Oxide-fed Liquid Thrust Vector Control system is proposed as an efficient method for vehicle attitude control during powered flight. Pulled from a N2O main...

  13. Identification and characterization of enhancer-blocking insulators to reduce retroviral vector genotoxicity.

    Directory of Open Access Journals (Sweden)

    Amy C Groth

    Full Text Available The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.

  14. Prediction of Banking Systemic Risk Based on Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Shouwei Li

    2013-01-01

    Full Text Available Banking systemic risk is a complex nonlinear phenomenon and has shed light on the importance of safeguarding financial stability by recent financial crisis. According to the complex nonlinear characteristics of banking systemic risk, in this paper we apply support vector machine (SVM to the prediction of banking systemic risk in an attempt to suggest a new model with better explanatory power and stability. We conduct a case study of an SVM-based prediction model for Chinese banking systemic risk and find the experiment results showing that support vector machine is an efficient method in such case.

  15. Baculovirus expression vector system: An efficient tool for the ...

    African Journals Online (AJOL)

    Baculovirus expression vector system is considered one of the most successful and widely acceptable means for the production of recombinant proteins in extremely large quantities. Proper posttranslational modifications of the expressed proteins in insect cells, the usual host of baculoviruses, get them soluble, correctly ...

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

    Science.gov (United States)

    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.

  17. Vector Disparity Sensor with Vergence Control for Active Vision Systems

    Science.gov (United States)

    Barranco, Francisco; Diaz, Javier; Gibaldi, Agostino; Sabatini, Silvio P.; Ros, Eduardo

    2012-01-01

    This paper presents an architecture for computing vector disparity for active vision systems as used on robotics applications. The control of the vergence angle of a binocular system allows us to efficiently explore dynamic environments, but requires a generalization of the disparity computation with respect to a static camera setup, where the disparity is strictly 1-D after the image rectification. The interaction between vision and motor control allows us to develop an active sensor that achieves high accuracy of the disparity computation around the fixation point, and fast reaction time for the vergence control. In this contribution, we address the development of a real-time architecture for vector disparity computation using an FPGA device. We implement the disparity unit and the control module for vergence, version, and tilt to determine the fixation point. In addition, two on-chip different alternatives for the vector disparity engines are discussed based on the luminance (gradient-based) and phase information of the binocular images. The multiscale versions of these engines are able to estimate the vector disparity up to 32 fps on VGA resolution images with very good accuracy as shown using benchmark sequences with known ground-truth. The performances in terms of frame-rate, resource utilization, and accuracy of the presented approaches are discussed. On the basis of these results, our study indicates that the gradient-based approach leads to the best trade-off choice for the integration with the active vision system. PMID:22438737

  18. Robust support vector machine-trained fuzzy system.

    Science.gov (United States)

    Forghani, Yahya; Yazdi, Hadi Sadoghi

    2014-02-01

    Because the SVM (support vector machine) classifies data with the widest symmetric margin to decrease the probability of the test error, modern fuzzy systems use SVM to tune the parameters of fuzzy if-then rules. But, solving the SVM model is time-consuming. To overcome this disadvantage, we propose a rapid method to solve the robust SVM model and use it to tune the parameters of fuzzy if-then rules. The robust SVM is an extension of SVM for interval-valued data classification. We compare our proposed method with SVM, robust SVM, ISVM-FC (incremental support vector machine-trained fuzzy classifier), BSVM-FC (batch support vector machine-trained fuzzy classifier), SOTFN-SV (a self-organizing TS-type fuzzy network with support vector learning) and SCLSE (a TS-type fuzzy system with subtractive clustering for antecedent parameter tuning and LSE for consequent parameter tuning) by using some real datasets. According to experimental results, the use of proposed approach leads to very low training and testing time with good misclassification rate. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. 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

    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...... directs efficient transposon mobilization from DNA circles in vector-transduced cells. Both transfected plasmid DNA and transduced IDLVs can serve as the source of active transposase. Most important, we demonstrate that the SB transposase overrides the natural lentiviral integration pathway and directs...... vector integration less frequently toward transcriptional units, resulting in a random genomic integration profile. The novel hybrid vector system combines the attractive features of efficient gene delivery by viral transduction and a safer genomic integration profile by DNA transposition....

  20. Orthogonal versus covariant Lyapunov vectors for rough hard disc systems

    Science.gov (United States)

    Bosetti, H.; Posch, H. A.

    2013-06-01

    The Oseledec splitting of the tangent space into covariant subspaces for a hyperbolic dynamical system is numerically accessible by computing the full set of covariant Lyapunov vectors. In this paper, the covariant Lyapunov vectors, the orthogonal Gram-Schmidt vectors and the corresponding local (time-dependent) Lyapunov exponents are analyzed for a planar system of rough hard discs (RHDS). These results are compared to respective results for a smooth-hard-disc system (SHDS). We find that the rotation of the discs deeply affects the Oseledec splitting and the structure of the tangent space. For both the smooth and rough hard discs, the stable, unstable and central manifolds are transverse to each other, although the minimal angle between the unstable and stable manifolds of the RHDS typically is very small. Both systems are hyperbolic. However, the central manifold is precisely orthogonal to the rest of the tangent space only for the smooth-particle case and not for the rough discs. We also demonstrate that the rotations destroy the Hamiltonian (symplectic) character for the RHDS. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Lyapunov analysis: from dynamical systems theory to applications’.

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

    Science.gov (United States)

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

    2017-03-18

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

  2. Fault Isolation for Nonlinear Systems Using Flexible Support Vector Regression

    Directory of Open Access Journals (Sweden)

    Yufang Liu

    2014-01-01

    Full Text Available While support vector regression is widely used as both a function approximating tool and a residual generator for nonlinear system fault isolation, a drawback for this method is the freedom in selecting model parameters. Moreover, for samples with discordant distributing complexities, the selection of reasonable parameters is even impossible. To alleviate this problem we introduce the method of flexible support vector regression (F-SVR, which is especially suited for modelling complicated sample distributions, as it is free from parameters selection. Reasonable parameters for F-SVR are automatically generated given a sample distribution. Lastly, we apply this method in the analysis of the fault isolation of high frequency power supplies, where satisfactory results have been obtained.

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

    African Journals Online (AJOL)

    ajl yemi

    2011-10-26

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

  4. Stability and Control of Large-Scale Dynamical Systems A Vector Dissipative Systems Approach

    CERN Document Server

    Haddad, Wassim M

    2011-01-01

    Modern complex large-scale dynamical systems exist in virtually every aspect of science and engineering, and are associated with a wide variety of physical, technological, environmental, and social phenomena, including aerospace, power, communications, and network systems, to name just a few. This book develops a general stability analysis and control design framework for nonlinear large-scale interconnected dynamical systems, and presents the most complete treatment on vector Lyapunov function methods, vector dissipativity theory, and decentralized control architectures. Large-scale dynami

  5. Application of modified vector fitting to grounding system modeling

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez, D.; Camargo, M.; Herrera, J.; Torres, H. [National University of Colombia (Colombia). Research Program on Acquisition and Analysis of Signals - PAAS], Emails: dyjimeneza@unal.edu.co, mpcamargom@unal.edu.co; Vargas, M. [Siemens S.A. - Power Transmission and Distribution - Energy Services (Colombia)

    2007-07-01

    The transient behavior of grounding systems (GS) influences greatly the performance of electrical networks under fault conditions. This fact has led the authors to present an application of the Modified Vector Fitting (MVF)1 methodology based upon the frequency response of the system, in order to find a rational function approximation and an equivalent electrical network whose transient behavior is similar to the original one of the GS. The obtained network can be introduced into the EMTP/ATP program for simulating the transient behavior of the GS. The MVF technique, which is a modification of the Vector Fitting (VF) technique, allows identifying state space models from the Frequency Domain Response for both single and multiple input-output systems. In this work, the methodology is used to fit the frequency response of a grounding grid, which is computed by means of the Hybrid Electromagnetic Model (HEM), finding the relation between voltages and input currents in two points of the grid in frequency domain. The model obtained with the MVF shows a good agreement with the frequency response of the GS. Besides, the model is tested in EMTP/ATP finding a good fitting with the calculated data, which demonstrates the validity and usefulness of the MVF. (author)

  6. Lyapunov, singular and bred vectors in a multi-scale system: an empirical exploration of vectors related to instabilities

    International Nuclear Information System (INIS)

    Norwood, Adrienne; Kalnay, Eugenia; Ide, Kayo; Yang, Shu-Chih; Wolfe, Christopher

    2013-01-01

    We compute and compare the three types of vectors frequently used to explore the instability properties of dynamical models, namely Lyapunov vectors (LVs), singular vectors (SVs) and bred vectors (BVs) in two systems, using the Wolfe–Samelson (2007 Tellus A 59 355–66) algorithm to compute all of the Lyapunov vectors. The first system is the Lorenz (1963 J. Atmos. Sci. 20 130–41) three-variable model. Although the leading Lyapunov vector, LV1, grows fastest globally, the second Lyapunov vector, LV2, which has zero growth globally, often grows faster than LV1 locally. Whenever this happens, BVs grow closer to LV2, suggesting that in larger atmospheric or oceanic models where several instabilities can grow in different areas of the world, BVs will grow toward the fastest growing local unstable mode. A comparison of their growth rates at different times shows that all three types of dynamical vectors have the ability to predict regime changes and the duration of the new regime based on their growth rates in the last orbit of the old regime, as shown for BVs by Evans et al (2004 Bull. Am. Meteorol. Soc. 520–4). LV1 and BVs have similar predictive skill, LV2 has a tendency to produce false alarms, and even LV3 shows that maximum decay is also associated with regime change. Initial and final SVs grow much faster and are the most accurate predictors of regime change, although the characteristics of the initial SVs are strongly dependent on the length of the optimization window. The second system is the toy ‘ocean-atmosphere’ model developed by Peña and Kalnay (2004 Nonlinear Process. Geophys. 11 319–27) coupling three Lorenz (1963 J. Atmos. Sci. 20 130–41) systems with different time scales, in order to test the effects of fast and slow modes of growth on the dynamical vectors. A fast ‘extratropical atmosphere’ is weakly coupled to a fast ‘tropical atmosphere’ which is, in turn, strongly coupled to a slow ‘ocean’ system, the latter coupling

  7. Lyapunov, singular and bred vectors in a multi-scale system: an empirical exploration of vectors related to instabilities

    Science.gov (United States)

    Norwood, Adrienne; Kalnay, Eugenia; Ide, Kayo; Yang, Shu-Chih; Wolfe, Christopher

    2013-06-01

    We compute and compare the three types of vectors frequently used to explore the instability properties of dynamical models, namely Lyapunov vectors (LVs), singular vectors (SVs) and bred vectors (BVs) in two systems, using the Wolfe-Samelson (2007 Tellus A 59 355-66) algorithm to compute all of the Lyapunov vectors. The first system is the Lorenz (1963 J. Atmos. Sci. 20 130-41) three-variable model. Although the leading Lyapunov vector, LV1, grows fastest globally, the second Lyapunov vector, LV2, which has zero growth globally, often grows faster than LV1 locally. Whenever this happens, BVs grow closer to LV2, suggesting that in larger atmospheric or oceanic models where several instabilities can grow in different areas of the world, BVs will grow toward the fastest growing local unstable mode. A comparison of their growth rates at different times shows that all three types of dynamical vectors have the ability to predict regime changes and the duration of the new regime based on their growth rates in the last orbit of the old regime, as shown for BVs by Evans et al (2004 Bull. Am. Meteorol. Soc. 520-4). LV1 and BVs have similar predictive skill, LV2 has a tendency to produce false alarms, and even LV3 shows that maximum decay is also associated with regime change. Initial and final SVs grow much faster and are the most accurate predictors of regime change, although the characteristics of the initial SVs are strongly dependent on the length of the optimization window. The second system is the toy ‘ocean-atmosphere’ model developed by Peña and Kalnay (2004 Nonlinear Process. Geophys. 11 319-27) coupling three Lorenz (1963 J. Atmos. Sci. 20 130-41) systems with different time scales, in order to test the effects of fast and slow modes of growth on the dynamical vectors. A fast ‘extratropical atmosphere’ is weakly coupled to a fast ‘tropical atmosphere’ which is, in turn, strongly coupled to a slow ‘ocean’ system, the latter coupling imitating the

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

    Science.gov (United States)

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

    2018-02-09

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

  9. Relativistic gravitation from massless systems of scalar and vector fields

    International Nuclear Information System (INIS)

    Fonseca Teixeira, A.F. da.

    1979-01-01

    Under the laws of Einstein's gravitational theory, a massless system consisting of the diffuse sources of two fields is discussed. One fields is scalar, of long range, the other is a vector field of short range. A proportionality between the sources is assumed. Both fields are minimally coupled to gravitation, and contribute positive definitely to the time component of the energy momentum tensor. A class of static, spherically symmetric solutions of the equations is obtained, in the weak field limit. The solutions are regular everywhere, stable, and can represent large or small physical systems. The gravitational field presents a Schwarzschild-type asymptotic behavior. The dependence of the energy on the various parameters characterizing the system is discussed in some detail. (Author) [pt

  10. Ultrasound Vector Flow Imaging: Part I: Sequential Systems

    DEFF Research Database (Denmark)

    Jensen, Jørgen Arendt; Nikolov, Svetoslav Ivanov; Yu, Alfred C. H.

    2016-01-01

    The paper gives a review of the most important methods for blood velocity vector flow imaging (VFI) for conventional, sequential data acquisition. This includes multibeam methods, speckle tracking, transverse oscillation, color flow mapping derived vector flow imaging, directional beamforming...

  11. Algevir: An Expression System for Microalgae Based on Viral Vectors

    Science.gov (United States)

    Bañuelos-Hernández, Bernardo; Monreal-Escalante, Elizabeth; González-Ortega, Omar; Angulo, Carlos; Rosales-Mendoza, Sergio

    2017-01-01

    The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins. PMID:28713333

  12. Algevir: An Expression System for Microalgae Based on Viral Vectors

    Directory of Open Access Journals (Sweden)

    Bernardo Bañuelos-Hernández

    2017-06-01

    Full Text Available The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin, which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture, which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

  13. Shuttle vector system for Methanococcus maripaludis with improved transformation efficiency.

    Science.gov (United States)

    Walters, Alison D; Smith, Sarah E; Chong, James P J

    2011-04-01

    We have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors in Methanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into the M. maripaludis genome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.

  14. Vectorization, parallelization and porting of nuclear codes on the VPP500 system (vectorization). Progress report fiscal 1997

    International Nuclear Information System (INIS)

    Kawasaki, Nobuo; Ogasawara, Shinobu; Adachi, Masaaki; Kume, Etsuo; Ishizuki, Shigeru; Tanabe, Hidenobu; Nemoto, Toshiyuki; Kawai, Wataru; Watanabe, Hideo

    1999-05-01

    Several computer codes in the nuclear field have been vectorized, parallelized and transported on the FUJITSU VPP500 system and/or the AP3000 system at Center for Promotion of Computational Science and Engineering in Japan Atomic Energy Research Institute. We dealt with 14 codes in fiscal 1997. These results are reported in 3 parts, i.e., the vectorization part, the parallelization part and the porting part. In this report, we describe the vectorization. In this vectorization part, the vectorization of multidimensional two-fluid model code ACE-3D for evaluation of constitutive equations, statistical decay code SD and three-dimensional thermal analysis code for in-core test section (T2) of HENDEL SSPHEAT are described. In the parallelization part, the parallelization of cylindrical direct numerical simulation code CYLDNS44N, worldwide version of system for prediction of environmental emergency dose information code WSPEEDI, extension of quantum molecular dynamics code EQMD and three-dimensional non-steady compressible fluid dynamics code STREAM are described. In the porting part, the porting of transient reactor analysis code TRAC-BF1 and Monte Carlo radiation transport code MCNP4A on the AP3000 are described. In addition, a modification of program libraries for command-driven interactive data analysis plotting program IPLOT is described. (author)

  15. Design and Modeling of a Novel Torque Vectoring Differential System

    Directory of Open Access Journals (Sweden)

    Chen Yu-Fan

    2017-01-01

    Full Text Available In this paper, a new concept torque vectoring differential (TVD system is presented. It is shown that the structure and the mechanism of the system, the operating methods, and the parameters design by a simulation program, i.e. SimulationX. First of all, the structure of the new TVD system is introduced, as well as the relevant mechanic equations. Second, we attempt to verify the feasibility and accuracy of SimulationX through establishing a simple mechanical model by MATLAB, so that the further modeling and simulation results of the new TVD system will be credible. Then, the simulation results at the setting conditions are presented. Finally, the sensitivity of the design parameters is analyzed, including adjusting the braking torque and the dimensions of the gear sets in the differential. According to these results, the characteristics of the new TVD system can be derived in order to develop the whole system with vehicle dynamic model in the next stage.

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

    Science.gov (United States)

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

    2016-04-20

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

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

    Directory of Open Access Journals (Sweden)

    Heather M Marshall

    2007-12-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  19. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  20. Upport vector machines for nonlinear kernel ARMA system identification.

    Science.gov (United States)

    Martínez-Ramón, Manel; Rojo-Alvarez, José Luis; Camps-Valls, Gustavo; Muñioz-Marí, Jordi; Navia-Vázquez, Angel; Soria-Olivas, Emilio; Figueiras-Vidal, Aníbal R

    2006-11-01

    Nonlinear system identification based on support vector machines (SVM) has been usually addressed by means of the standard SVM regression (SVR), which can be seen as an implicit nonlinear autoregressive and moving average (ARMA) model in some reproducing kernel Hilbert space (RKHS). The proposal of this letter is twofold. First, the explicit consideration of an ARMA model in an RKHS (SVM-ARMA2K) is proposed. We show that stating the ARMA equations in an RKHS leads to solving the regularized normal equations in that RKHS, in terms of the autocorrelation and cross correlation of the (nonlinearly) transformed input and output discrete time processes. Second, a general class of SVM-based system identification nonlinear models is presented, based on the use of composite Mercer's kernels. This general class can improve model flexibility by emphasizing the input-output cross information (SVM-ARMA4K), which leads to straightforward and natural combinations of implicit and explicit ARMA models (SVR-ARMA2K and SVR-ARMA4K). Capabilities of these different SVM-based system identification schemes are illustrated with two benchmark problems.

  1. An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

    Science.gov (United States)

    Anand, Ajith; Bass, Steven H; Wu, Emily; Wang, Ning; McBride, Kevin E; Annaluru, Narayana; Miller, Michael; Hua, Mo; Jones, Todd J

    2018-04-23

    A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.

  2. A Semisupervised Support Vector Machines Algorithm for BCI Systems

    Science.gov (United States)

    Qin, Jianzhao; Li, Yuanqing; Sun, Wei

    2007-01-01

    As an emerging technology, brain-computer interfaces (BCIs) bring us new communication interfaces which translate brain activities into control signals for devices like computers, robots, and so forth. In this study, we propose a semisupervised support vector machine (SVM) algorithm for brain-computer interface (BCI) systems, aiming at reducing the time-consuming training process. In this algorithm, we apply a semisupervised SVM for translating the features extracted from the electrical recordings of brain into control signals. This SVM classifier is built from a small labeled data set and a large unlabeled data set. Meanwhile, to reduce the time for training semisupervised SVM, we propose a batch-mode incremental learning method, which can also be easily applied to the online BCI systems. Additionally, it is suggested in many studies that common spatial pattern (CSP) is very effective in discriminating two different brain states. However, CSP needs a sufficient labeled data set. In order to overcome the drawback of CSP, we suggest a two-stage feature extraction method for the semisupervised learning algorithm. We apply our algorithm to two BCI experimental data sets. The offline data analysis results demonstrate the effectiveness of our algorithm. PMID:18368141

  3. A Semisupervised Support Vector Machines Algorithm for BCI Systems

    Directory of Open Access Journals (Sweden)

    Jianzhao Qin

    2007-07-01

    Full Text Available As an emerging technology, brain-computer interfaces (BCIs bring us new communication interfaces which translate brain activities into control signals for devices like computers, robots, and so forth. In this study, we propose a semisupervised support vector machine (SVM algorithm for brain-computer interface (BCI systems, aiming at reducing the time-consuming training process. In this algorithm, we apply a semisupervised SVM for translating the features extracted from the electrical recordings of brain into control signals. This SVM classifier is built from a small labeled data set and a large unlabeled data set. Meanwhile, to reduce the time for training semisupervised SVM, we propose a batch-mode incremental learning method, which can also be easily applied to the online BCI systems. Additionally, it is suggested in many studies that common spatial pattern (CSP is very effective in discriminating two different brain states. However, CSP needs a sufficient labeled data set. In order to overcome the drawback of CSP, we suggest a two-stage feature extraction method for the semisupervised learning algorithm. We apply our algorithm to two BCI experimental data sets. The offline data analysis results demonstrate the effectiveness of our algorithm.

  4. Some vector sequence transformations with applications to systems of equations

    Science.gov (United States)

    Brezinski, C.; Sadok, H.

    1992-12-01

    First, recursive algorithms for implementing some vector sequence transformations are given. In a particular case, these transformations are generalizations of Shanks transformation and the G-transformation. When the sequence of vectors under transformation is generated by linear fixed point iterations, Lanczos' method and the CGS are recovered respectively. In the case of a sequence generated by nonlinear fixed point iterations, a quadratically convergent method based on the ɛ-algorithm is recovered and a nonlinear analog of the CGS method is obtained.

  5. Vector spot compensation applied in a CNC system for laser cutting machine

    Science.gov (United States)

    Xie, Qiong; Li, Shimin; Xu, Zhene; Wu, Heqing; Yin, Quan

    1999-09-01

    The vector spot compensation (VSP) is discussed in CNC system for laser cutting machine. The accuracy of laser cutting will be improved by VSP. The modularized vector algorithm which is different from that of traditional geometry algorithm not only unites the symbol system and simplifies the programming complexity, but also improves the spot compensation speed and offers the essential data for the spline interpolation.

  6. Envelope determinants of equine lentiviral vaccine protection.

    Directory of Open Access Journals (Sweden)

    Jodi K Craigo

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

  7. Envelope Determinants of Equine Lentiviral Vaccine Protection

    Science.gov (United States)

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

    2013-01-01

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

  8. Antigen delivery systems for veterinary vaccine development. Viral-vector based delivery systems.

    Science.gov (United States)

    Brun, Alejandro; Albina, Emmanuel; Barret, Tom; Chapman, David A G; Czub, Markus; Dixon, Linda K; Keil, Günther M; Klonjkowski, Bernard; Le Potier, Marie-Frédérique; Libeau, Geneviève; Ortego, Javier; Richardson, Jennifer; Takamatsu, Haru-H

    2008-12-02

    The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems (ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.

  9. Energy Systems in the Era of Energy Vectors A Key to Define, Analyze and Design Energy Systems Beyond Fossil Fuels

    CERN Document Server

    Orecchini, Fabio

    2012-01-01

    What lies beyond the era of fossil fuels? While most answers focus on different primary energy resources, Energy Systems in the Era of Energy Vectors provides a completely new approach. Instead of providing a traditional consumption analysis of classical primary energy resources such as oil, coal, nuclear power and gas, Energy Systems in the Era of Energy Vectors describes and assesses energy technologies, markets and future strategies, focusing on their capacity to produce, exchange, and use energy vectors. Special attention is given to the renewable energy resources available in different areas of the world and made exploitable by the integration of energy vectors in the global energy system. Clear definitions of energy vectors and energy systems are used as the basis for a complete explanation and assessment of up-to-date, available technologies for energy resources, transport and storage systems, conversion and use. The energy vectors scheme allows the potential realisation of a worldwide sustainable ener...

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

    Science.gov (United States)

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

    2015-06-05

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Rahmberg Andrew R

    2011-05-01

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

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

    Science.gov (United States)

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

    2014-04-24

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

  14. An Oncotropic Adenovirus Vector System for Breast Cancer Treatment

    National Research Council Canada - National Science Library

    Dmitriev, Igor P

    2004-01-01

    ..., as compared to the control Ad5F4ls vector. These results suggest that generated Ad5F4ls6H indeed lacks CAR-mediated tropism while is able to utilize fiber-incorporated His tag for cell binding...

  15. Iterative linear system solvers with approximate matrix-vector products

    NARCIS (Netherlands)

    Eshof, J. van den; Sleijpen, G.L.G.; Gijzen, M.B. van

    2003-01-01

    There are classes of linear problems for which a matrix-vector product is a time consuming operation because an expensive approximation method is required to compute it to a given accuracy. One important example is simulations in lattice QCD with Neuberger fermions where a matrix multiply

  16. Biosensor method and system based on feature vector extraction

    Science.gov (United States)

    Greenbaum, Elias [Knoxville, TN; Rodriguez, Jr., Miguel; Qi, Hairong [Knoxville, TN; Wang, Xiaoling [San Jose, CA

    2012-04-17

    A method of biosensor-based detection of toxins comprises the steps of providing at least one time-dependent control signal generated by a biosensor in a gas or liquid medium, and obtaining a time-dependent biosensor signal from the biosensor in the gas or liquid medium to be monitored or analyzed for the presence of one or more toxins selected from chemical, biological or radiological agents. The time-dependent biosensor signal is processed to obtain a plurality of feature vectors using at least one of amplitude statistics and a time-frequency analysis. At least one parameter relating to toxicity of the gas or liquid medium is then determined from the feature vectors based on reference to the control signal.

  17. Test of nucleon-nucleon interaction by p vector-d vector polarization transfer in the three-nucleon system

    International Nuclear Information System (INIS)

    Sperisen, F.; Gruebler, W.; Koenig, V.; Schmelzbach, P.A.; Elsener, K.; Jenny, B.; Schweizer, C.; Ulbricht, J.; Doleschall, P.

    1982-01-01

    Measurements of three vector-to-vector and seven vector-to-tensor polarization transfer coefficients of the 2 H(p vector, d vector) 1 H elastic scattering at Esub(p) = 10 MeV are reported in the angular range between THETAsub(cm) = 92 0 and 180 0 . These second-order observables are compared with Faddeev calculations. It is shown that the vector-to-tensor components depend largely on the details of the N-N P-wave interaction. (orig.)

  18. Cyclophilin A interacts with diverse lentiviral capsids

    Directory of Open Access Journals (Sweden)

    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.

  19. Dynamic reduction of dimensions of a document vector in a document search and retrieval system

    Science.gov (United States)

    Jiao, Yu; Potok, Thomas E.

    2011-05-03

    The method and system of the invention involves processing each new document (20) coming into the system into a document vector (16), and creating a document vector with reduced dimensionality (17) for comparison with the data model (15) without recomputing the data model (15). These operations are carried out by a first computer (11) while a second computer (12) updates the data model (18), which can be comprised of an initial large group of documents (19) and is premised on the computing an initial data model (13, 14, 15) to provide a reference point for determining document vectors from documents processed from the data stream (20).

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  2. Vector perturbation based adaptive distributed precoding scheme with limited feedback for CoMP systems

    Directory of Open Access Journals (Sweden)

    Cuthbert Laurie

    2011-01-01

    Full Text Available Abstract A downlink adaptive distributed precoding scheme is proposed for coordinated multi-point (CoMP transmission systems. The serving base station (BS obtains the optimal precoding vector via user feedback. Meanwhile, the precoding vector of each coordinated BS is determined by adaptive gradient iteration according to the perturbation vector and the adjustment factor based on the vector perturbation method. In each transmission frame, the CoMP user feeds the precoding matrix index back to the serving BS, and feeds back the adjustment factor index to the coordinated BSs, which can reduce the uplink feedback overhead. The selected adjustment factor for each coordinated BS is obtained via the precoding vector of the coordinated BS used in the previous frame and the preferred precoding vector of the serving BS in this frame. The proposed scheme takes advantage of the spatial non-correlation and temporal correlation of the distributed MIMO channel. The design of the adjustment factor set is given and the channel feedback delay is considered. The system performance of the proposed scheme is verified with and without feedback delay respectively and the system feedback overhead is analyzed. Simulation results show that the proposed scheme has a good trade-off between system performance and the system control information overhead on feedback.

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

    Science.gov (United States)

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

    2007-01-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  5. An introduction to vectors, vector operators and vector analysis

    CERN Document Server

    Joag, Pramod S

    2016-01-01

    Ideal for undergraduate and graduate students of science and engineering, this book covers fundamental concepts of vectors and their applications in a single volume. The first unit deals with basic formulation, both conceptual and theoretical. It discusses applications of algebraic operations, Levi-Civita notation, and curvilinear coordinate systems like spherical polar and parabolic systems and structures, and analytical geometry of curves and surfaces. The second unit delves into the algebra of operators and their types and also explains the equivalence between the algebra of vector operators and the algebra of matrices. Formulation of eigen vectors and eigen values of a linear vector operator are elaborated using vector algebra. The third unit deals with vector analysis, discussing vector valued functions of a scalar variable and functions of vector argument (both scalar valued and vector valued), thus covering both the scalar vector fields and vector integration.

  6. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

    2002-01-01

    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  7. Strategic vectors of transformational shifts in the national tourism system of Ukraine

    Directory of Open Access Journals (Sweden)

    Alla OKHRIMENKO

    2017-10-01

    Full Text Available The article determines transformational factors, which influence a national tourism system (NTS of Ukraine and proposes strategical vectors of its development. Research of the NTS as an economic system is a pre-condition for formation of strategic vectors of development. Transformational driving forces principally change scales, components, and proportions between external and internal factors of development of the NTS. Correspondingly, the mentioned processes objectively encourage modernization of the national tourism system and application of innovative managerial methods. The following Strategical vectors of transformational shifts in the NTS were grounded: 1 Safety of tourists and investors; 2 The normative and legislative framework of the NTS development; 3 Development of infrastructure of the NTS component; 4 Human resources development; 5 A marketing policy of the NTS promotion; 6 Ecological and cultural policies. Their implementation will improve efficiency and competitiveness of the NTS and the national economy.

  8. VECTor: A Preclinical Imaging System for Simultaneous Submillimeter SPECT and PET

    NARCIS (Netherlands)

    Goorden, M.C.; van der Have, F.; Kreuger, R.; Ramakers, R.M.; Vastenhouw, B.; Burbach, J.P.H.; Booij, J.; Molthoff, C.F.M.; Beekman, F.J.

    2013-01-01

    Today, PET and SPECT tracers cannot be imaged simultaneously at high resolutions but require 2 separate imaging systems. This paper introduces a Versatile Emission Computed Tomography system (VECTor) for radionuclides that enables simultaneous submillimeter imaging of single-photon and positron-

  9. Small-Scale Production of Recombinant Proteins Using the Baculovirus Expression Vector System.

    Science.gov (United States)

    Yang, Jian-Ping

    2016-01-01

    Numerous technological improvements, including progress in vector design, simplification of virus isolation techniques, and advancements in molecular biology and cell culture technologies, have greatly facilitated the use of the baculovirus-insect cell system for routine production of recombinant proteins. This chapter outlines the basic techniques for small-scale protein production using the Baculovirus Expression Vector System (BEVS), including protocols for titer estimation in 96-well plates, expression optimization in 24-well plates, and recombinant protein expression from adherent and suspension cultures in six-well plates and in 50 mL insect cell cultures.

  10. Axial-vector gluons and the fine structure of heavy quark--antiquark systems

    International Nuclear Information System (INIS)

    Feinberg, G.; Lynn, B.; Sucher, J.

    1979-01-01

    We point out that two models of the origin of spin-dependent forces in heavy quark systems make very different predictions about the relative size of these forces in c-barc and b-barb. The model in which these forces are relativistic corrections to vector or scalar gluon exchange predicts smaller spin-dependent effects in b-barb than in c-barc while a model in which these forces are due to exchange of axial-vector gluons predicts a similar size for spin-dependent splittings in the two systems

  11. Developmental Testing of Electric Thrust Vector Control Systems for Manned Launch Vehicle Applications

    Science.gov (United States)

    Bates, Lisa B.; Young, David T.

    2012-01-01

    This paper describes recent developmental testing to verify the integration of a developmental electromechanical actuator (EMA) with high rate lithium ion batteries and a cross platform extensible controller. Testing was performed at the Thrust Vector Control Research, Development and Qualification Laboratory at the NASA George C. Marshall Space Flight Center. Electric Thrust Vector Control (ETVC) systems like the EMA may significantly reduce recurring launch costs and complexity compared to heritage systems. Electric actuator mechanisms and control requirements across dissimilar platforms are also discussed with a focus on the similarities leveraged and differences overcome by the cross platform extensible common controller architecture.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-18

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

  13. Wearable Vector Electrical Bioimpedance System to Assess Knee Joint Health.

    Science.gov (United States)

    Hersek, Sinan; Toreyin, Hakan; Teague, Caitlin N; Millard-Stafford, Mindy L; Jeong, Hyeon-Ki; Bavare, Miheer M; Wolkoff, Paul; Sawka, Michael N; Inan, Omer T

    2017-10-01

    We designed and validated a portable electrical bioimpedance (EBI) system to quantify knee joint health. Five separate experiments were performed to demonstrate the: 1) ability of the EBI system to assess knee injury and recovery; 2) interday variability of knee EBI measurements; 3) sensitivity of the system to small changes in interstitial fluid volume; 4) reducing the error of EBI measurements using acceleration signals; and 5) use of the system with dry electrodes integrated to a wearable knee wrap. 1) The absolute difference in resistance ( R) and reactance (X) from the left to the right knee was able to distinguish injured and healthy knees (p knee R was 2.5 Ω and for X was 1.2 Ω. 3) Local heating/cooling resulted in a significant decrease/increase in knee R (p knee R and X measured using the wet electrodes and the designed wearable knee wrap were highly correlated ( R 2 = 0.8 and 0.9, respectively). This study demonstrates the use of wearable EBI measurements in monitoring knee joint health. The proposed wearable system has the potential for assessing knee joint health outside the clinic/lab and help guide rehabilitation.

  14. Ultrasound Vector Flow Imaging: Part II: Parallel Systems

    DEFF Research Database (Denmark)

    Jensen, Jørgen Arendt; Nikolov, Svetoslav Ivanov; Yu, Alfred C. H.

    2016-01-01

    The paper gives a review of the current state-of-theart in ultrasound parallel acquisition systems for flow imaging using spherical and plane waves emissions. The imaging methods are explained along with the advantages of using these very fast and sensitive velocity estimators. These experimental...... ultrasound imaging for studying brain function in animals. The paper explains the underlying acquisition and estimation methods for fast 2-D and 3-D velocity imaging and gives a number of examples. Future challenges and the potentials of parallel acquisition systems for flow imaging are also discussed....

  15. A new method for distortion magnetic field compensation of a geomagnetic vector measurement system

    International Nuclear Information System (INIS)

    Liu, Zhongyan; Pan, Mengchun; Tang, Ying; Zhang, Qi; Geng, Yunling; Wan, Chengbiao; Chen, Dixiang; Tian, Wugang

    2016-01-01

    The geomagnetic vector measurement system mainly consists of three-axis magnetometer and an INS (inertial navigation system), which have many ferromagnetic parts on them. The magnetometer is always distorted by ferromagnetic parts and other electric equipments such as INS and power circuit module within the system, which can lead to geomagnetic vector measurement error of thousands of nT. Thus, the geomagnetic vector measurement system has to be compensated in order to guarantee the measurement accuracy. In this paper, a new distortion magnetic field compensation method is proposed, in which a permanent magnet with different relative positions is used to change the ambient magnetic field to construct equations of the error model parameters, and the parameters can be accurately estimated by solving linear equations. In order to verify effectiveness of the proposed method, the experiment is conducted, and the results demonstrate that, after compensation, the components errors of measured geomagnetic field are reduced significantly. It demonstrates that the proposed method can effectively improve the accuracy of the geomagnetic vector measurement system. (paper)

  16. Combinatorial Vector Fields for Piecewise Affine Control Systems

    DEFF Research Database (Denmark)

    Wisniewski, Rafal; Larsen, Jesper Abildgaard

    2008-01-01

    This paper is intended to be a continuation of Habets and van Schuppen (2004) and Habets, Collins and van Schuppen (2006), which address the control problem for piecewise-affine systems on an arbitrary polytope or a family of these. Our work deals with the underlying combinatorics of the underlyi...

  17. MODELING OF DYNAMIC SYSTEMS WITH MODULATION BY MEANS OF KRONECKER VECTOR-MATRIX REPRESENTATION

    Directory of Open Access Journals (Sweden)

    A. S. Vasilyev

    2015-09-01

    Full Text Available The paper deals with modeling of dynamic systems with modulation by the possibilities of state-space method. This method, being the basis of modern control theory, is based on the possibilities of vector-matrix formalism of linear algebra and helps to solve various problems of technical control of continuous and discrete nature invariant with respect to the dimension of their “input-output” objects. Unfortunately, it turned its back on the wide group of control systems, which hardware environment modulates signals. The marked system deficiency is partially offset by this paper, which proposes Kronecker vector-matrix representations for purposes of system representation of processes with signal modulation. The main result is vector-matrix representation of processes with modulation with no formal difference from continuous systems. It has been found that abilities of these representations could be effectively used in research of systems with modulation. Obtained model representations of processes with modulation are best adapted to the state-space method. These approaches for counting eigenvalues of Kronecker matrix summaries, that are matrix basis of model representations of processes described by Kronecker vector products, give the possibility to use modal direction in research of dynamics for systems with modulation. It is shown that the use of controllability for eigenvalues of general matrixes applied to Kronecker structures enabled to divide successfully eigenvalue spectrum into directed and not directed components. Obtained findings including design problems for models of dynamic processes with modulation based on the features of Kronecker vector and matrix structures, invariant with respect to the dimension of input-output relations, are applicable in the development of alternate current servo drives.

  18. A 35 GHz vector system for non destructive applications

    Science.gov (United States)

    Glay, D.; Lasri, T.; Mamouni, A.; Leroy, Y.

    2000-05-01

    It has been demonstrated that microwave techniques are well suited for nondestructive testing (NDT) of materials. The challenge now is to propose systems that can compete, in terms of reliability, precision and fabrication cost, with those already existing. Microwaves are expected to play a fundamental role if we are able to develop both methods and systems to fulfill the increasing demand of characterization in this dynamic field. We have developed devices operating at 2.45 and 10 GHz that have been used successfully for layer thickness and material parameters (permittivity, moisture,…) measurement. In order to tackle other problems met by the NDT community, that need to operate at higher frequencies we have conceived a sensor for the determination of the material under test reflection coefficient (magnitude and phase) at 35 GHz. This system conceived around a complex correlator (or IQ demodulator), made in microstrip technology, is used for the detection of flaws inside dielectric materials or on surface conductors. These investigations concern defects with various shapes, sizes, and dielectric properties.

  19. Usability Evaluation of an Augmented Reality System for Teaching Euclidean Vectors

    Science.gov (United States)

    Martin-Gonzalez, Anabel; Chi-Poot, Angel; Uc-Cetina, Victor

    2016-01-01

    Augmented reality (AR) is one of the emerging technologies that has demonstrated to be an efficient technological tool to enhance learning techniques. In this paper, we describe the development and evaluation of an AR system for teaching Euclidean vectors in physics and mathematics. The goal of this pedagogical tool is to facilitate user's…

  20. A classification system for one Killing vector solutions of Einstein's equations

    International Nuclear Information System (INIS)

    Hoenselaers, C.

    1978-01-01

    A double classification system for one Killing vector solutions in terms of the eigenvectors and eigenvalues of the Ricci and Bach tensor of the associated three manifold is proposed. The calculations of the Bach tensor are carried out for special cases. (author)

  1. Evaluation of systemic insecticides mixed in rodenticide baits for plague vector control

    DEFF Research Database (Denmark)

    Larsen, Kim Søholt; Lodal, Jens

    1997-01-01

    Rodenticide baits containing systemic insecticides were evaluated in the laboratory for their palatability to the house rat Rattus rattus and for their toxicity against the oriental rat flea Xenopsylla cheopis - both animals are important Vectors of plague in Africa. The test bait and a non...

  2. DSP-based vector controlled IM drive system with identification of secondary time constant

    Energy Technology Data Exchange (ETDEWEB)

    Iwasaki, Makoto; Matsui, Nobuyuki

    1988-09-20

    The development of the variable-speed driving technology for AC motors have put into practice the vector control of induction motors having the same high-speed torque response as that of DC motors. Several control systems are available but each of them has its own problems. A secondary time constant identification function vector control system has been developed in this background. It consists of processing all vectors by using DSP (digital signal processor) on the basis of non-interacting vector control conditions derived from a voltage model of an induction motor. The prototype system consists mainly of TMS 32010 to realize a softwere version of a full control system composed of a current control unit, speed control unit, and secondary time constant control unit. Experiments using a 2.2 kW induction motor have revealed a control precision of less than 3% at any torque up to rated torque. The operation time is 135 microseconds. (14 figs, 1 tab, 12 refs)

  3. A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram

    The development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engineering in mammalian cells, a n...... versatile expression vector system was developed. This vector system applies the ligation-free uracilexcision cloning technique to construct mammalian expression vectors of multiple parts and with maximum flexibility....

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

    Directory of Open Access Journals (Sweden)

    Xiaomei Wang

    2017-06-01

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

  5. Tractional Electric Drive with Non-Sensing Element Vector Control System

    Directory of Open Access Journals (Sweden)

    O. F. Opeyko

    2010-01-01

    Full Text Available The purpose of the paper is a structure formation and an analysis of non-sensing element vector control system developed for tractional electric drive with the help of mathematical simulation method. The paper presents a functional diagram of the electric drive with non-sensing element vector control system  operated by an asynchronous short-circuited electric motor.  Main expressions used for evaluation of variables of system conditions and parameters are cited in the paper. The paper provides results of mathematical simulation method for electric drive system taking into consideration various parameter values which confirm serviceability of the developed control system within the whole range of possible parameter chnges.

  6. Fault Identification in an Unbalanced Distribution System Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Sophi Shilpa Gururajapathy

    2016-12-01

    Full Text Available Fast and effective fault location in distribution system is important to improve the power system reliability. Most of the researches rarely mention about effective fault location consisting of faulted phase, fault type, faulty section and fault distance identification. This work presents a method using support vector machine to identify the faulted phase, fault type, faulty section and distance at the same time. Support vector classification and regression analysis are performed to locate fault. The method uses the voltage sag data during fault condition measured at the primary substation. The faulted phase and the fault type are identified using three-dimensional support vector classification. The possible faulty sections are identified by matching voltage sag at fault condition to the voltage sag in database and the possible sections are ranked using shortest distance principle. The fault distance for the possible faulty sections isthen identified using support vector regression analysis. The performance of the proposed method was tested on an unbalanced distribution system from SaskPower, Canada. The results show that the accuracy of the proposed method is satisfactory.

  7. Improved pFastBac™ donor plasmid vectors for higher protein production using the Bac-to-Bac® baculovirus expression vector system.

    Science.gov (United States)

    Shang, Hui; Garretson, Tyler A; Kumar, C M Senthil; Dieter, Robert F; Cheng, Xiao-Wen

    2017-08-10

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-based Bac-to-Bac ® expression system consists of a bacmid and five pFastBac™ donor transfer vectors. It has been widely used for eukaryotic gene expression in insect cells to elucidate gene function in biotechnology laboratories. The pFastBac™ vectors contain a 50bp AcMNPV polyhedrin (polh) promoter and a 127bp SV40 polyadenylation (pA) signal for cloning a gene of interest into the bacmid, resulting in unsolved lower gene expression levels than the wild type (wt) AcMNPV in insect cells. Therefore, the purpose of this research is to understand why the Bac-to-Bac system produces lower gene expression levels. Here, we determined that bacmids transposed with pFastBac™ vectors produced 3-4 fold lower levels of certain proteins than the wt AcMNPV. We found that an 80bp cis element 147bp upstream of the 50bp polh promoter and a 134bp polh pA signal are required in pFastBac™ to achieve bacmid protein expression levels equivalent to wt AcMNPV in High Five insect cells. Therefore, researchers currently using pFastBac™ vectors for protein expression can transfer their genes of interest into the improved vectors in this report to elevate protein expression yields in insect cells to reduce protein production costs. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Modeling the control of the central nervous system over the cardiovascular system using support vector machines.

    Science.gov (United States)

    Díaz, José; Acosta, Jesús; González, Rafael; Cota, Juan; Sifuentes, Ernesto; Nebot, Àngela

    2018-02-01

    The control of the central nervous system (CNS) over the cardiovascular system (CS) has been modeled using different techniques, such as fuzzy inductive reasoning, genetic fuzzy systems, neural networks, and nonlinear autoregressive techniques; the results obtained so far have been significant, but not solid enough to describe the control response of the CNS over the CS. In this research, support vector machines (SVMs) are used to predict the response of a branch of the CNS, specifically, the one that controls an important part of the cardiovascular system. To do this, five models are developed to emulate the output response of five controllers for the same input signal, the carotid sinus blood pressure (CSBP). These controllers regulate parameters such as heart rate, myocardial contractility, peripheral and coronary resistance, and venous tone. The models are trained using a known set of input-output response in each controller; also, there is a set of six input-output signals for testing each proposed model. The input signals are processed using an all-pass filter, and the accuracy performance of the control models is evaluated using the percentage value of the normalized mean square error (MSE). Experimental results reveal that SVM models achieve a better estimation of the dynamical behavior of the CNS control compared to others modeling systems. The main results obtained show that the best case is for the peripheral resistance controller, with a MSE of 1.20e-4%, while the worst case is for the heart rate controller, with a MSE of 1.80e-3%. These novel models show a great reliability in fitting the output response of the CNS which can be used as an input to the hemodynamic system models in order to predict the behavior of the heart and blood vessels in response to blood pressure variations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Global Positioning Systems (GPS) Technology to Study Vector-Pathogen-Host Interactions

    Science.gov (United States)

    2016-12-01

    Award Number: W81XWH-11-2-0175 TITLE: Global Positioning Systems (GPS) Technology to Study Vector-Pathogen-Host Interactions PRINCIPAL...sophisticated Global Positioning Systems (GPS) technology of isolated viruses and genetic characterization, spatial and temporal analysis are being...REPORT DATE December 2016 2. REPORT TYPE Final 3. DATES COVERED 27 Sept 2011 - 26 Sept 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Global

  10. Deformation vector measurement by means of ground based interferometric radar system

    Science.gov (United States)

    Michelini, Alberto; Coppi, Francesco

    2017-10-01

    Ground Based Interferometric Radar (GBInRad) is a class of terrestrial remote sensing imaging system, based on microwave interferometric techniques. The principal application of GBInRad system is deformation monitoring, since respect to other techniques they can provide remote sensing, high sensitivity to small deformations, long range of measurements, imaging capability and fast scan time. The main limitation of standard GBInRad system is their capability of detecting movements only along the Line of Sight (LoS) of the sensor, although actual targets may show deformations in any direction of space; this represents an important limitation with respect to other techniques able to estimate the full 3D deformation vector. If the displacement direction is not known a priori, combining together LoS displacement measured from different spatial positions, it is possible to reconstruct the actual 3D displacement vector of monitored targets. In this paper are introduced and analysed the various aspect of the displacement vector measurement with multiple GBInRad system that work both in a monostatic and in a bistatic configuration. In the monostatic configuration every system transmits and receives the signal independently from the others; this approach requires multiple GBInRad system deployed to monitoring the same scenario and therefore its main limitations lie in the costs, power consumption and maintenance. A possible cost-effective evolution of the monostatic configuration is to exploit GBInRad system in a multiple bistatic configuration; a multiple bistatic Radar is a system in which a transmitter operates together with multiple receivers located in different positions in space. In this paper, the deformation vector measurement by means of bistatic GBInRad is proposed.

  11. A Fiber-Optic Borehole Seismic Vector Sensor System for Geothermal Site Characterization and Monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Paulsson, Bjorn N.P. [Paulsson, Inc., Van Nuys, CA (United States); Thornburg, Jon A. [Paulsson, Inc., Van Nuys, CA (United States); He, Ruiqing [Paulsson, Inc., Van Nuys, CA (United States)

    2015-04-21

    Seismic techniques are the dominant geophysical techniques for the characterization of subsurface structures and stratigraphy. The seismic techniques also dominate the monitoring and mapping of reservoir injection and production processes. Borehole seismology, of all the seismic techniques, despite its current shortcomings, has been shown to provide the highest resolution characterization and most precise monitoring results because it generates higher signal to noise ratio and higher frequency data than surface seismic techniques. The operational environments for borehole seismic instruments are however much more demanding than for surface seismic instruments making both the instruments and the installation much more expensive. The current state-of-the-art borehole seismic instruments have not been robust enough for long term monitoring compounding the problems with expensive instruments and installations. Furthermore, they have also not been able to record the large bandwidth data available in boreholes or having the sensitivity allowing them to record small high frequency micro seismic events with high vector fidelity. To reliably achieve high resolution characterization and long term monitoring of Enhanced Geothermal Systems (EGS) sites a new generation of borehole seismic instruments must therefore be developed and deployed. To address the critical site characterization and monitoring needs for EGS programs, US Department of Energy (DOE) funded Paulsson, Inc. in 2010 to develop a fiber optic based ultra-large bandwidth clamped borehole seismic vector array capable of deploying up to one thousand 3C sensor pods suitable for deployment into ultra-high temperature and high pressure boreholes. Tests of the fiber optic seismic vector sensors developed on the DOE funding have shown that the new borehole seismic sensor technology is capable of generating outstanding high vector fidelity data with extremely large bandwidth: 0.01 – 6,000 Hz. Field tests have shown

  12. Multi-channel analog-to-digital converter in the VECTOR-CAMAC systems

    International Nuclear Information System (INIS)

    Borodyanskij, M.E.; Moravskij, E.I.

    1986-01-01

    A four-channel analog-to-digital converter (ADC) realized on microcircuits of high level of integration is described. Refinement of the result of conversion from one tact to another is ensured at the expense of introducing redemdancy of signal conversion at intermediate tacts. The ADC represents a unit of the Vector system: a controller is placed in the second unit. The ADS is compatibl with the Vector system and after negligible alternations it can be performed in the CAMAC system. Time of ADSsub(n) conversion in one channel is 5μs, output code is 14-digit, binary maximum value of integral nonlinearity <= 0.05% from the dynamic range of measured signals is +- 10V, capacity of the immediate memonry is 1024 14-digit words

  13. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9

    Directory of Open Access Journals (Sweden)

    Ming Yuan

    Full Text Available The current method for creation of vaccinia virus (VACV vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (∼90% in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.

  14. Investigation of Closed Vector Control System for Asynchronous Motor Drive of Shipboard Tow Winch

    Directory of Open Access Journals (Sweden)

    Н. A. Mehdiyev

    2012-01-01

    Full Text Available The paper considers a closed vector control system for induction motor rotation speed of a shipboard tow winch. Structural schemes and transfer functions of the system at control and disturbing influences are presented in the paper. The system with asynchronous motor drive parameters of a shipboard tow winch has been investigated in the paper. It has been revealed that transit process duration of the given system is insignificant. The fact is rather important to prevent accidents in case of vessel towing.

  15. Parameter identification based synchronization for a class of chaotic systems with offset vectors

    International Nuclear Information System (INIS)

    Chen Cailian; Feng Gang; Guan Xinping

    2004-01-01

    Based on a parameter identification scheme, a novel synchronization method is presented for a class of chaotic systems with offset vectors which can be represented by the so-called T-S fuzzy model. It is shown that the slave system can synchronize the master system and the unknown parameters of the master system can be identified simultaneously. The delayed feedback technique is also developed in order to reduce the energy and time required for the identification and synchronization. Numerical simulations demonstrate the effectiveness of the proposed method

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

    Science.gov (United States)

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

    2010-08-01

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

  17. Mapping of courses on vector biology and vector-borne diseases systems: time for a worldwide effort

    Directory of Open Access Journals (Sweden)

    Jérôme Casas

    Full Text Available Major emergency efforts are being mounted for each vector-borne disease epidemiological crisis anew, while knowledge about the biology of arthropods vectors is dwindling slowly but continuously, as is the number of field entomologists. The discrepancy between the rates of production of knowledge and its use and need for solving crises is widening, in particular due to the highly differing time spans of the two concurrent processes. A worldwide web based search using multiple key words and search engines of onsite and online courses in English, Spanish, Portuguese, French, Italian and German concerned with the biology of vectors identified over 140 courses. They are geographically and thematically scattered, the vast majority of them are on-site, with very few courses using the latest massive open online course (MOOC powerfulness. Over two third of them is given in English and Western Africa is particularity poorly represented. The taxonomic groups covered are highly unbalanced towards mosquitoes. A worldwide unique portal to guide students of all grades and levels of expertise, in particular those in remote locations, is badly needed. This is the objective a new activity supported by the Special Programme for Research and Training in Tropical Diseases (TDR.

  18. A simple vector system to improve performance and utilisation of recombinant antibodies

    Directory of Open Access Journals (Sweden)

    Vincent Karen J

    2006-12-01

    Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

  19. PREPARATION OF PLANT TRANSFORMATION VECTOR CONTAINING “SELF-EXCISION” CRE/LOXP SYSTEM

    Directory of Open Access Journals (Sweden)

    Jana Moravčíková

    2012-02-01

    Full Text Available This work is focused on preparation of the plant transformation vector pZP6 containing “self-excision” Cre/loxP system. The T-DNA of binary vector consists of the cre recombinase gene driven by the Arabidopsis DLL promoter and the nptII expression unit flanked by two loxP sites in direct orientation. The gus reporter gene controlled by the double CaMV 35S promoter was placed out of the loxP embedded DNA. To confirm functionality of the Cre/loxP system, the pZP6 was analyzed for correct removal of the loxP embedded sequence in E. coli. The pZP6 was transformed into two bacterial strains A. tumefaciens AGLO and LBA 4404. Its stability in agrobacteria was evaluated by restriction analyses.

  20. Product demand forecasts using wavelet kernel support vector machine and particle swarm optimization in manufacture system

    Science.gov (United States)

    Wu, Qi

    2010-03-01

    Demand forecasts play a crucial role in supply chain management. The future demand for a certain product is the basis for the respective replenishment systems. Aiming at demand series with small samples, seasonal character, nonlinearity, randomicity and fuzziness, the existing support vector kernel does not approach the random curve of the sales time series in the space (quadratic continuous integral space). In this paper, we present a hybrid intelligent system combining the wavelet kernel support vector machine and particle swarm optimization for demand forecasting. The results of application in car sale series forecasting show that the forecasting approach based on the hybrid PSOWv-SVM model is effective and feasible, the comparison between the method proposed in this paper and other ones is also given, which proves that this method is, for the discussed example, better than hybrid PSOv-SVM and other traditional methods.

  1. Prospects for Foamy Viral Vector Anti-HIV Gene Therapy

    Directory of Open Access Journals (Sweden)

    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.

  2. Performance monitoring for coherent DP-QPSK systems based on stokes vectors analysis

    Science.gov (United States)

    Louchet, Hadrien; Koltchanov, Igor; Richter, André

    2010-12-01

    We show how to estimate accurately the Jones matrix of the transmission line by analyzing the Stokes vectors of DP-QPSK signals. This method can be used to perform in-situ PMD measurement in dual-polarization QPSK systems, and in addition to the constant modulus algorithm (CMA) to mitigate polarization-induced impairments. The applicability of this method to other modulation formats is discussed.

  3. Adaptive vector quantization in SVD MIMO system backward link with limited number of active sub channels

    Directory of Open Access Journals (Sweden)

    Ivaniš Predrag

    2004-01-01

    Full Text Available This paper presents combination of Channel Optimized Vector Quantization based on LBG algorithm and sub channel power allocation for MIMO systems with Singular Value Decomposition and limited number of active sub channels. Proposed algorithm is designed to enable maximal throughput with bit error rate bellow some tar- get level in case of backward channel capacity limitation. Presence of errors effect in backward channel is also considered.

  4. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    Directory of Open Access Journals (Sweden)

    Javier López-Vidal

    Full Text Available Vaccines based on virus-like particles (VLPs have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60 were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

  5. Combination of grafted Schwann cells and lentiviral-mediated prevention of glial scar formation improve recovery of spinal cord injured rats.

    Science.gov (United States)

    Do-Thi, Anh; Perrin, Florence E; Desclaux, Mathieu; Saillour, Paulette; Amar, Lahouari; Privat, Alain; Mallet, Jacques

    2016-10-01

    The present study was intended to combine three therapeutic approaches in a well-defined rat model of spinal cord injury, a lateral hemisection at thoracic level. A guidance channel was implanted at the lesion site. This channel was seeded with native Schwann cells or Schwann cells that had been previously transduced with a lentiviral vector carrying the GDNF gene. Thereafter, these experiences were reproduced in animals injected with lentiviral vectors carrying a shRNA for GFAP (Lv-shGFAP), which has recently been shown to block glial scar formation. Functional evaluations showed that Lv-shGFAP induced a significant improvement in recovery in animals grafted with Schwann cells. Histological studies demonstrated the outgrowth of axons in the guidance channel containing Schwann cells transduced or not with GDNF. This axonal growth was enhanced in rats receiving Lv-shGFAP vector. Also, a significant increase of serotonergic innervation of the injured hemicord, distal to the lesion, was found only in animals treated with Lv-shGFAP vectors. Importantly, this study confirms that glial scar formation is a major impediment for axonal sprouting after spinal cord injury, and emphasizes the importance of serotonergic innervation for locomotor function. Moreover we show a significant additive effect of a combinatorial approach to axonal regeneration in the injured spinal cord. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Anticipatory Monitoring and Control of Complex Systems using a Fuzzy based Fusion of Support Vector Regressors

    Energy Technology Data Exchange (ETDEWEB)

    Miltiadis Alamaniotis; Vivek Agarwal

    2014-10-01

    This paper places itself in the realm of anticipatory systems and envisions monitoring and control methods being capable of making predictions over system critical parameters. Anticipatory systems allow intelligent control of complex systems by predicting their future state. In the current work, an intelligent model aimed at implementing anticipatory monitoring and control in energy industry is presented and tested. More particularly, a set of support vector regressors (SVRs) are trained using both historical and observed data. The trained SVRs are used to predict the future value of the system based on current operational system parameter. The predicted values are then inputted to a fuzzy logic based module where the values are fused to obtain a single value, i.e., final system output prediction. The methodology is tested on real turbine degradation datasets. The outcome of the approach presented in this paper highlights the superiority over single support vector regressors. In addition, it is shown that appropriate selection of fuzzy sets and fuzzy rules plays an important role in improving system performance.

  7. Modelling and Simulation of SVPWM Based Vector Controlled HVDC Light Systems

    Directory of Open Access Journals (Sweden)

    Ajay Kumar MOODADLA

    2012-11-01

    Full Text Available Recent upgrades in power electronics technology have lead to the improvements of insulated gate bipolar transistor (IGBT based Voltage source converter High voltage direct current (VSC HVDC transmission systems. These are also commercially known as HVDC Light systems, which are popular in renewable, micro grid, and electric power systems. Out of different pulse width modulation (PWM schemes, Space vector PWM (SVPWM control scheme finds growing importance in power system applications because of its better dc bus utilization. In this paper, modelling of the converter is described, and SVPWM scheme is utilized to control the HVDC Light system in order to achieve better DC bus utilization, harmonic reduction, and for reduced power fluctuations. The simulations are carried out in the MATLAB/SIMULINK environment and the results are provided for steady state and dynamic conditions. Finally, the performance of SVPWM based vector controlled HVDC Light transmission system is compared with sinusoidal pulse width modulation (SPWM based HVDC Light system in terms of output voltage and total harmonic distortion (THD.

  8. Real Time Monitoring System of Pollution Waste on Musi River Using Support Vector Machine (SVM) Method

    Science.gov (United States)

    Fachrurrozi, Muhammad; Saparudin; Erwin

    2017-04-01

    Real-time Monitoring and early detection system which measures the quality standard of waste in Musi River, Palembang, Indonesia is a system for determining air and water pollution level. This system was designed in order to create an integrated monitoring system and provide real time information that can be read. It is designed to measure acidity and water turbidity polluted by industrial waste, as well as to show and provide conditional data integrated in one system. This system consists of inputting and processing the data, and giving output based on processed data. Turbidity, substances, and pH sensor is used as a detector that produce analog electrical direct current voltage (DC). Early detection system works by determining the value of the ammonia threshold, acidity, and turbidity level of water in Musi River. The results is then presented based on the level group pollution by the Support Vector Machine classification method.

  9. TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells.

    Science.gov (United States)

    Mashko, S V; Veiko, V P; Lapidus, A L; Lebedeva, M I; Mochulsky, A V; Shechter, I I; Trukhan, M E; Ratmanova, K I; Rebentish, B A; Kaluzhsky, V E

    1990-03-30

    A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. 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.

  11. Immunogenetics of Small Ruminant Lentiviral Infections

    Directory of Open Access Journals (Sweden)

    Nancy Stonos

    2014-08-01

    Full Text Available The small ruminant lentiviruses (SRLV include the caprine arthritis encephalitis virus (CAEV and the Maedi-Visna virus (MVV. Both of these viruses limit production and can be a major source of economic loss to producers. Little is known about how the immune system recognizes and responds to SRLVs, but due to similarities with the human immunodeficiency virus (HIV, HIV research can shed light on the possible immune mechanisms that control or lead to disease progression. This review will focus on the host immune response to HIV-1 and SRLV, and will discuss the possibility of breeding for enhanced SRLV disease resistance.

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

    DEFF Research Database (Denmark)

    Maiorino, C; Khorooshi, R; Ruffini, F

    2013-01-01

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

  13. The genus Amycolatopsis: Indigenous plasmids, cloning vectors and gene transfer systems.

    Science.gov (United States)

    Malhotra, S; Lal, R

    2007-03-01

    The genus Amycolatopsis is a member of the phylogenetic group nocardioform actinomycetes. Most of the members of the genus Amycolatopsis are known to produce antibiotics. Additionally, members of this genus have been reported to metabolize aromatic compounds as the sole sources of carbon and energy. Development of genetic manipulation in Amycolatopsis has progressed slowly due to paucity of genetic tools and methods. The occurrence of indigenous plasmids in different species of Amycolatopsis is not very common. Till date, only three indigenous plasmids viz., pMEA100, pMEA300 and pA387 have been reported in Amycolatopsis species. Various vectors based on the indigenous plasmids, pMEA100, pMEA300 and pA387, have been constructed. These vectors have proved useful for molecular genetics studies of actinomycetes. Molecular genetic work with Amycolatopsis strains is not easy, since transformation methods have to be developed, or at least optimized, for each particular strain. Nonetheless, methods for efficient transformation (polyethyleneglycol (PEG) induced protoplast transformation, transformation by electroporation and direct transformation) have been developed and used successfully for the introduction of DNA into several Amycolatopsis species. The construction of plasmid cloning vectors and the development of gene transfer systems has opened up possibilities for studying the molecular genetics of these bacteria.

  14. Implementation of algorithms based on support vector machine (SVM for electric systems: topic review

    Directory of Open Access Journals (Sweden)

    Jefferson Jara Estupiñan

    2016-06-01

    Full Text Available Objective: To perform a review of implementation of algorithms based on support vectore machine applied to electric systems. Method: A paper search is done mainly on Biblio­graphic Indexes (BI and Bibliographic Bases with Selection Committee (BBSC about support vector machine. This work shows a qualitative and/or quan­titative description about advances and applications in the electrical environment, approaching topics such as: electrical market prediction, demand predic­tion, non-technical losses (theft, alternative energy source and transformers, among others, in each work the respective citation is done in order to guarantee the copy right and allow to the reader a dynamic mo­vement between the reading and the cited works. Results: A detailed review is done, focused on the searching of implemented algorithms in electric sys­tems and innovating application areas. Conclusion: Support vector machines have a lot of applications due to their multiple benefits, however in the electric energy area; they have not been tota­lly applied, this allow to identify a promising area of researching.

  15. Parameter Improved Particle Swarm Optimization Based Direct-Current Vector Control Strategy for Solar PV System

    Directory of Open Access Journals (Sweden)

    NAMMALVAR, P.

    2018-02-01

    Full Text Available This paper projects Parameter Improved Particle Swarm Optimization (PIPSO based direct current vector control technology for the integration of photovoltaic array in an AC micro-grid to enhance the system performance and stability. A photovoltaic system incorporated with AC micro-grid is taken as the pursuit of research study. The test system features two power converters namely, PV side converter which consists of DC-DC boost converter with Perturbation and Observe (P&O MPPT control to reap most extreme power from the PV array, and grid side converter which consists of Grid Side-Voltage Source Converter (GS-VSC with proposed direct current vector control strategy. The gain of the proposed controller is chosen from a set of three values obtained using apriori test and tuned through the PIPSO algorithm so that the Integral of Time multiplied Absolute Error (ITAE between the actual and the desired DC link capacitor voltage reaches a minimum and allows the system to extract maximum power from PV system, whereas the existing d-q control strategy is found to perform slowly to control the DC link voltage under varying solar insolation and load fluctuations. From simulation results, it is evident that the proposed optimal control technique provides robust control and improved efficiency.

  16. A static investigation of the thrust vectoring system of the F/A-18 high-alpha research vehicle

    Science.gov (United States)

    Mason, Mary L.; Capone, Francis J.; Asbury, Scott C.

    1992-01-01

    A static (wind-off) test was conducted in the static test facility of the Langley 16-foot Transonic Tunnel to evaluate the vectoring capability and isolated nozzle performance of the proposed thrust vectoring system of the F/A-18 high alpha research vehicle (HARV). The thrust vectoring system consisted of three asymmetrically spaced vanes installed externally on a single test nozzle. Two nozzle configurations were tested: A maximum afterburner-power nozzle and a military-power nozzle. Vane size and vane actuation geometry were investigated, and an extensive matrix of vane deflection angles was tested. The nozzle pressure ratios ranged from two to six. The results indicate that the three vane system can successfully generate multiaxis (pitch and yaw) thrust vectoring. However, large resultant vector angles incurred large thrust losses. Resultant vector angles were always lower than the vane deflection angles. The maximum thrust vectoring angles achieved for the military-power nozzle were larger than the angles achieved for the maximum afterburner-power nozzle.

  17. Sentinel surveillance systems with special focus on vector-borne diseases.

    Science.gov (United States)

    Racloz, V; Griot, C; Stärk, K D C

    2006-01-01

    In the past few decades, vector-borne diseases have been spreading into countries previously free of these agents. It is necessary for a surveillance method to be tailored to the biology of these agents in order to detect their incursion. Using a sentinel herd system, it is possible to target high-risk areas where occurrence is most probably due to vector presence. Since the 1970s, diseases such as Akabane, vesicular stomatitis and Bluetongue disease have successfully been monitored using cattle herds as sentinels in many countries such as Saudi Arabia, Australia, China, Indonesia, Sultanate of Oman and most recently in countries in Western Europe. This paper reviews the strengths and weaknesses of sentinel herd surveillance systems in general. In order to determine their efficacy, the following criteria were found to be essential: the choice of sentinel locations, sentinel animal, seasonality of sampling and diagnostic testing methods. We conclude that due to its ability to focus on a specific disease, sentinel herd systems have been successful in the early detection of the spread of a targeted agent. This review is used as a basis for recommendations for the development of future sentinel herd systems.

  18. Misalignment calibration of geomagnetic vector measurement system using parallelepiped frame rotation method

    International Nuclear Information System (INIS)

    Pang, Hongfeng; Zhu, XueJun; Pan, Mengchun; Zhang, Qi; Wan, Chengbiao; Luo, Shitu; Chen, Dixiang; Chen, Jinfei; Li, Ji; Lv, Yunxiao

    2016-01-01

    Misalignment error is one key factor influencing the measurement accuracy of geomagnetic vector measurement system, which should be calibrated with the difficulties that sensors measure different physical information and coordinates are invisible. A new misalignment calibration method by rotating a parallelepiped frame is proposed. Simulation and experiment result show the effectiveness of calibration method. The experimental system mainly contains DM-050 three-axis fluxgate magnetometer, INS (inertia navigation system), aluminium parallelepiped frame, aluminium plane base. Misalignment angles are calculated by measured data of magnetometer and INS after rotating the aluminium parallelepiped frame on aluminium plane base. After calibration, RMS error of geomagnetic north, vertical and east are reduced from 349.441 nT, 392.530 nT and 562.316 nT to 40.130 nT, 91.586 nT and 141.989 nT respectively. - Highlights: • A new misalignment calibration method by rotating a parallelepiped frame is proposed. • It does not need to know sensor attitude information or local dip angle. • The calibration system attitude change angle is not strictly required. • It can be widely used when sensors measure different physical information. • Geomagnetic vector measurement error is reduced evidently.

  19. Misalignment calibration of geomagnetic vector measurement system using parallelepiped frame rotation method

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Hongfeng [Academy of Equipment, Beijing 101416 (China); College of Mechatronics Engineering and Automation, National University of Defense Technology, Changsha 410073 (China); Zhu, XueJun, E-mail: zhuxuejun1990@126.com [College of Mechatronics Engineering and Automation, National University of Defense Technology, Changsha 410073 (China); Pan, Mengchun; Zhang, Qi; Wan, Chengbiao; Luo, Shitu; Chen, Dixiang; Chen, Jinfei; Li, Ji; Lv, Yunxiao [College of Mechatronics Engineering and Automation, National University of Defense Technology, Changsha 410073 (China)

    2016-12-01

    Misalignment error is one key factor influencing the measurement accuracy of geomagnetic vector measurement system, which should be calibrated with the difficulties that sensors measure different physical information and coordinates are invisible. A new misalignment calibration method by rotating a parallelepiped frame is proposed. Simulation and experiment result show the effectiveness of calibration method. The experimental system mainly contains DM-050 three-axis fluxgate magnetometer, INS (inertia navigation system), aluminium parallelepiped frame, aluminium plane base. Misalignment angles are calculated by measured data of magnetometer and INS after rotating the aluminium parallelepiped frame on aluminium plane base. After calibration, RMS error of geomagnetic north, vertical and east are reduced from 349.441 nT, 392.530 nT and 562.316 nT to 40.130 nT, 91.586 nT and 141.989 nT respectively. - Highlights: • A new misalignment calibration method by rotating a parallelepiped frame is proposed. • It does not need to know sensor attitude information or local dip angle. • The calibration system attitude change angle is not strictly required. • It can be widely used when sensors measure different physical information. • Geomagnetic vector measurement error is reduced evidently.

  20. Kochen-Specker vectors

    International Nuclear Information System (INIS)

    Pavicic, Mladen; Merlet, Jean-Pierre; McKay, Brendan; Megill, Norman D

    2005-01-01

    We give a constructive and exhaustive definition of Kochen-Specker (KS) vectors in a Hilbert space of any dimension as well as of all the remaining vectors of the space. KS vectors are elements of any set of orthonormal states, i.e., vectors in an n-dimensional Hilbert space, H n , n≥3, to which it is impossible to assign 1s and 0s in such a way that no two mutually orthogonal vectors from the set are both assigned 1 and that not all mutually orthogonal vectors are assigned 0. Our constructive definition of such KS vectors is based on algorithms that generate MMP diagrams corresponding to blocks of orthogonal vectors in R n , on algorithms that single out those diagrams on which algebraic (0)-(1) states cannot be defined, and on algorithms that solve nonlinear equations describing the orthogonalities of the vectors by means of statistically polynomially complex interval analysis and self-teaching programs. The algorithms are limited neither by the number of dimensions nor by the number of vectors. To demonstrate the power of the algorithms, all four-dimensional KS vector systems containing up to 24 vectors were generated and described, all three-dimensional vector systems containing up to 30 vectors were scanned, and several general properties of KS vectors were found

  1. Vector control of three-phase AC machines system development in the practice

    CERN Document Server

    Quang, Nguyen Phung; Dittrich, J

    2015-01-01

    This book addresses the vector control of three-phase AC machines, in particular induction motors with squirrel-cage rotors (IM), permanent magnet synchronous motors (PMSM) and doubly-fed induction machines (DFIM), from a practical design and development perspective. The main focus is on the application of IM and PMSM in electrical drive systems, where field-orientated control has been successfully established in practice. It also discusses the use of grid-voltage oriented control of DFIMs in wind power plants. This second, enlarged edition includes new insights into flatness-based  nonlinear

  2. Bio-signal analysis system design with support vector machines based on cloud computing service architecture.

    Science.gov (United States)

    Shen, Chia-Ping; Chen, Wei-Hsin; Chen, Jia-Ming; Hsu, Kai-Ping; Lin, Jeng-Wei; Chiu, Ming-Jang; Chen, Chi-Huang; Lai, Feipei

    2010-01-01

    Today, many bio-signals such as Electroencephalography (EEG) are recorded in digital format. It is an emerging research area of analyzing these digital bio-signals to extract useful health information in biomedical engineering. In this paper, a bio-signal analyzing cloud computing architecture, called BACCA, is proposed. The system has been designed with the purpose of seamless integration into the National Taiwan University Health Information System. Based on the concept of. NET Service Oriented Architecture, the system integrates heterogeneous platforms, protocols, as well as applications. In this system, we add modern analytic functions such as approximated entropy and adaptive support vector machine (SVM). It is shown that the overall accuracy of EEG bio-signal analysis has increased to nearly 98% for different data sets, including open-source and clinical data sets.

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

    Directory of Open Access Journals (Sweden)

    Siew Ching Ngai

    2015-01-01

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

  4. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.

    Science.gov (United States)

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji

    2016-10-24

    Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

  5. Cloud Monitoring for Solar Plants with Support Vector Machine Based Fault Detection System

    Directory of Open Access Journals (Sweden)

    Hong-Chan Chang

    2014-01-01

    Full Text Available This study endeavors to develop a cloud monitoring system for solar plants. This system incorporates numerous subsystems, such as a geographic information system, an instantaneous power-consumption information system, a reporting system, and a failure diagnosis system. Visual C# was integrated with ASP.NET and SQL technologies for the proposed monitoring system. A user interface for database management system was developed to enable users to access solar power information and management systems. In addition, by using peer-to-peer (P2P streaming technology and audio/video encoding/decoding technology, real-time video data can be transmitted to the client end, providing instantaneous and direct information. Regarding smart failure diagnosis, the proposed system employs the support vector machine (SVM theory to train failure mathematical models. The solar power data are provided to the SVM for analysis in order to determine the failure types and subsequently eliminate failures at an early stage. The cloud energy-management platform developed in this study not only enhances the management and maintenance efficiency of solar power plants but also increases the market competitiveness of solar power generation and renewable energy.

  6. Using geographic information systems and decision support systems for the prediction, prevention, and control of vector-borne diseases.

    Science.gov (United States)

    Eisen, Lars; Eisen, Rebecca J

    2011-01-01

    Emerging and resurging vector-borne diseases cause significant morbidity and mortality, especially in the developing world. We focus on how advances in mapping, Geographic Information System, and Decision Support System technologies, and progress in spatial and space-time modeling, can be harnessed to prevent and control these diseases. Major themes, which are addressed using examples from tick-borne Lyme borreliosis; flea-borne plague; and mosquito-borne dengue, malaria, and West Nile virus disease, include (a) selection of spatial and space-time modeling techniques, (b) importance of using high-quality and biologically or epidemiologically relevant data, (c) incorporation of new technologies into operational vector and disease control programs, (d) transfer of map-based information to stakeholders, and (e) adaptation of technology solutions for use in resource-poor environments. We see great potential for the use of new technologies and approaches to more effectively target limited surveillance, prevention, and control resources and to reduce vector-borne and other infectious diseases.

  7. Prototype early warning systems for vector-borne diseases in Europe.

    Science.gov (United States)

    Semenza, Jan C

    2015-06-02

    Globalization and environmental change, social and demographic determinants and health system capacity are significant drivers of infectious diseases which can also act as epidemic precursors. Thus, monitoring changes in these drivers can help anticipate, or even forecast, an upsurge of infectious diseases. The European Environment and Epidemiology (E3) Network has been built for this purpose and applied to three early warning case studies: (1) The environmental suitability of malaria transmission in Greece was mapped in order to target epidemiological and entomological surveillance and vector control activities. Malaria transmission in these areas was interrupted in 2013 through such integrated preparedness and response activities. (2) Since 2010, recurrent West Nile fever outbreaks have ensued in South/eastern Europe. Temperature deviations from a thirty year average proved to be associated with the 2010 outbreak. Drivers of subsequent outbreaks were computed through multivariate logistic regression models and included monthly temperature anomalies for July and a normalized water index. (3) Dengue is a tropical disease but sustained transmission has recently emerged in Madeira. Autochthonous transmission has also occurred repeatedly in France and in Croatia mainly due to travel importation. The risk of dengue importation into Europe in 2010 was computed with the volume of international travelers from dengue affected areas worldwide.These prototype early warning systems indicate that monitoring drivers of infectious diseases can help predict vector-borne disease threats.

  8. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems.

    Science.gov (United States)

    Guan, S; Rosenecker, J

    2017-03-01

    Because of its safe and effective protein expression profile, in vitro transcribed messenger RNA (IVT-mRNA) represents a promising candidate in the development of novel therapeutics for genetic diseases, vaccines or gene editing strategies, especially when its inherent shortcomings (for example, instability and immunogenicity) have been partially addressed via structural modifications. However, numerous unsolved technical difficulties in successful in vivo delivery of IVT-mRNA have greatly hindered the applications of IVT-mRNA in clinical development. Recent advances in nanotechnology and material science have yielded many promising nonviral delivery systems, some of which were able to efficiently facilitate targeted in vivo delivery of IVT-mRNA in safe and noninvasive manners. The diversity and flexibility of these delivery systems highlight the recent progress of IVT-mRNA-based therapy using nonviral vectors. In this review, we summarize recent advances of existing and emerging nonviral vector-based nanotechnologies for IVT-mRNA delivery and briefly summarize the interesting but rarely discussed applications on simultaneous delivery of IVT-mRNA with DNA.

  9. Output-only modal parameter estimator of linear time-varying structural systems based on vector TAR model and least squares support vector machine

    Science.gov (United States)

    Zhou, Si-Da; Ma, Yuan-Chen; Liu, Li; Kang, Jie; Ma, Zhi-Sai; Yu, Lei

    2018-01-01

    Identification of time-varying modal parameters contributes to the structural health monitoring, fault detection, vibration control, etc. of the operational time-varying structural systems. However, it is a challenging task because there is not more information for the identification of the time-varying systems than that of the time-invariant systems. This paper presents a vector time-dependent autoregressive model and least squares support vector machine based modal parameter estimator for linear time-varying structural systems in case of output-only measurements. To reduce the computational cost, a Wendland's compactly supported radial basis function is used to achieve the sparsity of the Gram matrix. A Gamma-test-based non-parametric approach of selecting the regularization factor is adapted for the proposed estimator to replace the time-consuming n-fold cross validation. A series of numerical examples have illustrated the advantages of the proposed modal parameter estimator on the suppression of the overestimate and the short data. A laboratory experiment has further validated the proposed estimator.

  10. A novel and highly efficient production system for recombinant adeno-associated virus vector.

    Science.gov (United States)

    Wu, Zhijian; Wu, Xiaobing; Cao, Hui; Dong, Xiaoyan; Wang, Hong; Hou, Yunde

    2002-02-01

    Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/DeltaUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/DeltaUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28x10(4) particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

  11. Efficiency improvement of the maximum power point tracking for PV systems using support vector machine technique

    Science.gov (United States)

    Kareim, Ameer A.; Mansor, Muhamad Bin

    2013-06-01

    The aim of this paper is to improve efficiency of maximum power point tracking (MPPT) for PV systems. The Support Vector Machine (SVM) was proposed to achieve the MPPT controller. The theoretical, the perturbation and observation (P&O), and incremental conductance (IC) algorithms were used to compare with proposed SVM algorithm. MATLAB models for PV module, theoretical, SVM, P&O, and IC algorithms are implemented. The improved MPPT uses the SVM method to predict the optimum voltage of the PV system in order to extract the maximum power point (MPP). The SVM technique used two inputs which are solar radiation and ambient temperature of the modeled PV module. The results show that the proposed SVM technique has less Root Mean Square Error (RMSE) and higher efficiency than P&O and IC methods.

  12. Implementation of the Orbital Maneuvering Systems Engine and Thrust Vector Control for the European Service Module

    Science.gov (United States)

    Millard, Jon

    2014-01-01

    The European Space Agency (ESA) has entered into a partnership with the National Aeronautics and Space Administration (NASA) to develop and provide the Service Module (SM) for the Orion Multipurpose Crew Vehicle (MPCV) Program. The European Service Module (ESM) will provide main engine thrust by utilizing the Space Shuttle Program Orbital Maneuvering System Engine (OMS-E). Thrust Vector Control (TVC) of the OMS-E will be provided by the Orbital Maneuvering System (OMS) TVC, also used during the Space Shuttle Program. NASA will be providing the OMS-E and OMS TVC to ESA as Government Furnished Equipment (GFE) to integrate into the ESM. This presentation will describe the OMS-E and OMS TVC and discuss the implementation of the hardware for the ESM.

  13. Quantization of Hamiltonian systems with a position dependent mass: Killing vector fields and Noether momenta approach

    Science.gov (United States)

    Cariñena, José F.; Rañada, Manuel F.; Santander, Mariano

    2017-11-01

    The quantization of systems with a position dependent mass (PDM) is studied. We present a method that starts with the study of the existence of Killing vector fields for the PDM geodesic motion (Lagrangian with a PDM kinetic term but without any potential) and the construction of the associated Noether momenta. Then the method considers, as the appropriate Hilbert space, the space of functions that are square integrable with respect to a measure related with the PDM and, after that, it establishes the quantization, not of the canonical momenta p, but of the Noether momenta P instead. The quantum Hamiltonian, that depends on the Noether momenta, is obtained as an Hermitian operator defined on the PDM Hilbert space. In the second part several systems with position-dependent mass, most of them related with nonlinear oscillators, are quantized by making use of the method proposed in the first part.

  14. Classification of polynomial integrable systems of mixed scalar and vector evolution equations: I

    International Nuclear Information System (INIS)

    Tsuchida, Takayuki; Wolf, Thomas

    2005-01-01

    We perform a classification of integrable systems of mixed scalar and vector evolution equations with respect to higher symmetries. We consider polynomial systems that are homogeneous under a suitable weighting of variables. This paper deals with the KdV weighting, the Burgers (or potential KdV or modified KdV) weighting, the Ibragimov-Shabat weighting and two unfamiliar weightings. The case of other weightings will be studied in a subsequent paper. Making an ansatz for undetermined coefficients and using a computer package for solving bilinear algebraic systems, we give the complete lists of second-order systems with a third-order or a fourth-order symmetry and third-order systems with a fifth-order symmetry. For all but a few systems in the lists, we show that the system (or, at least a subsystem of it) admits either a Lax representation or a linearizing transformation. A thorough comparison with recent work of Foursov and Olver is made

  15. Genetic modification of human sural nerve segments by a lentiviral vector encoding nerve growth factor

    NARCIS (Netherlands)

    Tannemaat, Martijn R; Boer, Gerard J; Verhaagen, J.; Malessy, Martijn J A

    2007-01-01

    OBJECTIVE: Autologous nerve grafts are used to treat severe peripheral nerve injury, but recovery of nerve function after grafting is rarely complete. Exogenous application of neurotrophic factors may enhance regeneration, but thus far the application of neurotrophic factors has been hampered by

  16. Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.

    Science.gov (United States)

    Han, Seung-Soo; Lee, Jinjoo; Jung, Yideul; Kang, Myeong-Ho; Hong, Jung-Hyub; Cha, Min-Suk; Park, Yu-Jin; Lee, Ezra; Yoon, Cheol-Hee; Bae, Yong-Soo

    2015-09-11

    We developed a CTL vaccine vector by modification of the RPS-Vax system, a mucosal vaccine vector derived from a poliovirus Sabin 1 strain, and generated an oral CTL vaccine against HIV-1. A DNA fragment encoding a cytoplasmic transduction peptide (CTP) was integrated into the RPS-Vax system to generate RPS-CTP, a CTL vaccine vector. An HIV-1 p24 cDNA fragment was introduced into the RPS-CTP vector system and a recombinant poliovirus (rec-PV) named vRPS-CTP/p24 was produced. vRPS-CTP/p24 was genetically stable and efficiently induced Th1 immunity and p24-specific CTLs in immunized poliovirus receptor-transgenic (PVR-Tg) mice. In challenge experiments, PVR-Tg mice that were pre-immunized orally with vRPS-CTP/p24 were resistant to challenge with a lethal dose of p24-expressing recombinant vaccinia virus (rMVA-p24). These results suggested that the RPS-CTP vector system had potential for developing oral CTL vaccines against infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Yu-Ping Zheng

    2016-01-01

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

  18. Biology of Adeno-Associated Viral Vectors in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Giridhar eMurlidharan

    2014-09-01

    Full Text Available Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS. In particular, Adeno-Associated Viruses (AAV have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic.

  19. Calibration Performance and Capabilities of the New Compact Ocean Wind Vector Radiometer System

    Science.gov (United States)

    Brown, S. T.; Focardi, P.; Kitiyakara, A.; Maiwald, F.; Montes, O.; Padmanabhan, S.; Redick, R.; Russell, D.; Wincentsen, J.

    2014-12-01

    The paper describes performance and capabilities of a new satellite conically imaging microwave radiometer system, the Compact Ocean Wind Vector Radiometer (COWVR), being built by the Jet Propulsion Laboratory (JPL) for an Air Force demonstration mission. COWVR is an 18-34 GHz fully polarimetric radiometer designed to provide measurements of ocean vector winds with an accuracy that meets or exceeds that provided by WindSat, but using a simpler design which has both calibration and cost advantages. Heritage conical radiometer systems, such as WindSat, AMSR, GMI or SSMI(S), all have a similar overall architecture and have exhibited significant intra-channel and inter-sensor calibration biases, due in part to the relative independence of the radiometers between the different polarizations and frequencies in the system. The COWVR system uses a broadband compact hybrid combining architecture and Electronic Polarization Basis Rotation to minimize the number of free calibration parameters between polarization and frequencies, as well as providing a definitive calibration reference from the modulation of the mean polarized signal from the Earth. This second calibration advantage arises because the sensor modulates the incoming polarized signal at the input antenna aperture in a known way based only on the instrument geometry which forces relative calibration consistency between the polarimetric channels of the sensor and provides a gain and offset calibration independent of a model or other ancillary data source, which has typically been a weakness in the calibration and inter-calibration of heritage microwave sensors. This paper will give a description of the COWVR instrument and an overview of the technology demonstration mission. We will discuss the overall calibration approach for this system, its advantages over existing systems and how many of the calibration issues that impact existing satellite radiometers can be eliminated in future operational systems based on

  20. Genetically modifying the insect gut microbiota to control Chagas disease vectors through systemic RNAi.

    Directory of Open Access Journals (Sweden)

    Mabel L Taracena

    2015-02-01

    Full Text Available Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3 expressing dsRNA for the Rhodnius heme-binding protein (RHBP and for catalase (CAT were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all developmental stages. The RNA interference effect was systemic and temporal. Concentrations of E. coli HT115(DE3 above 3.35 × 10(7 CFU/mL produced a significant RHBP and CAT gene knockdown in nymphs and adults. RHBP expression in the fat body was reduced by 99% three days after feeding, returning to normal levels 10 days after feeding. CAT expression was reduced by 99% and 96% in the ovary and the posterior midgut, respectively, five days after ingestion. Mortality rates increased by 24-30% in first instars fed RHBP and CAT bacteria. Molting rates were reduced by 100% in first instars and 80% in third instars fed bacteria producing RHBP or CAT dsRNA. Oviposition was reduced by 43% (RHBP and 84% (CAT. Embryogenesis was arrested in 16% (RHBP and 20% (CAT of laid eggs. Feeding females 105 CFU/mL of the natural symbiont, Rhodococcus rhodnii, transformed to express RHBP-specific hairpin RNA reduced RHBP expression by 89% and reduced oviposition. Modifying the insect microbiota to induce systemic RNAi in R. prolixus may result in a paratransgenic strategy for sustainable vector control.

  1. Support vector machine based diagnostic system for breast cancer using swarm intelligence.

    Science.gov (United States)

    Chen, Hui-Ling; Yang, Bo; Wang, Gang; Wang, Su-Jing; Liu, Jie; Liu, Da-You

    2012-08-01

    Breast cancer is becoming a leading cause of death among women in the whole world, meanwhile, it is confirmed that the early detection and accurate diagnosis of this disease can ensure a long survival of the patients. In this paper, a swarm intelligence technique based support vector machine classifier (PSO_SVM) is proposed for breast cancer diagnosis. In the proposed PSO-SVM, the issue of model selection and feature selection in SVM is simultaneously solved under particle swarm (PSO optimization) framework. A weighted function is adopted to design the objective function of PSO, which takes into account the average accuracy rates of SVM (ACC), the number of support vectors (SVs) and the selected features simultaneously. Furthermore, time varying acceleration coefficients (TVAC) and inertia weight (TVIW) are employed to efficiently control the local and global search in PSO algorithm. The effectiveness of PSO-SVM has been rigorously evaluated against the Wisconsin Breast Cancer Dataset (WBCD), which is commonly used among researchers who use machine learning methods for breast cancer diagnosis. The proposed system is compared with the grid search method with feature selection by F-score. The experimental results demonstrate that the proposed approach not only obtains much more appropriate model parameters and discriminative feature subset, but also needs smaller set of SVs for training, giving high predictive accuracy. In addition, Compared to the existing methods in previous studies, the proposed system can also be regarded as a promising success with the excellent classification accuracy of 99.3% via 10-fold cross validation (CV) analysis. Moreover, a combination of five informative features is identified, which might provide important insights to the nature of the breast cancer disease and give an important clue for the physicians to take a closer attention. We believe the promising result can ensure that the physicians make very accurate diagnostic decision in

  2. Object Recognition System-on-Chip Using the Support Vector Machines

    Directory of Open Access Journals (Sweden)

    Houzet Dominique

    2005-01-01

    Full Text Available The first aim of this work is to propose the design of a system-on-chip (SoC platform dedicated to digital image and signal processing, which is tuned to implement efficiently multiply-and-accumulate (MAC vector/matrix operations. The second aim of this work is to implement a recent promising neural network method, namely, the support vector machine (SVM used for real-time object recognition, in order to build a vision machine. With such a reconfigurable and programmable SoC platform, it is possible to implement any SVM function dedicated to any object recognition problem. The final aim is to obtain an automatic reconfiguration of the SoC platform, based on the results of the learning phase on an objects' database, which makes it possible to recognize practically any object without manual programming. Recognition can be of any kind that is from image to signal data. Such a system is a general-purpose automatic classifier. Many applications can be considered as a classification problem, but are usually treated specifically in order to optimize the cost of the implemented solution. The cost of our approach is more important than a dedicated one, but in a near future, hundreds of millions of gates will be common and affordable compared to the design cost. What we are proposing here is a general-purpose classification neural network implemented on a reconfigurable SoC platform. The first version presented here is limited in size and thus in object recognition performances, but can be easily upgraded according to technology improvements.

  3. Genetically modifying the insect gut microbiota to control Chagas disease vectors through systemic RNAi.

    Science.gov (United States)

    Taracena, Mabel L; Oliveira, Pedro L; Almendares, Olivia; Umaña, Claudia; Lowenberger, Carl; Dotson, Ellen M; Paiva-Silva, Gabriela O; Pennington, Pamela M

    2015-02-01

    Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all developmental stages. The RNA interference effect was systemic and temporal. Concentrations of E. coli HT115(DE3) above 3.35 × 10(7) CFU/mL produced a significant RHBP and CAT gene knockdown in nymphs and adults. RHBP expression in the fat body was reduced by 99% three days after feeding, returning to normal levels 10 days after feeding. CAT expression was reduced by 99% and 96% in the ovary and the posterior midgut, respectively, five days after ingestion. Mortality rates increased by 24-30% in first instars fed RHBP and CAT bacteria. Molting rates were reduced by 100% in first instars and 80% in third instars fed bacteria producing RHBP or CAT dsRNA. Oviposition was reduced by 43% (RHBP) and 84% (CAT). Embryogenesis was arrested in 16% (RHBP) and 20% (CAT) of laid eggs. Feeding females 105 CFU/mL of the natural symbiont, Rhodococcus rhodnii, transformed to express RHBP-specific hairpin RNA reduced RHBP expression by 89% and reduced oviposition. Modifying the insect microbiota to induce systemic RNAi in R. prolixus may result in a paratransgenic strategy for sustainable vector control.

  4. BaBar experiment: CP violation in the B0 B-bar0 system. Vector-vector channels of charmonium K*0 type

    International Nuclear Information System (INIS)

    Roussot, E.

    1999-04-01

    Following a summary of CP violation phenomenology in the context of the Standard Model, we focus on the particular case of CP violation by interference between B 0 B-bar 0 mixing and decay to a CP final state. We study the feasibility of the unitary triangle parameter sin (2β) measurement in the vector-vector like decay channel Charmonium K * . A full angular analysis is required in order to separate contribution between final states of opposite CP. With simulated data, we develop a kinematic selection of signal events. A likelihood allows to extract the value of sin (2β) with an expecting resolution of 0.39 for the first 30 fb -1 . This contribution constitutes the first quantitative analysis on this particular hadronic decay channel, in the framework of the BaBar experiment at the PEPII e + /e - asymmetric collider situated at SLAC (California). Besides, particle identification is assumed by a new type of Cherenkov detector (the DIRC), the principle, the architecture, the data acquisition and the online control system of which are fully described. (author)

  5. Combining nonlinear multiresolution system and vector quantization for still image compression

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Y.

    1993-12-17

    It is popular to use multiresolution systems for image coding and compression. However, general-purpose techniques such as filter banks and wavelets are linear. While these systems are rigorous, nonlinear features in the signals cannot be utilized in a single entity for compression. Linear filters are known to blur the edges. Thus, the low-resolution images are typically blurred, carrying little information. We propose and demonstrate that edge-preserving filters such as median filters can be used in generating a multiresolution system using the Laplacian pyramid. The signals in the detail images are small and localized to the edge areas. Principal component vector quantization (PCVQ) is used to encode the detail images. PCVQ is a tree-structured VQ which allows fast codebook design and encoding/decoding. In encoding, the quantization error at each level is fed back through the pyramid to the previous level so that ultimately all the error is confined to the first level. With simple coding methods, we demonstrate that images with PSNR 33 dB can be obtained at 0.66 bpp without the use of entropy coding. When the rate is decreased to 0.25 bpp, the PSNR of 30 dB can still be achieved. Combined with an earlier result, our work demonstrate that nonlinear filters can be used for multiresolution systems and image coding.

  6. Analysis of Vector Quantizers Using Transformed Codebooks with Application to Feedback-Based Multiple Antenna Systems

    Directory of Open Access Journals (Sweden)

    Bhaskar D. Rao

    2008-07-01

    Full Text Available Transformed codebooks are obtained by a transformation of a given codebook to best match the statistical environment at hand. The procedure, though suboptimal, has recently been suggested for feedback of channel state information (CSI in multiple antenna systems with correlated channels because of their simplicity and effectiveness. In this paper, we first consider the general distortion analysis of vector quantizers with transformed codebooks. Bounds on the average system distortion of this class of quantizers are provided. It exposes the effects of two kinds of suboptimality introduced by the transformed codebook, namely, the loss caused by suboptimal point density and the loss caused by mismatched Voronoi shape. We then focus our attention on the application of the proposed general framework to providing capacity analysis of a feedback-based MISO system over spatially correlated fading channels. In particular, with capacity loss as an objective function, upper and lower bounds on the average distortion of MISO systems with transformed codebooks are provided and compared to that of the optimal channel quantizers. The expressions are examined to provide interesting insights in the high and low SNR regime. Numerical and simulation results are presented which confirm the tightness of the distortion bounds.

  7. Analysis of Vector Quantizers Using Transformed Codebooks with Application to Feedback-Based Multiple Antenna Systems

    Directory of Open Access Journals (Sweden)

    Zheng Jun

    2008-01-01

    Full Text Available Transformed codebooks are obtained by a transformation of a given codebook to best match the statistical environment at hand. The procedure, though suboptimal, has recently been suggested for feedback of channel state information (CSI in multiple antenna systems with correlated channels because of their simplicity and effectiveness. In this paper, we first consider the general distortion analysis of vector quantizers with transformed codebooks. Bounds on the average system distortion of this class of quantizers are provided. It exposes the effects of two kinds of suboptimality introduced by the transformed codebook, namely, the loss caused by suboptimal point density and the loss caused by mismatched Voronoi shape. We then focus our attention on the application of the proposed general framework to providing capacity analysis of a feedback-based MISO system over spatially correlated fading channels. In particular, with capacity loss as an objective function, upper and lower bounds on the average distortion of MISO systems with transformed codebooks are provided and compared to that of the optimal channel quantizers. The expressions are examined to provide interesting insights in the high and low SNR regime. Numerical and simulation results are presented which confirm the tightness of the distortion bounds.

  8. Estimation of Nonlinear Functions of State Vector for Linear Systems with Time-Delays and Uncertainties

    Directory of Open Access Journals (Sweden)

    Il Young Song

    2015-01-01

    Full Text Available This paper focuses on estimation of a nonlinear function of state vector (NFS in discrete-time linear systems with time-delays and model uncertainties. The NFS represents a multivariate nonlinear function of state variables, which can indicate useful information of a target system for control. The optimal nonlinear estimator of an NFS (in mean square sense represents a function of the receding horizon estimate and its error covariance. The proposed receding horizon filter represents the standard Kalman filter with time-delays and special initial horizon conditions described by the Lyapunov-like equations. In general case to calculate an optimal estimator of an NFS we propose using the unscented transformation. Important class of polynomial NFS is considered in detail. In the case of polynomial NFS an optimal estimator has a closed-form computational procedure. The subsequent application of the proposed receding horizon filter and nonlinear estimator to a linear stochastic system with time-delays and uncertainties demonstrates their effectiveness.

  9. A vector radiative transfer model for coupled atmosphere and ocean systems with a rough interface

    International Nuclear Information System (INIS)

    Zhai Pengwang; Hu Yongxiang; Chowdhary, Jacek; Trepte, Charles R.; Lucker, Patricia L.; Josset, Damien B.

    2010-01-01

    We report on an exact vector (polarized) radiative transfer (VRT) model for coupled atmosphere and ocean systems. This VRT model is based on the successive order of scattering (SOS) method, which virtually takes all the multiple scattering processes into account, including atmospheric scattering, oceanic scattering, reflection and transmission through the rough ocean surface. The isotropic Cox-Munk wave model is used to derive the ref and transmission matrices for the rough ocean surface. Shadowing effects are included by the shadowing function. We validated the SOS results by comparing them with those calculated by two independent codes based on the doubling/adding and Monte Carlo methods. Two error analyses related to the ocean color remote sensing are performed in the coupled atmosphere and ocean systems. One is the scalar error caused by ignoring the polarization in the whole system. The other is the error introduced by ignoring the polarization of the light transmitted through the ocean interface. Both errors are significant for the cases studied. This code fits for the next generation of ocean color study because it converges fast for absorbing medium as, for instance, ocean.

  10. A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

    NARCIS (Netherlands)

    Kuhlemeier, C. J.; Thomas, A. A.; van der Ende, A.; van Leen, R. W.; Borrias, W. E.; van den Hondel, C. A.; van Arkel, G. A.

    1983-01-01

    We describe the construction of a series of vectors suitable for gene cloning in the cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector

  11. 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

  12. A novel balanced-lethal host-vector system based on glmS.

    Directory of Open Access Journals (Sweden)

    Kwangsoo Kim

    Full Text Available During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(- mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN or N-acetyl-D-glucosamine (GlcNAc, components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+p complemented the phenotype of the GlmS(- mutant, and that GlmS(+ p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.

  13. Short term prediction of the horizontal wind vector within a wake vortex warning system

    Energy Technology Data Exchange (ETDEWEB)

    Frech, M.; Holzaepfel, F.; Gerz, T. [DLR Deutsches Zentrum fuer Luft- und Raumfahrt e.V., Wessling (Germany). Inst. fuer Physik der Atmosphaere; Konopka, J. [Deutsche Flugsicherung (DFS) GmbH, Langen (Germany)

    2000-07-14

    A wake vortex warning system (WVWS) has been developed for Frankfurt airport. This airport has two parallel runways which are separated by 518 m, a distance too short to operate them independently because wake vortices may be advected to the adjacent runway. The objective of the WVWS is to enable operation with reduced separation between two aircraft approaching the parallel runways at appropriate wind conditions. The WVWS applies a statistical persistence model to predict the crosswind within a 20 minute period. One of the main problems identified in the old WVWS are discontinuities between successive forecasts. These forecast breakdowns were not acceptable to airtraffic controllers. At least part of the problem was related to the fact that the forecast was solely based on the prediction of crosswind. A new method is developed on the basis of 523 days of sonic anemometer measurements at Frankfurt airport. It is demonstrated that the prediction of the horizontal wind vector avoids these difficulties and significantly improves the system's performance. (orig.)

  14. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami

    2017-05-08

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  15. Comparison of a fimbrial versus an autotransporter display system for viral epitopes on an attenuated Salmonella vaccine vector.

    Science.gov (United States)

    Chen, Huaiqing; Schifferli, Dieter M

    2007-02-19

    Attenuated Salmonella have been used as vectors to deliver foreign antigens as live vaccines. We have previously developed an efficient surface-display system by genetically engineering 987P fimbriae to present transmissible gastroenteritis virus (TGEV) C and A epitopes for the induction of anti-TGEV antibodies with a Salmonella vaccine vector. Here, this system was compared with an autotransporter protein surface display system. The TGEV C and A epitopes were fused to the passenger domain of the MisL autotransporter of Salmonella. Expression of both the MisL- and 987P subunit FasA-fusions to the TGEV epitopes were under the control of in vivo-induced promoters. Expression of the TGEV epitopes from the Salmonella typhimurium CS4552 (crp cya asd pgtE) vaccine strain was greater when the epitopes were fused to MisL than when they were fused to the 987P FasA subunit. However, when BALB/c mice were orally immunized with the Salmonella vector expressing the TGEV epitopes from either one of the fusion constructs or both together, the highest level of anti-TGEV antibody was obtained with the 987P-TGEV immunogen-displaying vector. This result suggested that better immune responses towards specific epitopes could be obtained by using a polymeric display system such as fimbriae.

  16. Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system

    NARCIS (Netherlands)

    Patel, R.M.; Kan, van J.A.L.; Bailey, A.M.; Foster, G.D.

    2010-01-01

    For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the ß-tubulin (tubA)

  17. Structural basis for functional tetramerization of lentiviral integrase.

    Directory of Open Access Journals (Sweden)

    Stephen Hare

    2009-07-01

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

  18. Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2016-08-01

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Large Animal Models for Foamy Virus Vector Gene Therapy

    Directory of Open Access Journals (Sweden)

    Peter A. Horn

    2012-12-01

    Full Text Available Foamy virus (FV vectors have shown great promise for hematopoietic stem cell (HSC gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit.

  1. Vectorization, parallelization and porting of nuclear codes on the VPP500 system (porting). Progress report fiscal 1997

    International Nuclear Information System (INIS)

    Ishizuki, Shigeru; Nemoto, Toshiyuki; Kawai, Wataru; Watanabe, Hideo; Tanabe, Hidenobu; Kawasaki, Nobuo; Adachi, Masaaki; Ogasawara, Shinobu; Kume, Etsuo

    1999-05-01

    Several computer codes in the nuclear field have been vectorized, parallelized and transported on the FUJITSU VPP500 system and/or the AP3000 system at Center for Promotion of Computational Science and Engineering in Japan Atomic Energy Research Institute. We dealt with 14 codes in fiscal 1997. These results are reported in 3 parts, i.e., the vectorization part, the parallelization part and the porting part. In this report, we describe the porting. In this porting part, the porting of transient reactor analysis code TRAC-BF1 and Monte Carlo radiation transport code MCNP4A on the AP3000 are described. In addition, a modification of program libraries for command-driven interactive data analysis plotting program IPLOT is described. In the vectorization part, the vectorization of multidimensional two-fluid model code ACE-3D for evaluation of constitutive equations, statistical decay code SD and three-dimensional thermal analysis code for in-core test section (T2) of HENDEL SSPHEAT are described. In the parallelization part, the parallelization of cylindrical direct numerical simulation code CYLDNS44N, worldwide version of system for prediction of environmental emergency dose information code WSPEEDI, extension of quantum molecular dynamics code EQMD and three-dimensional non-steady compressible fluid dynamics code STREAM are described. (author)

  2. Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and strategies for improvement.

    Science.gov (United States)

    Wang, Yong; Yuan, Fan

    2006-01-01

    It is a challenge to deliver therapeutic genes to tumor cells using viral vectors because (i) the size of these vectors are close to or larger than the space between fibers in extracellular matrix and (ii) viral proteins are potentially toxic in normal tissues. In general, gene delivery is hindered by various physiological barriers to virus transport from the site of injection to the nucleus of tumor cells and is limited by normal tissue tolerance of toxicity determined by local concentrations of transgene products and viral proteins. To illustrate the obstacles encountered in the delivery and yet limit the scope of discussion, this review focuses only on extracellular transport in solid tumors and distribution of viral vectors in normal organs after they are injected intravenously or intratumorally. This review also discusses current strategies for improving intratumoral transport and specificity of viral vectors.

  3. Intelligent Diagnosis Method for Centrifugal Pump System Using Vibration Signal and Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Hongtao Xue

    2014-01-01

    Full Text Available This paper proposed an intelligent diagnosis method for a centrifugal pump system using statistic filter, support vector machine (SVM, possibility theory, and Dempster-Shafer theory (DST on the basis of the vibration signals, to diagnose frequent faults in the centrifugal pump at an early stage, such as cavitation, impeller unbalance, and shaft misalignment. Firstly, statistic filter is used to extract the feature signals of pump faults from the measured vibration signals across an optimum frequency region, and nondimensional symptom parameters (NSPs are defined to represent the feature signals for distinguishing fault types. Secondly, the optimal classification hyperplane for distinguishing two states is obtained by SVM and NSPs, and its function is defined as synthetic symptom parameter (SSP in order to increase the diagnosis’ sensitivity. Finally, the possibility functions of the SSP are used to construct a sequential fuzzy diagnosis for fault detection and fault-type identification by possibility theory and DST. The proposed method has been applied to detect the faults of the centrifugal pump, and the efficiency of the method has been verified using practical examples.

  4. An MR Brain Images Classifier System via Particle Swarm Optimization and Kernel Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Yudong Zhang

    2013-01-01

    Full Text Available Automated abnormal brain detection is extremely of importance for clinical diagnosis. Over last decades numerous methods had been presented. In this paper, we proposed a novel hybrid system to classify a given MR brain image as either normal or abnormal. The proposed method first employed digital wavelet transform to extract features then used principal component analysis (PCA to reduce the feature space. Afterwards, we constructed a kernel support vector machine (KSVM with RBF kernel, using particle swarm optimization (PSO to optimize the parameters C and σ. Fivefold cross-validation was utilized to avoid overfitting. In the experimental procedure, we created a 90 images dataset brain downloaded from Harvard Medical School website. The abnormal brain MR images consist of the following diseases: glioma, metastatic adenocarcinoma, metastatic bronchogenic carcinoma, meningioma, sarcoma, Alzheimer, Huntington, motor neuron disease, cerebral calcinosis, Pick’s disease, Alzheimer plus visual agnosia, multiple sclerosis, AIDS dementia, Lyme encephalopathy, herpes encephalitis, Creutzfeld-Jakob disease, and cerebral toxoplasmosis. The 5-folded cross-validation classification results showed that our method achieved 97.78% classification accuracy, higher than 86.22% by BP-NN and 91.33% by RBF-NN. For the parameter selection, we compared PSO with those of random selection method. The results showed that the PSO is more effective to build optimal KSVM.

  5. Fault diagnosis of automobile hydraulic brake system using statistical features and support vector machines

    Science.gov (United States)

    Jegadeeshwaran, R.; Sugumaran, V.

    2015-02-01

    Hydraulic brakes in automobiles are important components for the safety of passengers; therefore, the brakes are a good subject for condition monitoring. The condition of the brake components can be monitored by using the vibration characteristics. On-line condition monitoring by using machine learning approach is proposed in this paper as a possible solution to such problems. The vibration signals for both good as well as faulty conditions of brakes were acquired from a hydraulic brake test setup with the help of a piezoelectric transducer and a data acquisition system. Descriptive statistical features were extracted from the acquired vibration signals and the feature selection was carried out using the C4.5 decision tree algorithm. There is no specific method to find the right number of features required for classification for a given problem. Hence an extensive study is needed to find the optimum number of features. The effect of the number of features was also studied, by using the decision tree as well as Support Vector Machines (SVM). The selected features were classified using the C-SVM and Nu-SVM with different kernel functions. The results are discussed and the conclusion of the study is presented.

  6. Vector analysis

    CERN Document Server

    Newell, Homer E

    2006-01-01

    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  7. About vectors

    CERN Document Server

    Hoffmann, Banesh

    1975-01-01

    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

  8. Vectorization, parallelization and porting of nuclear codes on the VPP500 system (porting). Progress report fiscal 1996

    International Nuclear Information System (INIS)

    Nemoto, Toshiyuki; Kawasaki, Nobuo; Tanabe, Hidenobu

    1998-01-01

    Several computer codes in the nuclear field have been vectorized, parallelized and transported on the FUJITSU VPP500 system at Center for Promotion of Computational Science and Engineering in Japan Atomic Energy Research Institute. These results are reported in 3 parts, i.e., the vectorization part, the parallelization part and the porting part. In this report, we describe the porting. In this porting part, the porting of reactor safety analysis code RELAP5/MOD3.2 and RELAP5/MOD3.2.1.2, nuclear data processing system NJOY and 2-D multigroup discrete ordinate transport code TWOTRAN-II are described. And also, a survey for the porting of command-driven interactive data analysis plotting program IPLOT are described. In the parallelization part, the parallelization of 2-Dimensional relativistic electromagnetic particle code EM2D, Cylindrical Direct Numerical Simulation code CYLDNS and molecular dynamics code for simulating radiation damages in diamond crystals DGR are described. And then, in the vectorization part, the vectorization of two and three dimensional discrete ordinates simulation code DORT-TORT, gas dynamics analysis code FLOWGR and relativistic Boltzmann-Uehling-Uhlenbeck simulation code RBUU are described. (author)

  9. Vectorization, parallelization and porting of nuclear codes on the VPP500 system (parallelization). Progress report fiscal 1996

    International Nuclear Information System (INIS)

    Watanabe, Hideo; Kawai, Wataru; Nemoto, Toshiyuki

    1997-12-01

    Several computer codes in the nuclear field have been vectorized, parallelized and transported on the FUJITSU VPP500 system at Center for Promotion of Computational Science and Engineering in Japan Atomic Energy Research Institute. These results are reported in 3 parts, i.e., the vectorization part, the parallelization part and the porting part. In this report, we describe the parallelization. In this parallelization part, the parallelization of 2-Dimensional relativistic electromagnetic particle code EM2D, Cylindrical Direct Numerical Simulation code CYLDNS and molecular dynamics code for simulating radiation damages in diamond crystals DGR are described. In the vectorization part, the vectorization of two and three dimensional discrete ordinates simulation code DORT-TORT, gas dynamics analysis code FLOWGR and relativistic Boltzmann-Uehling-Uhlenbeck simulation code RBUU are described. And then, in the porting part, the porting of reactor safety analysis code RELAP5/MOD3.2 and RELAP5/MOD3.2.1.2, nuclear data processing system NJOY and 2-D multigroup discrete ordinate transport code TWOTRAN-II are described. And also, a survey for the porting of command-driven interactive data analysis plotting program IPLOT are described. (author)

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

    Science.gov (United States)

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

    2018-01-03

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

  11. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Science.gov (United States)

    Salem, Tamer Z; Seaborn, Craig P; Turney, Colin M; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).

  12. Elementary vectors

    CERN Document Server

    Wolstenholme, E Œ

    1978-01-01

    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

  13. On-line transient stability assessment of large-scale power systems by using ball vector machines

    International Nuclear Information System (INIS)

    Mohammadi, M.; Gharehpetian, G.B.

    2010-01-01

    In this paper ball vector machine (BVM) has been used for on-line transient stability assessment of large-scale power systems. To classify the system transient security status, a BVM has been trained for all contingencies. The proposed BVM based security assessment algorithm has very small training time and space in comparison with artificial neural networks (ANN), support vector machines (SVM) and other machine learning based algorithms. In addition, the proposed algorithm has less support vectors (SV) and therefore is faster than existing algorithms for on-line applications. One of the main points, to apply a machine learning method is feature selection. In this paper, a new Decision Tree (DT) based feature selection technique has been presented. The proposed BVM based algorithm has been applied to New England 39-bus power system. The simulation results show the effectiveness and the stability of the proposed method for on-line transient stability assessment procedure of large-scale power system. The proposed feature selection algorithm has been compared with different feature selection algorithms. The simulation results demonstrate the effectiveness of the proposed feature algorithm.

  14. CW-THz vector spectroscopy and imaging system based on 1.55-µm fiber-optics.

    Science.gov (United States)

    Kim, Jae-Young; Song, Ho-Jin; Yaita, Makoto; Hirata, Akihiko; Ajito, Katsuhiro

    2014-01-27

    We present a continuous-wave terahertz (THz) vector spectroscopy and imaging system based on a 1.5-µm fiber optic uni-traveling-carrier photodiode and InGaAs photo-conductive receiver. Using electro-optic (EO) phase modulators for THz phase control with shortened optical paths, the system achieves fast vector measurement with effective phase stabilization. Dynamic ranges of 100 dB · Hz and 75 dB · Hz at 300 GHz and 1 THz, and phase stability of 1.5° per minute are obtained. With the simultaneous measurement of absorbance and relative permittivity, we demonstrate non-destructive analyses of pharmaceutical cocrystals inside tablets within a few minutes.

  15. Beam steering and coordinate system rotation improves accuracy of ultrasonic measurements of tissue displacement vector and lateral displacement

    Directory of Open Access Journals (Sweden)

    Sumi C

    2011-11-01

    Full Text Available Chikayoshi Sumi1,2, Kento Ichimaru2, Yusuke Shinozuka21Department of Information and Communication Science, 2Department of Electrical and Electronics Engineering, Faculty of Science and Technology, Sophia University, Kioi-cho, Chiyoda-ku, Tokyo, JapanAbstract: With the proper use of beam steering and apodization, a higher resolution lateral echo image is obtained than with conventional imaging. This is achieved by superimposing crossed, steered beams, which is referred to as “lateral modulation” (LM. This type of beamforming achieves almost the same accuracy in lateral displacement measurements as in axial displacement measurements, ie, displacement vector measurements. The steering angle (ASTA can also be used instead of LM, which uses only beams with a steering angle. In this report, the accuracy of the displacement vectors and lateral displacement measurements for LM and ASTA were evaluated using simulations and agar phantom experiments. The parameters used were direction of a displacement vector, steering angles, and rotation angle for the coordinate system. Changes in the steering angle and rotation angle of the coordinate system permit control of frequencies in the respective directions of the coordinate axes. As shown, when performing a simple ASTA for displacement vector measurement, a spectra frequency division should be performed using a previously developed multidimensional autocorrelation or Doppler method instead of block-matching methods. In this version of ASTA, the combination of nonsteering and rotation of the coordinate system is also effective, because the lateral bandwidth does not decrease. In such a case, transmission of a laterally wide wave will also be effective, particularly for three-dimensional measurement/imaging using a two-dimensional array transducer. ASTA can also be used for accurate lateral displacement measurements. Although a proper beam steering and/or a proper coordinate rotation improves the

  16. Existence and Hadamard well-posedness of a system of simultaneous generalized vector quasi-equilibrium problems

    Directory of Open Access Journals (Sweden)

    Wenyan Zhang

    2017-03-01

    Full Text Available Abstract An existence result for the solution set of a system of simultaneous generalized vector quasi-equilibrium problems (for short, (SSGVQEP is obtained, which improves Theorem 3.1 of the work of Ansari et al. (J. Optim. Theory Appl. 127:27-44, 2005. Moreover, a definition of Hadamard-type well-posedness for (SSGVQEP is introduced and sufficient conditions for Hadamard well-posedness of (SSGVQEP are established.

  17. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...... or meromorphic (allowing poles as singularities) functions. There already exists a well-developed theory for iterative holomorphic dynamical systems, and successful relations found between iteration theory and flows of vector fields have been one of the main motivations for the recent interest in holomorphic...... vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...

  18. Vector control of three-phase AC machines system development in the practice

    CERN Document Server

    Quang, Nguyen Phung

    2008-01-01

    Covers the area of vector control of 3-phase AC machines, in particular induction motors with squirrel-cage rotor, permanent excited synchronous motors and doubly-fed induction machines. This title summarizes the basic structure of a field-oriented controlled 3-phase AC drive and grid voltage orientated controlled wind power plant.

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

    Directory of Open Access Journals (Sweden)

    Michelle Millington

    2009-07-01

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

  20. A reliable in vitro fruiting system for Armillaria mellea for evaluation of Agrobacterium tumefaciens transformation vectors.

    Science.gov (United States)

    Ford, Kathryn L; Baumgartner, Kendra; Henricot, Béatrice; Bailey, Andy M; Foster, Gary D

    2015-10-01

    Armillaria mellea is a serious pathogen of horticultural and agricultural systems in Europe and North America. The lack of a reliable in vitro fruiting system for heterothallic A. mellea has hindered research and required dependence on intermittently available wild-collected basidiospores of endemic genotypes, necessitating the use of variable genetic material in transformation studies. Here we describe a reliable, reproducible in vitro fruiting method for heterothallic A. mellea from the western US. Isolates and growth conditions were evaluated to determine effective fruiting conditions. Following medium colonisation for 4 weeks, cultures were incubated under warm/bright conditions for 4-6 weeks before incubation in dim/cool conditions. Primordia emerged within 3-4 weeks following a temperature decrease and this was most efficient when coupled with a light reduction. Basidiocarps matured within 3-4 weeks and produced viable basidiospores. Agrobacterium tumefaciens and vectors were evaluated by transformation of in vitro-produced basidiospores and a versatile transformation vector was constructed to simplify promoter and marker gene exchange using homologous recombination in yeast. Fruiting bodies and viable basidiospores of A. mellea have been reliably produced in vitro which, coupled with the enhanced knowledge of suitable A. tumefaciens strains and vectors for transformation, will assist future genetic research into this important pathogen. Copyright © 2015 The British Mycological Society. All rights reserved.

  1. Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

    Science.gov (United States)

    Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

    2013-01-01

    We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

  2. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...... of parameter spaces into structurally stable domains, and a description of the bifurcations. For this reason, the talk will focus on these questions for complex polynomial vector fields.......The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...

  3. Synchronous optical transmission data link integrated with FPGA for TESLA FEL SIMCON system: long data vector optical transceiver module tests

    Science.gov (United States)

    Zielinski, Jerzy S.; Pozniak, Krzysztof T.; Romaniuk, Ryszard S.

    2006-10-01

    The X-ray free-electron laser X-FEL that is being planned at the DESY research center in cooperation with European partners will produce high-intensity ultra-short X-ray flashes with the properties of laser light. This new light source, which can only be described in terms of superlatives, will open up a whole range of new possibilities for the natural sciences. It could also offer very promising opportunities for industrial users. SIMCON (SIMulator and CONtroller) is the project of the fast, low latency digital controller dedicated to the LLRF system in VUV FEL experiment. The main purpose of the project is to create a controller to stabilize the vector sum of fields in cavities of one cryo-module in the experiment. The device can be also used as the simulator of the cavity and test bench for other devices. The synchronic, optical link project was made for the accelerator X-FEL laser TESLA, the LLRF control system experiment at DESY, Hamburg. The control and diagnostic data is transmitted up to 2.5Gbit/s through a plastic fiber in a distance up to a few hundred meters. The link is synchronized once after power up, and never resynchronized when data is transmitted with maximum speed. The one way link bit error rate is less then 10 -15. The transceiver component written in VHDL that works in the dedicated Altera® Stratix® GX FPGA circuit. During the work in the PERG laboratory a 2.5Gbit/s serial link with the long vector parallel interface transceiver was created. Long-Data-Vector transceiver transmits 16bit vector each 8ns with 120ns latency.

  4. Vector analysis

    CERN Document Server

    Brand, Louis

    2006-01-01

    The use of vectors not only simplifies treatments of differential geometry, mechanics, hydrodynamics, and electrodynamics, but also makes mathematical and physical concepts more tangible and easy to grasp. This text for undergraduates was designed as a short introductory course to give students the tools of vector algebra and calculus, as well as a brief glimpse into these subjects' manifold applications. The applications are developed to the extent that the uses of the potential function, both scalar and vector, are fully illustrated. Moreover, the basic postulates of vector analysis are brou

  5. Hierarchical Modulation with Vector Rotation for E-MBMS Transmission in LTE Systems

    Directory of Open Access Journals (Sweden)

    Hui Zhao

    2010-01-01

    Full Text Available Enhanced Multimedia Broadcast and Multicast Service (E-MBMS is considered of key importance for the proliferation of Long-Term Evolution (LTE network in mobile market. Hierarchical modulation (HM, which involves a “base-layer” (BL and an “enhancement-layer” (EL bit streams, is a simple technique for achieving tradeoff between service quality and radio coverage. Therefore, it is appealing for MBMS. Generally, HM suffers from the severe performance degradation of the less protected EL stream. In this paper, HM with vector rotation operation introduced to EL stream is proposed, in order to improve EL's performance. With the proper interleaving in frequency domain, this operation can exploit the inherent diversity gain from the multipath channel. In this way, HM with vector rotation can effectively enhance multimedia broadcasting on quality video and coverage. The simulation results with scalable video coding (SVC as source show the significant benefits in comparison with the conventional HM and alternative schemes.

  6. STAT3/NF-κB-Regulated Lentiviral TK/GCV Suicide Gene Therapy for Cisplatin-Resistant Triple-Negative Breast Cancer.

    Science.gov (United States)

    Kuo, Wei-Ying; Hwu, Luen; Wu, Chun-Yi; Lee, Jhih-Shian; Chang, Chi-Wei; Liu, Ren-Shyan

    2017-01-01

    Triple-negative breast cancer (TNBC) represents approximately 20% of all breast cancers and appears resistance to conventional cytotoxic chemotherapy, demonstrating a particularly poor prognosis and a significantly worse clinical outcome than other types of cancer. Suicide gene therapy has been used for the in vivo treatment of various solid tumors in recent clinical trials. In tumor microenvironment, STAT3/NF-κB pathways are constitutively activated in stromal cells as well as in cancer stem cells (CSCs). In this study, we have cloned a novel STAT3/NF-κB-based reporter system to drive the expression of herpes simplex virus thymidine kinase (HSV-TK) against breast cancer. Lentiviral vector expressing HSV-TK under the regulation of STAT3/NF-κB fused response element was developed. In this setting, we exploited the constitutive STAT3/NF-κB activation in tumors to achieve higher transgene expression than that driven by a constitutively active CMV promotor in vivo . An orthotropic MDA-MB-231 triple negative breast cancer mouse model was used for evaluating the feasibility of STAT3-NF-κB-TK/GCV suicide gene therapy system. The basal promoter activity of Lenti-CMV-TK and Lenti-STAT3-NF-κB-TK in MDA-MB-231 cells was compared by 3 H-FEAU uptake assay. The Lenti-CMV-TK showed ~5 fold higher 3 H-FEAU uptake then Lenti -STAT3-NF-κB-TK. In clonogenic assay, cells expressing Lenti-CMV-TK were 2-fold more sensitive to GCV than Lenti-STAT3-NF-κB-TK transduced cells. In vitro effect of STAT3-NF-κB-induced transgene expression was determined by 10ng/mL TNF-α induction and confirmed by western blot analysis and DsRedm fluorescent microscopy. In vivo evaluation of therapeutic effect by bioluminescence and [ 18 F]FHBG microPET imaging indicated that Lenti-STAT3-NF-κB-TK showed more tumor growth retardation than Lenti-CMV-TK when GCV (20 mg/kg) was administered. The invasiveness and expression of cancer stem cell markers were both decreased after STAT3/NF-κB-regulated HSV

  7. An H-band Vector Vortex Coronagraph for the Subaru Coronagraphic Extreme-adaptive Optics System

    Science.gov (United States)

    Kühn, J.; Serabyn, E.; Lozi, J.; Jovanovic, N.; Currie, T.; Guyon, O.; Kudo, T.; Martinache, F.; Liewer, K.; Singh, G.; Tamura, M.; Mawet, D.; Hagelberg, J.; Defrere, D.

    2018-03-01

    The vector vortex is a coronagraphic imaging mode of the recently commissioned Subaru Coronagraphic Extreme Adaptive Optics (SCExAO) platform on the 8 m Subaru Telescope. This multi-purpose high-contrast visible and near-infrared (R- to K-band) instrument is not only intended to serve as a VLT-class “planet-imager” instrument in the northern hemisphere, but also to operate as a technology demonstration testbed ahead of the ELTs-era, with a particular emphasis on small inner-working angle (IWA) coronagraphic capabilities. The given priority to small-IWA imaging led to the early design choice to incorporate focal-plane phase-mask coronagraphs. In this context, a test H-band vector vortex liquid crystal polymer waveplate was provided to SCExAO, to allow a one-to-one comparison of different small-IWA techniques on the same telescope instrument, before considering further steps. Here we present a detailed overview of the vector vortex coronagraph, from its installation and performances on the SCExAO optical bench, to the on-sky results in the extreme AO regime, as of late 2016/early 2017. To this purpose, we also provide a few recent on-sky imaging examples, notably high-contrast ADI detection of the planetary-mass companion κ Andromedae b, with a signal-to-noise ratio above 100 reached in less than 10 mn exposure time.

  8. A Fast Computation for the State Vector in a Max-Plus Algebraic System with an Adjacency Matrix of a Directed Acyclic Graph

    Science.gov (United States)

    Goto, Hiroyuki

    We provide a useful method for calculating the state vector of a state equation efficiently in a max-plus algebraic system. For a discrete event system whose precedence relationships are represented by a directed acyclic graph, computing the transition matrix, which includes the Kleene star operation of a weighted adjacency matrix, is occasionally the bottleneck. On the other hand, the common objective is to compute the state equation, rather than the transition matrix itself. Since the state equation is essentially the multiplication of the transition matrix and vector, we propose algorithms for efficiently calculating the multiplication and left division of the Kleene star of an adjacency matrix and a vector.

  9. Vector velocimeter

    DEFF Research Database (Denmark)

    2012-01-01

    The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...

  10. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes.

    Science.gov (United States)

    Kang, W; Wang, L; Harrell, H; Liu, J; Thomas, D L; Mayfield, T L; Scotti, M M; Ye, G J; Veres, G; Knop, D R

    2009-02-01

    Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.

  11. Revisiting the study of pedophilia phenomenon and related issues in Russia and in the world on the basis of Yuri Burlan’s System vector Psychology

    OpenAIRE

    Vinevskaya, Anna; Ochirova, Valentina

    2014-01-01

    The paper is about pedophilia phenomenon. Pedophilia is a phenomenon that should be studied through innovative approaches in science. The causes of pedophilia and the early prophylaxis methods have been discovered in Yuri Burlan’s System-Vector Psychology.

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

    Science.gov (United States)

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

    2018-04-10

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

  13. Malaria in Kakuma refugee camp, Turkana, Kenya: facilitation of Anopheles arabiensis vector populations by installed water distribution and catchment systems

    Directory of Open Access Journals (Sweden)

    Cetron Martin S

    2011-06-01

    Full Text Available Abstract Background Malaria is a major health concern for displaced persons occupying refugee camps in sub-Saharan Africa, yet there is little information on the incidence of infection and nature of transmission in these settings. Kakuma Refugee Camp, located in a dry area of north-western Kenya, has hosted ca. 60,000 to 90,000 refugees since 1992, primarily from Sudan and Somalia. The purpose of this study was to investigate malaria prevalence and attack rate and sources of Anopheles vectors in Kakuma refugee camp, in 2005-2006, after a malaria epidemic was observed by staff at camp clinics. Methods Malaria prevalence and attack rate was estimated from cases of fever presenting to camp clinics and the hospital in August 2005, using rapid diagnostic tests and microscopy of blood smears. Larval habitats of vectors were sampled and mapped. Houses were sampled for adult vectors using the pyrethrum knockdown spray method, and mapped. Vectors were identified to species level and their infection with Plasmodium falciparum determined. Results Prevalence of febrile illness with P. falciparum was highest among the 5 to 17 year olds (62.4% while malaria attack rate was highest among the two to 4 year olds (5.2/1,000/day. Infected individuals were spatially concentrated in three of the 11 residential zones of the camp. The indoor densities of Anopheles arabiensis, the sole malaria vector, were similar during the wet and dry seasons, but were distributed in an aggregated fashion and predominantly in the same zones where malaria attack rates were high. Larval habitats and larval populations were also concentrated in these zones. Larval habitats were man-made pits of water associated with tap-stands installed as the water delivery system to residents with year round availability in the camp. Three percent of A. arabiensis adult females were infected with P. falciparum sporozoites in the rainy season. Conclusions Malaria in Kakuma refugee camp was due mainly

  14. An Adaptive Supervisory Sliding Fuzzy Cerebellar Model Articulation Controller for Sensorless Vector-Controlled Induction Motor Drive Systems

    Directory of Open Access Journals (Sweden)

    Shun-Yuan Wang

    2015-03-01

    Full Text Available This paper presents the implementation of an adaptive supervisory sliding fuzzy cerebellar model articulation controller (FCMAC in the speed sensorless vector control of an induction motor (IM drive system. The proposed adaptive supervisory sliding FCMAC comprised a supervisory controller, integral sliding surface, and an adaptive FCMAC. The integral sliding surface was employed to eliminate steady-state errors and enhance the responsiveness of the system. The adaptive FCMAC incorporated an FCMAC with a compensating controller to perform a desired control action. The proposed controller was derived using the Lyapunov approach, which guarantees learning-error convergence. The implementation of three intelligent control schemes—the adaptive supervisory sliding FCMAC, adaptive sliding FCMAC, and adaptive sliding CMAC—were experimentally investigated under various conditions in a realistic sensorless vector-controlled IM drive system. The root mean square error (RMSE was used as a performance index to evaluate the experimental results of each control scheme. The analysis results indicated that the proposed adaptive supervisory sliding FCMAC substantially improved the system performance compared with the other control schemes.

  15. Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

    Science.gov (United States)

    Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

    2015-02-10

    Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    Science.gov (United States)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  17. Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi

    OpenAIRE

    Taracena, Mabel L.; Oliveira, Pedro L.; Almendares, Olivia; Uma?a, Claudia; Lowenberger, Carl; Dotson, Ellen M.; Paiva-Silva, Gabriela O.; Pennington, Pamela M.

    2015-01-01

    Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all de...

  18. Robust anti-synchronization of uncertain chaotic systems based on multiple-kernel least squares support vector machine modeling

    International Nuclear Information System (INIS)

    Chen Qiang; Ren Xuemei; Na Jing

    2011-01-01

    Highlights: Model uncertainty of the system is approximated by multiple-kernel LSSVM. Approximation errors and disturbances are compensated in the controller design. Asymptotical anti-synchronization is achieved with model uncertainty and disturbances. Abstract: In this paper, we propose a robust anti-synchronization scheme based on multiple-kernel least squares support vector machine (MK-LSSVM) modeling for two uncertain chaotic systems. The multiple-kernel regression, which is a linear combination of basic kernels, is designed to approximate system uncertainties by constructing a multiple-kernel Lagrangian function and computing the corresponding regression parameters. Then, a robust feedback control based on MK-LSSVM modeling is presented and an improved update law is employed to estimate the unknown bound of the approximation error. The proposed control scheme can guarantee the asymptotic convergence of the anti-synchronization errors in the presence of system uncertainties and external disturbances. Numerical examples are provided to show the effectiveness of the proposed method.

  19. The Automation System Censor Speech for the Indonesian Rude Swear Words Based on Support Vector Machine and Pitch Analysis

    Science.gov (United States)

    Endah, S. N.; Nugraheni, D. M. K.; Adhy, S.; Sutikno

    2017-04-01

    According to Law No. 32 of 2002 and the Indonesian Broadcasting Commission Regulation No. 02/P/KPI/12/2009 & No. 03/P/KPI/12/2009, stated that broadcast programs should not scold with harsh words, not harass, insult or demean minorities and marginalized groups. However, there are no suitable tools to censor those words automatically. Therefore, researches to develop a system of intelligent software to censor the words automatically are needed. To conduct censor, the system must be able to recognize the words in question. This research proposes the classification of speech divide into two classes using Support Vector Machine (SVM), first class is set of rude words and the second class is set of properly words. The speech pitch values as an input in SVM, it used for the development of the system for the Indonesian rude swear word. The results of the experiment show that SVM is good for this system.

  20. Cloning vector

    Science.gov (United States)

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  1. Cloning vector

    Science.gov (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  2. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    International Nuclear Information System (INIS)

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B.

    1990-01-01

    The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli β-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses β-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system

  3. A novel minicircle vector based system for inhibting the replication and gene expression of enterovirus 71 and coxsackievirus A16.

    Science.gov (United States)

    Yang, Zhuo; Li, Guodong; Zhang, Yingqiu; Liu, Xiaoman; Tien, Po

    2012-11-01

    Enterovirus 71 (EV 71) and Coxsackievirus A16 (CA 16) are two major causative agents of hand, foot and mouth disease (HFMD). They have been associated with severe neurological and cardiological complications worldwide, and have caused significant mortalities during large-scale outbreaks in China. Currently, there are no effective treatments against EV 71 and CA 16 infections. We now describe the development of a novel minicircle vector based RNA interference (RNAi) system as a therapeutic approach to inhibiting EV 71 and CA 16 replication. Small interfering RNA (siRNA) molecules targeting the conserved regions of the 3C(pro) and 3D(pol) function gene of the EV 71 and CA 16 China strains were designed based on their nucleotide sequences available in GenBank. This RNAi system was found to effectively block the replication and gene expression of these viruses in rhabdomyosarcoma (RD) cells and virus-infected mice model. The inhibitory effects were confirmed by a corresponding decrease in viral RNA, viral protein, and progeny virus production. In addition, no significant adverse off-target silencing or cytotoxic effects were observed. These results demonstrated the potential and feasibility of this novel minicircle vector based RNAi system for antiviral therapy against EV 71 and CA 16 infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. The production of viral vectors designed to express large and difficult to express transgenes within neurons.

    Science.gov (United States)

    Holehonnur, Roopashri; Lella, Srihari K; Ho, Anthony; Luong, Jonathan A; Ploski, Jonathan E

    2015-02-24

    Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons. We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains. We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express

  5. A least square support vector machine-based approach for contingency classification and ranking in a large power system

    Directory of Open Access Journals (Sweden)

    Bhanu Pratap Soni

    2016-12-01

    Full Text Available This paper proposes an effective supervised learning approach for static security assessment of a large power system. Supervised learning approach employs least square support vector machine (LS-SVM to rank the contingencies and predict the system severity level. The severity of the contingency is measured by two scalar performance indices (PIs: line MVA performance index (PIMVA and Voltage-reactive power performance index (PIVQ. SVM works in two steps. Step I is the estimation of both standard indices (PIMVA and PIVQ that is carried out under different operating scenarios and Step II contingency ranking is carried out based on the values of PIs. The effectiveness of the proposed methodology is demonstrated on IEEE 39-bus (New England system. The approach can be beneficial tool which is less time consuming and accurate security assessment and contingency analysis at energy management center.

  6. Vector Control System Design for Four Degree-of-Freedom Dynamic Flexible Simulation of the Variable-Frequency Drive

    Directory of Open Access Journals (Sweden)

    Kladiev Sergey N.

    2017-01-01

    Full Text Available In the present work we investigate the control system development of the drive simulators to train driver/operator driving skills, taking into account the ever-changing terrain. In order to meet the required response of the four degree-of-freedom motion platform servomotor current studies have been focused on the vector control of the resistance motor angular velocity from the sensor being incremental encoder. In proposed system the standard security of the frequency converter is realized. It leads to overload capacity of two times within minutes determined by servomotor inertia. Further, we represent the algorithms: positional limitation, reliable acceleration and restraint, frequency break. As well as we demonstrate the position switches implement in software. As a result, the control system commands the control of the angular position of the platform in coordinates.

  7. Signal Detection for QPSK Based Cognitive Radio Systems using Support Vector Machines

    Directory of Open Access Journals (Sweden)

    M. T. Mushtaq

    2015-04-01

    Full Text Available Cognitive radio based network enables opportunistic dynamic spectrum access by sensing, adopting and utilizing the unused portion of licensed spectrum bands. Cognitive radio is intelligent enough to adapt the communication parameters of the unused licensed spectrum. Spectrum sensing is one of the most important tasks of the cognitive radio cycle. In this paper, the auto-correlation function kernel based Support Vector Machine (SVM classifier along with Welch's Periodogram detector is successfully implemented for the detection of four QPSK (Quadrature Phase Shift Keying based signals propagating through an AWGN (Additive White Gaussian Noise channel. It is shown that the combination of statistical signal processing and machine learning concepts improve the spectrum sensing process and spectrum sensing is possible even at low Signal to Noise Ratio (SNR values up to -50 dB.

  8. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Deng, Ling; Zhu, Haojun; Chen, Zhengjun

    2009-01-01

    , and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were...... mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out...... constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers....

  9. 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.

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

    Science.gov (United States)

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

    2011-07-16

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

  11. Generation of retroviral particles for the spleen necrosis virus (SNV)-based vector system and their use in transduction of various cell types.

    Science.gov (United States)

    Parveen, Zahida; Mukhtar, Muhammad; Pomerantz, Roger J

    2010-06-01

    Genetically engineered retroviruses are widely used for gene delivery into human cells. A number of investigators have studied spleen necrosis virus (SNV) as a vehicle for gene delivery. Vectors developed from SNV and its closely associated avian reticuloendotheliosis virus strain A (REV-A) can be used for gene transfer into a variety of cells, including primary hematopoietic cells and human brain and post-mitotic neuronal cells that are difficult to transduce with other vector systems. SNV-based vector systems have the advantage of being quite safe, because wild-type SNV is unable to infect human cells and has less preference for integration into transcriptionally active sites or genes. However, the generation of retroviral vectors requires cotransfection of more than one plasmid into a packaging cell line, which is a tedious process. The development of stable packaging cell lines expressing envelope (Env) proteins and the structural proteins Gag-Pol will enhance mass production of retroviral vectors for future gene therapy experiments both in vitro and in vivo. This protocol describes the generation of retroviral particles for the SNV-based vector system. These particles can then be used for transduction of various cell types; as an example, a technique for transduction of post-mitotic neurons is also presented.

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

    Directory of Open Access Journals (Sweden)

    Dismuke Adria D

    2009-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Kohn Aimee

    2009-01-01

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

  14. Quantitative Assessment of Pap Smear Cells by PC-Based Cytopathologic Image Analysis System and Support Vector Machine

    Science.gov (United States)

    Huang, Po-Chi; Chan, Yung-Kuan; Chan, Po-Chou; Chen, Yung-Fu; Chen, Rung-Ching; Huang, Yu-Ruei

    Cytologic screening has been widely used for controlling the prevalence of cervical cancer. Errors from sampling, screening and interpretation, still concealed some unpleasant results. This study aims at designing a cellular image analysis system based on feasible and available software and hardware for a routine cytologic laboratory. Totally 1814 cellular images from the liquid-based cervical smears with Papanicolaou stain in 100x, 200x, and 400x magnification were captured by a digital camera. Cell images were reviewed by pathologic experts with peer agreement and only 503 images were selected for further study. The images were divided into 4 diagnostic categories. A PC-based cellular image analysis system (PCCIA) was developed for computing morphometric parameters. Then support vector machine (SVM) was used to classify signature patterns. The results show that the selected 13 morphometric parameters can be used to correctly differentiate the dysplastic cells from the normal cells (pgynecologic cytologic specimens.

  15. Information security of industrial control systems: possible attack vectors and protection methods

    Directory of Open Access Journals (Sweden)

    Ignatiy A. Grachkov

    2018-03-01

    obtaining unauthorized access to industrial control systems using the Shodan search engine is described and recommendations how to ensure information security of the industrial control system are given.

  16. Molecular mechanism of mutagenesis induced by olaquindox using a shuttle vector pSP189/mammalian cell system

    International Nuclear Information System (INIS)

    Hao Lihua; Chen Qian; Xiao Xilong

    2006-01-01

    Olaquindox, a quinoxaline 1,4-dioxide derivative from quindoxin, is widely used as an animal growth promoter in China. We tested olaquindox as a mutagen in a SV40-based shuttle vector pSP189 and African green kidney cell (Vero E6 cell line) system to define the safety of olaquindox as a food-additive for animals. When applied at 6.6 μg/ml, olaquindox caused 12 times higher mutation frequency in comparison to untreated controls. More than 70% of base substitutions happened at G:C base pairs featuring G:C to T:A or G:C to A:T conversions. Frequency of point mutations for in vitro modified plasmids was also dramatically increased from the spontaneous background level. Olaquindox-induced mutations did not occur randomly along the supF shuttle vector, but instead, had a hot spot at base pair no. 155 which accounts for 37% of total mutations. Olaquindox-induced mutations also showed sequence-specificity in which most point mutations occurred at site N in a 5'-NNTTNN-3' sequence while most tandem bases deletion and rearrangement were seen at the 5'-ANGGCCNAAA-3' sequence. We conclude that olaquindox induces DNA mutation, therefore, should not be used as an additive to promote animal growth

  17. The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector.

    Science.gov (United States)

    Correa, Valdir R; Majerczak, Doris R; Ammar, El-Desouky; Merighi, Massimo; Pratt, Richard C; Hogenhout, Saskia A; Coplin, David L; Redinbaugh, Margaret G

    2012-09-01

    Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host.

  18. Systemic Errors in Quantitative Polymerase Chain Reaction Titration of Self-Complementary Adeno-Associated Viral Vectors and Improved Alternative Methods

    Science.gov (United States)

    Fagone, Paolo; Wright, J. Fraser; Nathwani, Amit C.; Nienhuis, Arthur W.; Davidoff, Andrew M.

    2012-01-01

    Abstract Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat that connects the two monomer single-stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. In addition, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has low interassay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities. PMID:22428975

  19. Application of Diagnostic Analysis Tools to the Ares I Thrust Vector Control System

    Science.gov (United States)

    Maul, William A.; Melcher, Kevin J.; Chicatelli, Amy K.; Johnson, Stephen B.

    2010-01-01

    The NASA Ares I Crew Launch Vehicle is being designed to support missions to the International Space Station (ISS), to the Moon, and beyond. The Ares I is undergoing design and development utilizing commercial-off-the-shelf tools and hardware when applicable, along with cutting edge launch technologies and state-of-the-art design and development. In support of the vehicle s design and development, the Ares Functional Fault Analysis group was tasked to develop an Ares Vehicle Diagnostic Model (AVDM) and to demonstrate the capability of that model to support failure-related analyses and design integration. One important component of the AVDM is the Upper Stage (US) Thrust Vector Control (TVC) diagnostic model-a representation of the failure space of the US TVC subsystem. This paper first presents an overview of the AVDM, its development approach, and the software used to implement the model and conduct diagnostic analysis. It then uses the US TVC diagnostic model to illustrate details of the development, implementation, analysis, and verification processes. Finally, the paper describes how the AVDM model can impact both design and ground operations, and how some of these impacts are being realized during discussions of US TVC diagnostic analyses with US TVC designers.

  20. Nighttime Fire/Smoke Detection System Based on a Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Chao-Ching Ho

    2013-01-01

    Full Text Available Currently, video surveillance-based early fire smoke detection is crucial to the prevention of large fires and the protection of life and goods. To overcome the nighttime limitations of video smoke detection methods, a laser light can be projected into the monitored field of view, and the returning projected light section image can be analyzed to detect fire and/or smoke. If smoke appears within the monitoring zone created from the diffusion or scattering of light in the projected path, the camera sensor receives a corresponding signal. The successive processing steps of the proposed real-time algorithm use the spectral, diffusing, and scattering characteristics of the smoke-filled regions in the image sequences to register the position of possible smoke in a video. Characterization of smoke is carried out by a nonlinear classification method using a support vector machine, and this is applied to identify the potential fire/smoke location. Experimental results in a variety of nighttime conditions demonstrate that the proposed fire/smoke detection method can successfully and reliably detect fires by identifying the location of smoke.

  1. Vector geometry

    CERN Document Server

    Robinson, Gilbert de B

    2011-01-01

    This brief undergraduate-level text by a prominent Cambridge-educated mathematician explores the relationship between algebra and geometry. An elementary course in plane geometry is the sole requirement for Gilbert de B. Robinson's text, which is the result of several years of teaching and learning the most effective methods from discussions with students. Topics include lines and planes, determinants and linear equations, matrices, groups and linear transformations, and vectors and vector spaces. Additional subjects range from conics and quadrics to homogeneous coordinates and projective geom

  2. Engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells

    Science.gov (United States)

    Mehalko, Jennifer L.; Esposito, Dominic

    2016-01-01

    One of the most common methods for producing recombinant baculovirus for insect cell protein production involves a transposition mediated system invented over 2 decades ago. This Tn7-mediated system, commercially sold as Bac-to-Bac, has proven highly useful for construction of high quality baculovirus, but suffers from a number of drawbacks which reduce the efficiency of the process and limit its utility for high throughput protein production processes. We describe here the creation of Bac-2-the-Future, a 2nd generation Tn7-based system for recombinant baculovirus production which uses optimized expression vectors, new E. coli strains, and enhanced protocols to dramatically enhance the efficiency of the baculovirus production process. The new system which we describe eliminates the need for additional screening of positive clones, improves the efficiency of transposition, and reduces the cost and time required for high throughput baculovirus production. The system is compatible with multiple cloning methodologies, and has been demonstrated to produce baculovirus with equal or better titer and protein productivity than the currently available systems. PMID:27616621

  3. Modified Lentiviral LTRs Allow Flp Recombinase–mediated Cassette Exchange and In Vivo Tracing of “Factor-free” Induced Pluripotent Stem Cells

    Science.gov (United States)

    Kuehle, Johannes; Turan, Soeren; Cantz, Tobias; Hoffmann, Dirk; Suerth, Julia D; Maetzig, Tobias; Zychlinski, Daniela; Klein, Christoph; Steinemann, Doris; Baum, Christopher; Bode, Juergen; Schambach, Axel

    2014-01-01

    Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for “safe harbor” integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy. PMID:24434935

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

    Directory of Open Access Journals (Sweden)

    Andreas Holmgaard

    2017-12-01

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

  5. [Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

    Science.gov (United States)

    Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

    2011-08-01

    We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

  6. An Improved Azimuth Angle Estimation Method with a Single Acoustic Vector Sensor Based on an Active Sonar Detection System

    Science.gov (United States)

    Zhao, Anbang; Ma, Lin; Ma, Xuefei; Hui, Juan

    2017-01-01

    In this paper, an improved azimuth angle estimation method with a single acoustic vector sensor (AVS) is proposed based on matched filtering theory. The proposed method is mainly applied in an active sonar detection system. According to the conventional passive method based on complex acoustic intensity measurement, the mathematical and physical model of this proposed method is described in detail. The computer simulation and lake experiments results indicate that this method can realize the azimuth angle estimation with high precision by using only a single AVS. Compared with the conventional method, the proposed method achieves better estimation performance. Moreover, the proposed method does not require complex operations in frequency-domain and achieves computational complexity reduction. PMID:28230763

  7. An Improved Azimuth Angle Estimation Method with a Single Acoustic Vector Sensor Based on an Active Sonar Detection System.

    Science.gov (United States)

    Zhao, Anbang; Ma, Lin; Ma, Xuefei; Hui, Juan

    2017-02-20

    In this paper, an improved azimuth angle estimation method with a single acoustic vector sensor (AVS) is proposed based on matched filtering theory. The proposed method is mainly applied in an active sonar detection system. According to the conventional passive method based on complex acoustic intensity measurement, the mathematical and physical model of this proposed method is described in detail. The computer simulation and lake experiments results indicate that this method can realize the azimuth angle estimation with high precision by using only a single AVS. Compared with the conventional method, the proposed method achieves better estimation performance. Moreover, the proposed method does not require complex operations in frequencydomain and achieves computational complexity reduction.

  8. Combining support vector machines with linear quadratic regulator adaptation for the online design of an automotive active suspension system

    International Nuclear Information System (INIS)

    Chiou, J-S; Liu, M-T

    2008-01-01

    As a powerful machine-learning approach to pattern recognition problems, the support vector machine (SVM) is known to easily allow generalization. More importantly, it works very well in a high-dimensional feature space. This paper presents a nonlinear active suspension controller which achieves a high level performance by compensating for actuator dynamics. We use a linear quadratic regulator (LQR) to ensure optimal control of nonlinear systems. An LQR is used to solve the problem of state feedback and an SVM is used to address the question of the estimation and examination of the state. These two are then combined and designed in a way that outputs feedback control. The real-time simulation demonstrates that an active suspension using the combined SVM-LQR controller provides passengers with a much more comfortable ride and better road handling

  9. VECTOR INTEGRATION

    NARCIS (Netherlands)

    Thomas, E. G. F.

    2012-01-01

    This paper deals with the theory of integration of scalar functions with respect to a measure with values in a, not necessarily locally convex, topological vector space. It focuses on the extension of such integrals from bounded measurable functions to the class of integrable functions, proving

  10. The Key Role of the Vector Optimization Algorithm and Robust Design Approach for the Design of Polygeneration Systems

    Directory of Open Access Journals (Sweden)

    Alfredo Gimelli

    2018-04-01

    Full Text Available In recent decades, growing concerns about global warming and climate change effects have led to specific directives, especially in Europe, promoting the use of primary energy-saving techniques and renewable energy systems. The increasingly stringent requirements for carbon dioxide reduction have led to a more widespread adoption of distributed energy systems. In particular, besides renewable energy systems for power generation, one of the most effective techniques used to face the energy-saving challenges has been the adoption of polygeneration plants for combined heating, cooling, and electricity generation. This technique offers the possibility to achieve a considerable enhancement in energy and cost savings as well as a simultaneous reduction of greenhouse gas emissions. However, the use of small-scale polygeneration systems does not ensure the achievement of mandatory, but sometimes conflicting, aims without the proper sizing and operation of the plant. This paper is focused on a methodology based on vector optimization algorithms and developed by the authors for the identification of optimal polygeneration plant solutions. To this aim, a specific calculation algorithm for the study of cogeneration systems has also been developed. This paper provides, after a detailed description of the proposed methodology, some specific applications to the study of combined heat and power (CHP and organic Rankine cycle (ORC plants, thus highlighting the potential of the proposed techniques and the main results achieved.

  11. An Orbital Feedback Linearization Approach to Solving Terminal Problems for Affine Systems with Vector Control

    Directory of Open Access Journals (Sweden)

    D. A. Fetisov

    2015-01-01

    Full Text Available State-feedback linearization is widely used to solve various problems of the control theory. An affine system is said to be state-feedback linearizable if there are a smooth change of variables in the space of states and an invertible change of controls, which transform the system to the system of a regular canonical form. However if a system is not state-feedback linearizable it yet can be orbitally feedback linearized, i.e. the system can be transformed to a regular canonical form after a change of the independent variable.The article solves the following terminal problem for multi-dimensional stationary affine systems: for given two states, find controls and a time T such that the corresponding trajectory of the system joins these states for the time T. We make an integrable change of the independent variable depending on controls. As a result, we obtain a non-stationary affine system, its dimension being one less than dimension of the original system. The new terminal problem with the restriction on controls is formulated for the transformed system. We prove the relation between solutions of the original terminal problem and solutions of the terminal problem for the transformed system. It is shown that to solve the original terminal problem it is sufficient to solve terminal problem for the transformed system. Then, we check whether the transformed system can be state-feedback linearized. For this purpose, we check the necessary and sufficient conditions of state-feedback linearization for non-stationary affine systems. If the conditions are met then we transform the system to a regular canonical form for which the concept of inverse dynamics problems can be used to solve terminal problems. However, due to the restriction on controls an additional check is necessary whether the found controls meet the restriction.An example of the terminal problem for the five-dimensional affine system with two controls is given. We prove that the system in

  12. Sampling strategies based on singular vectors for assimilated models in ocean forecasting systems

    Science.gov (United States)

    Fattorini, Maria; Brandini, Carlo; Ortolani, Alberto

    2016-04-01

    Meteorological and oceanographic models do need observations, not only as a ground truth element to verify the quality of the models, but also to keep model forecast error acceptable: through data assimilation techniques which merge measured and modelled data, natural divergence of numerical solutions from reality can be reduced / controlled and a more reliable solution - called analysis - is computed. Although this concept is valid in general, its application, especially in oceanography, raises many problems due to three main reasons: the difficulties that have ocean models in reaching an acceptable state of equilibrium, the high measurements cost and the difficulties in realizing them. The performances of the data assimilation procedures depend on the particular observation networks in use, well beyond the background quality and the used assimilation method. In this study we will present some results concerning the great impact of the dataset configuration, in particular measurements position, on the evaluation of the overall forecasting reliability of an ocean model. The aim consists in identifying operational criteria to support the design of marine observation networks at regional scale. In order to identify the observation network able to minimize the forecast error, a methodology based on Singular Vectors Decomposition of the tangent linear model is proposed. Such a method can give strong indications on the local error dynamics. In addition, for the purpose of avoiding redundancy of information contained in the data, a minimal distance among data positions has been chosen on the base of a spatial correlation analysis of the hydrodynamic fields under investigation. This methodology has been applied for the choice of data positions starting from simplified models, like an ideal double-gyre model and a quasi-geostrophic one. Model configurations and data assimilation are based on available ROMS routines, where a variational assimilation algorithm (4D-var) is

  13. A reliable in vitro fruiting system for armillaria mellea for evaluation of agrobacterium tumefaciens transformation vectors

    Science.gov (United States)

    Armillaria mellea is a serious pathogen of horticultural and agricultural systems in Europe and North America. The lack of a reliable in vitro fruiting system has hindered research, and necessitated dependence on intermittently available wild-collected basidiospores. Here we describe a reliable, rep...

  14. Enhancement of Antituberculosis Immunity in a Humanized Model System by a Novel Virus-Vectored Respiratory Mucosal Vaccine.

    Science.gov (United States)

    Yao, Yushi; Lai, Rocky; Afkhami, Sam; Haddadi, Siamak; Zganiacz, Anna; Vahedi, Fatemeh; Ashkar, Ali A; Kaushic, Charu; Jeyanathan, Mangalakumari; Xing, Zhou

    2017-07-01

    The translation of preclinically promising novel tuberculosis vaccines to ultimate human applications has been challenged by the lack of animal models with an immune system equivalent to the human immune system in its genetic diversity and level of susceptibility to tuberculosis. We have developed a humanized mice (Hu-mice) tuberculosis model system to investigate the clinical relevance of a novel virus-vectored (VV) tuberculosis vaccine administered via respiratory mucosal or parenteral route. We find that VV vaccine activates T cells in Hu-mice as it does in human vaccinees. The respiratory mucosal route for delivery of VV vaccine in Hu-mice, but not the parenteral route, significantly reduces the humanlike lung tuberculosis outcomes in a human T-cell-dependent manner. Our results suggest that the Hu-mouse can be used to predict the protective efficacy of novel tuberculosis vaccines/strategies before they proceed to large, expensive human trials. This new vaccine testing system will facilitate the global pace of clinical tuberculosis vaccine development. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  15. Performance improvement of 64-QAM coherent optical communication system by optimizing symbol decision boundary based on support vector machine

    Science.gov (United States)

    Chen, Wei; Zhang, Junfeng; Gao, Mingyi; Shen, Gangxiang

    2018-03-01

    High-order modulation signals are suited for high-capacity communication systems because of their high spectral efficiency, but they are more vulnerable to various impairments. For the signals that experience degradation, when symbol points overlap on the constellation diagram, the original linear decision boundary cannot be used to distinguish the classification of symbol. Therefore, it is advantageous to create an optimum symbol decision boundary for the degraded signals. In this work, we experimentally demonstrated the 64-quadrature-amplitude modulation (64-QAM) coherent optical communication system using support-vector machine (SVM) decision boundary algorithm to create the optimum symbol decision boundary for improving the system performance. We investigated the influence of various impairments on the 64-QAM coherent optical communication systems, such as the impairments caused by modulator nonlinearity, phase skew between in-phase (I) arm and quadrature-phase (Q) arm of the modulator, fiber Kerr nonlinearity and amplified spontaneous emission (ASE) noise. We measured the bit-error-ratio (BER) performance of 75-Gb/s 64-QAM signals in the back-to-back and 50-km transmission. By using SVM to optimize symbol decision boundary, the impairments caused by I/Q phase skew of the modulator, fiber Kerr nonlinearity and ASE noise are greatly mitigated.

  16. Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.

    Science.gov (United States)

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T; Skilton, Rachel J; Lambden, Paul R; Clarke, Ian N

    2011-09-01

    Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by

  17. Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.

    Directory of Open Access Journals (Sweden)

    Yibing Wang

    2011-09-01

    Full Text Available Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB. Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP. As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome

  18. The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system.

    Science.gov (United States)

    Kim, Cha Young; Ahn, Young Ock; Kim, Sun Ha; Kim, Yun-Hee; Lee, Haeng-Soon; Catanach, Andrew S; Jacobs, Jeanne M E; Conner, Anthony J; Kwak, Sang-Soo

    2010-07-01

    MYB transcription factors play important roles in transcriptional regulation of many secondary metabolites including anthocyanins. We cloned the R2R3-MYB type IbMYB1 complementary DNAs from the purple-fleshed sweet potato (Ipomoea batatas L. cv Sinzami) and investigated the expression patterns of IbMYB1 gene with IbMYB1a and IbMYB1b splice variants in leaf and root tissues of various sweet potato cultivars by reverse transcription-polymerase chain reaction. The transcripts of IbMYB1 were predominantly expressed in the purple-fleshed storage roots and they were also detectable in the leaf tissues accumulating anthocyanin pigments. In addition, transcript levels of IbMYB1 gene were up-regulated by treatment with methyl jasmonate or salicylic acid in leaf and root tissues of cv. White Star. To set up the intragenic vector system in sweet potato, we first evaluated the utilization of the IbMYB1 gene as a visible selectable marker. The IbMYB1a was transiently expressed in tobacco leaves under the control of a constitutive cauliflower mosaic virus 35S promoter, a root-specific and sucrose-inducible sporamin promoter, and an oxidative stress-inducible sweet potato anionic peroxidase2 promoter. We also showed that overexpression of IbMYB1a induced massive anthocyanin pigmentation in tobacco leaves and up-regulated the transcript levels of the structural genes in anthocyanin biosynthetic pathway. Furthermore, high-performance liquid chromatography analysis revealed that the expression of IbMYB1a led to production of cyanidin as a major core molecule of anthocyanidins in tobacco leaves. These results suggest that the IbMYB1 gene can be applicable to a visible marker for sweet potato transformation with intragenic vectors, as well as the production of anthocyanin as important nutritive value in other plant species.

  19. 3D Modelling of a Vectored Water Jet-Based Multi-Propeller Propulsion System for a Spherical Underwater Robot

    Directory of Open Access Journals (Sweden)

    Xichuan Lin

    2013-01-01

    Full Text Available This paper presents an improved modelling method for a water jet-based multi-propeller propulsion system. In our previous work, the modelling experiments were only carried out in 2D planes, whose experimental results had poor agreement when we wanted to control the propulsive forces in 3D space directly. This research extends the 2D modelling described in the authors' previous work into 3D space. By doing this, the model could include 3D space information, which is more useful than that of 2D space. The effective propulsive forces and moments in 3D space can be obtained directly by synthesizing the propulsive vectors of propellers. For this purpose, a novel experimental mechanism was developed to achieve the proposed 3D modelling. This mechanism was designed with the mass distribution centred for the robot. By installing a six-axis load-cell sensor at the equivalent mass centre, we obtained the direct propulsive effect of the system for the robot. Also, in this paper, the orientation surface and propulsive surfaces are developed to provide the 3D information of the propulsive system. Experiments for each propeller were first carried out to establish the models. Then, further experiments were carried out with all of the propellers working together to validate the models. Finally, we compared the various experimental results with the simulation data. The utility of this modelling method is discussed at length.

  20. A Short-Term and High-Resolution System Load Forecasting Approach Using Support Vector Regression with Hybrid Parameters Optimization

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Huaiguang [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2017-08-25

    This work proposes an approach for distribution system load forecasting, which aims to provide highly accurate short-term load forecasting with high resolution utilizing a support vector regression (SVR) based forecaster and a two-step hybrid parameters optimization method. Specifically, because the load profiles in distribution systems contain abrupt deviations, a data normalization is designed as the pretreatment for the collected historical load data. Then an SVR model is trained by the load data to forecast the future load. For better performance of SVR, a two-step hybrid optimization algorithm is proposed to determine the best parameters. In the first step of the hybrid optimization algorithm, a designed grid traverse algorithm (GTA) is used to narrow the parameters searching area from a global to local space. In the second step, based on the result of the GTA, particle swarm optimization (PSO) is used to determine the best parameters in the local parameter space. After the best parameters are determined, the SVR model is used to forecast the short-term load deviation in the distribution system.

  1. Independent Component Analysis-Support Vector Machine-Based Computer-Aided Diagnosis System for Alzheimer's with Visual Support.

    Science.gov (United States)

    Khedher, Laila; Illán, Ignacio A; Górriz, Juan M; Ramírez, Javier; Brahim, Abdelbasset; Meyer-Baese, Anke

    2017-05-01

    Computer-aided diagnosis (CAD) systems constitute a powerful tool for early diagnosis of Alzheimer's disease (AD), but limitations on interpretability and performance exist. In this work, a fully automatic CAD system based on supervised learning methods is proposed to be applied on segmented brain magnetic resonance imaging (MRI) from Alzheimer's disease neuroimaging initiative (ADNI) participants for automatic classification. The proposed CAD system possesses two relevant characteristics: optimal performance and visual support for decision making. The CAD is built in two stages: a first feature extraction based on independent component analysis (ICA) on class mean images and, secondly, a support vector machine (SVM) training and classification. The obtained features for classification offer a full graphical representation of the images, giving an understandable logic in the CAD output, that can increase confidence in the CAD support. The proposed method yields classification results up to 89% of accuracy (with 92% of sensitivity and 86% of specificity) for normal controls (NC) and AD patients, 79% of accuracy (with 82% of sensitivity and 76% of specificity) for NC and mild cognitive impairment (MCI), and 85% of accuracy (with 85% of sensitivity and 86% of specificity) for MCI and AD patients.

  2. Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

    Directory of Open Access Journals (Sweden)

    Todo Tomoki

    2006-09-01

    Full Text Available Abstract Background Oncolytic herpes simplex virus (HSV vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. Results We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39

  3. Modeling and Development of Medical Information System Based on Support Vector Machine in Web Network

    Directory of Open Access Journals (Sweden)

    Chuanfu Hu

    2017-12-01

    Full Text Available This paper aims at improving and utilizing the ontology information in ontology design of FOAF and vCard in real time, and the application of open relational data technology, SPARQL query information results and sending RDF/JSON data format. In addition, improve the effectiveness and efficiency of patient information extraction from the medical information website. This article includes two web search engines that are used to inform patients about medical care information. The experiment uses Drupal as the main software tool, and the Drupal RDF extension module provides some meaningful mapping. In the evaluation part, the structure of the experimental test platform is established and the system function test is carried out. The evaluation results include consumers or patients retrieving the latest doctor information and comparing search capabilities and techniques, between our system and existing systems.

  4. Potato virus X and Tobacco mosaic virus-based vectors compatible with the Gateway-TM cloning system

    NARCIS (Netherlands)

    Lacorte, C.C.; Ribeiro, S.G.; Lohuis, H.; Goldbach, R.W.; Prins, M.W.

    2010-01-01

    Virus-based expression vectors are important tools for high-level production of foreign proteins and for gene function analysis through virus induced gene silencing. To exploit further their advantages as fast, high yield replicons, a set of vectors was produced by converting and adapting Potato

  5. Hepatic lentiviral gene transfer prevents the long-term onset of hepatic tumours of glycogen storage disease type 1a in mice.

    Science.gov (United States)

    Clar, Julie; Mutel, Elodie; Gri, Blandine; Creneguy, Alison; Stefanutti, Anne; Gaillard, Sophie; Ferry, Nicolas; Beuf, Olivier; Mithieux, Gilles; Nguyen, Tuan Huy; Rajas, Fabienne

    2015-04-15

    Glycogen storage disease type 1a (GSD1a) is a rare disease due to the deficiency in the glucose-6-phosphatase (G6Pase) catalytic subunit (encoded by G6pc), which is essential for endogenous glucose production. Despite strict diet control to maintain blood glucose, patients with GSD1a develop hepatomegaly, steatosis and then hepatocellular adenomas (HCA), which can undergo malignant transformation. Recently, gene therapy has attracted attention as a potential treatment for GSD1a. In order to maintain long-term transgene expression, we developed an HIV-based vector, which allowed us to specifically express the human G6PC cDNA in the liver. We analysed the efficiency of this lentiviral vector in the prevention of the development of the hepatic disease in an original GSD1a mouse model, which exhibits G6Pase deficiency exclusively in the liver (L-G6pc(-/-) mice). Recombinant lentivirus were injected in B6.G6pc(ex3lox/ex3lox). SA(creERT2/w) neonates and G6pc deletion was induced by tamoxifen treatment at weaning. Magnetic resonance imaging was then performed to follow up the development of hepatic tumours. Lentiviral gene therapy restored glucose-6 phosphatase activity sufficient to correct fasting hypoglycaemia during 9 months. Moreover, lentivirus-treated L-G6pc(-/-) mice presented normal hepatic triglyceride levels, whereas untreated mice developed steatosis. Glycogen stores were also decreased although liver weight remained high. Interestingly, lentivirus-treated L-G6pc(-/-) mice were protected against the development of hepatic tumours after 9 months of gene therapy while most of untreated L-G6pc(-/-) mice developed millimetric HCA. Thus the treatment of newborns by recombinant lentivirus appears as an attractive approach to protect the liver from the development of steatosis and hepatic tumours associated to GSD1a pathology. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. The duality in the topological vector spaces and the linear physical system theory

    International Nuclear Information System (INIS)

    Oliveira Castro, F.M. de.

    1980-01-01

    The excitation-response relation in a linear, passive, and causal physical system who has the property of this relation be invariant for a time translation is univocally determined by the general form of the linear and continuous functionals defined on the linear topological space chosen for the representation of the excitations. (L.C.) [pt

  7. B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome

    NARCIS (Netherlands)

    M.C. Castiello (Maria Carmina); S. Scaramuzza (Samantha); G. Pala (Gianni); F. Ferrua (Francesca); P. Uva (Paolo); I. Brigida (Immacolata); L. Sereni (Lucia); M. van der Burg (Mirjam); G. Ottaviano (Giorgio); M. Albert (Michael); M. Grazia Roncarolo (Maria); L. Naldini (Luigi); A. Aiuti (Alessandro); A. Anna (Villa); M. Bosticardo (Marita)

    2015-01-01

    textabstractBackground Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however,

  8. Schwann Cells Transduced with a Lentiviral Vector Encoding Fgf-2 Promote Motor Neuron Regeneration Following Sciatic Nerve Injury

    NARCIS (Netherlands)

    Allodi, I.; Mecollari, V.; Gonzalez-Perez, F.; Eggers, R.; Hoyng, S.; Verhaagen, J.; Navarro, X.; Udina, E.

    2014-01-01

    Fibroblast growth factor 2 (FGF-2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF-2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF-2 specifically increases motor neuron axonal growth. To further explore the potential

  9. Schwann cells transduced with a lentiviral vector encoding Fgf-2 promote motor neuron regeneration following sciatic nerve injury

    NARCIS (Netherlands)

    Allodi, Ilary; Mecollari, Vasil; González-Pérez, Francisco; Eggers, R.; Hoyng, S.; Verhaagen, J.; Navarro, Xavier; Udina, Esther

    2014-01-01

    Fibroblast growth factor 2 (FGF-2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF-2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF-2 specifically increases motor neuron axonal growth. To further explore the potential

  10. Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC)

    International Nuclear Information System (INIS)

    Levitzki, Alexander

    2012-01-01

    Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody.

  11. Cell Phone-Based System (Chaak) for Surveillance of Immatures of Dengue Virus Mosquito Vectors

    Science.gov (United States)

    LOZANO–FUENTES, SAUL; WEDYAN, FADI; HERNANDEZ–GARCIA, EDGAR; SADHU, DEVADATTA; GHOSH, SUDIPTO; BIEMAN, JAMES M.; TEP-CHEL, DIANA; GARCÍA–REJÓN, JULIÁN E.; EISEN, LARS

    2014-01-01

    Capture of surveillance data on mobile devices and rapid transfer of such data from these devices into an electronic database or data management and decision support systems promote timely data analyses and public health response during disease outbreaks. Mobile data capture is used increasingly for malaria surveillance and holds great promise for surveillance of other neglected tropical diseases. We focused on mosquito-borne dengue, with the primary aims of: 1) developing and field-testing a cell phone-based system (called Chaak) for capture of data relating to the surveillance of the mosquito immature stages, and 2) assessing, in the dengue endemic setting of Mérida, México, the cost-effectiveness of this new technology versus paper-based data collection. Chaak includes a desktop component, where a manager selects premises to be surveyed for mosquito immatures, and a cell phone component, where the surveyor receives the assigned tasks and captures the data. Data collected on the cell phone can be transferred to a central database through different modes of transmission, including near-real time where data are transferred immediately (e.g., over the Internet) or by first storing data on the cell phone for future transmission. Spatial data are handled in a novel, semantically driven, geographic information system. Compared with a pen-and-paper-based method, use of Chaak improved the accuracy and increased the speed of data transcription into an electronic database. The cost-effectiveness of using the Chaak system will depend largely on the up-front cost of purchasing cell phones and the recurring cost of data transfer over a cellular network. PMID:23926788

  12. A Linear System of Differential Equations Related to Vector-Valued Jack Polynomials on the Torus

    Science.gov (United States)

    Dunkl, Charles F.

    2017-06-01

    For each irreducible module of the symmetric group S_{N} there is a set of parametrized nonsymmetric Jack polynomials in N variables taking values in the module. These polynomials are simultaneous eigenfunctions of a commutative set of operators, self-adjoint with respect to two Hermitian forms, one called the contravariant form and the other is with respect to a matrix-valued measure on the N-torus. The latter is valid for the parameter lying in an interval about zero which depends on the module. The author in a previous paper [SIGMA 12 (2016), 033, 27 pages] proved the existence of the measure and that its absolutely continuous part satisfies a system of linear differential equations. In this paper the system is analyzed in detail. The N-torus is divided into (N-1)! connected components by the hyperplanes x_{i}=x_{j}, isystem. The main result is that the orthogonality ! measure has no singular part with respect to Haar measure, and thus is given by a matrix function times Haar measure. This function is analytic on each of the connected components.

  13. Lentiviral Transfer of γ-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine β-Thalassemia.

    Science.gov (United States)

    Zhao, Hui Fen; Abraham, Allistair; Kim, Yoon-Sang; Wang, Yong-Dong; Pestina, Tamara; Zhan, Jun; Humphries, Keith; Nienhuis, Arthur W; Persons, Derek A

    2017-03-01

    Recently, an engineered Homeobox-nucleoporin fusion gene, NUP98-HOXA10HD or NA10HD, was reported to expand and maintain murine hematopoietic stem cells (HSCs). We postulated that NA10HD would increase the number of human γ-globin-expressing cells to therapeutic levels. We developed a double gene lentiviral vector encoding both human γ-globin and NA10HD, which was used to transduce human peripheral blood CD34 + cells and increased engraftment 2- to 2.5-fold at 15 weeks post-transplantation in immunodeficient mice. In β-thalassemic mice transplanted with β-thalassemic HSCs transduced with the γ-globin/NA10HD vector, the number of fetal hemoglobin (HbF)-expressing cells was significantly increased after 3 months, leading to resolution of the anemia. Furthermore, the increases in HbF were maintained at 6 months and persisted after secondary transplantation. In addition, NA10HD enrichment of transduced HSCs led to HbF increases without affecting homeostasis of the white blood cell lineages. Our results suggest that NA10HD increases the number of γ-globin-transduced HSCs that engraft, leading to an elevated number of fetal hemoglobin-containing red cells. These effects of NA10HD provide an improved platform for testing of the therapeutic efficacy of novel globin vectors and provide further impetus to develop safe and effective methods for selective expansion of genetically modified cells. Copyright © 2017. Published by Elsevier Inc.

  14. Design of Online Monitoring and Fault Diagnosis System for Belt Conveyors Based on Wavelet Packet Decomposition and Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-01-01

    Full Text Available Belt conveyors are the equipment widely used in coal mines and other manufacturing factories, whose main components are a number of idlers. The faults of belt conveyors can directly influence the daily production. In this paper, a fault diagnosis method combining wavelet packet decomposition (WPD and support vector machine (SVM is proposed for monitoring belt conveyors with the focus on the detection of idler faults. Since the number of the idlers could be large, one acceleration sensor is applied to gather the vibration signals of several idlers in order to reduce the number of sensors. The vibration signals are decomposed with WPD, and the energy of each frequency band is extracted as the feature. Then, the features are employed to train an SVM to realize the detection of idler faults. The proposed fault diagnosis method is firstly tested on a testbed, and then an online monitoring and fault diagnosis system is designed for belt conveyors. An experiment is also carried out on a belt conveyor in service, and it is verified that the proposed system can locate the position of the faulty idlers with a limited number of sensors, which is important for operating belt conveyors in practices.

  15. Simultaneous-Fault Diagnosis of Automotive Engine Ignition Systems Using Prior Domain Knowledge and Relevance Vector Machine

    Directory of Open Access Journals (Sweden)

    Chi-Man Vong

    2013-01-01

    Full Text Available Engine ignition patterns can be analyzed to identify the engine fault according to both the specific prior domain knowledge and the shape features of the patterns. One of the challenges in ignition system diagnosis is that more than one fault may appear at a time. This kind of problem refers to simultaneous-fault diagnosis. Another challenge is the acquisition of a large amount of costly simultaneous-fault ignition patterns for constructing the diagnostic system because the number of the training patterns depends on the combination of different single faults. The above problems could be resolved by the proposed framework combining feature extraction, probabilistic classification, and decision threshold optimization. With the proposed framework, the features of the single faults in a simultaneous-fault pattern are extracted and then detected using a new probabilistic classifier, namely, pairwise coupling relevance vector machine, which is trained with single-fault patterns only. Therefore, the training dataset of simultaneous-fault patterns is not necessary. Experimental results show that the proposed framework performs well for both single-fault and simultaneous-fault diagnoses and is superior to the existing approach.

  16. Noospheric vector of management applied to the system of higher professional education

    Directory of Open Access Journals (Sweden)

    Yulia A. Mishina

    2017-01-01

    Full Text Available The aim of the research is to substantiate the necessity, expediency of the wide use of the noospheric management approaches, and technologies applied to the system of higher professional education in modern conditions. The materials and methods. Information base of the research is formed, based on education laws in Ukraine, Russia and the Donetsk People’s Republic, educational standards of higher professional education, as well as publications of scientists on the subject matter under study. In the given research it was used such methods: textological, analysis, generalization and modeling. The results. In the research, it is comprehensively examined the concept of noosphere, analyzed the modern theories and concepts of its development in relation to the development of society, an integral part of which is the system of education. It is indicated that there is the relationship between the noosphere and management in general, and with modern system of education, in particular. The concept of noospheric education is introduced and studied its impact on holistic  development of personality. The comparative characteristic of the structure and levels of the educational system of Ukraine, the Russian Federation and the Donetsk People’s Republic is performed; the types of educational institutions of the abovementioned states are compared. The structure of educational institution of higher professional education is proposed as a socio-economic system, managed from the perspective of the concept of noospheric education. The factors of competitiveness of educational institution of the higher school from the point of view of noospheric management are considered. Special attention is given to the internal factor of competitiveness of educational institutions, such as the quality of student life, because this position, particularly from the point of view of noospheric management, has a significant influence on the rating among consumers. The

  17. Convergence to Equilibrium in Energy-Reaction-Diffusion Systems Using Vector-Valued Functional Inequalities

    Science.gov (United States)

    Mielke, Alexander; Mittnenzweig, Markus

    2018-04-01

    We discuss how the recently developed energy dissipation methods for reaction diffusion systems can be generalized to the non-isothermal case. For this, we use concave entropies in terms of the densities of the species and the internal energy, where the importance is that the equilibrium densities may depend on the internal energy. Using the log-Sobolev estimate and variants for lower-order entropies as well as estimates for the entropy production of the nonlinear reactions, we give two methods to estimate the relative entropy by the total entropy production, namely a somewhat restrictive convexity method, which provides explicit decay rates, and a very general, but weaker compactness method.

  18. Systemic insecticides used in dogs: potential candidates for phlebotomine vector control?

    Science.gov (United States)

    Gomez, Sonia Ares; Picado, Albert

    2017-06-01

    Zoonotic visceral leishmaniasis (ZVL) is a public health problem endemic in some countries. Current control measures, in particular culling infected dogs, have not reduced ZVL incidence in humans. We evaluated the use of five systemic insecticides (spinosad, fluralaner, afoxolaner, sarolaner and moxidectin) currently used in dogs for other purposes (e.g. tick, flea control) in controlling ZVL transmission. The anti-phlebotomine capacity of these compounds confirmed in experimental studies makes their use in ZVL control programmes very promising. Limitations and benefits of using this new control tool are compared to current practices. © 2017 The Authors. Tropical Medicine & International Health published by John Wiley & Sons Ltd.

  19. A UNIX Based Device Driver for the Vector General 3404 Graphics Display System.

    Science.gov (United States)

    1982-03-01

    lines of code, the system can easily be understood and maintained by one person. Of the 10,000 lines Qf UNIX code, less than ten percent is written in...user is granted special access rights and priviledges that other users do not receive. These rights and priviledges allow the super- user to make any...inhibited as long as the processor priority is greater than or equal to the interrupt priority (Ref 10:9-2). When the processor priority becomes less

  20. Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector.

    Science.gov (United States)

    Rincon, Melvin Y; de Vin, Filip; Duqué, Sandra I; Fripont, Shelly; Castaldo, Stephanie A; Bouhuijzen-Wenger, Jessica; Holt, Matthew G

    2018-03-09

    Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood-brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-03-01

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

  3. A novel Ti-plasmid-convertible lambda phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants.

    Science.gov (United States)

    Fuse, T; Kodama, H; Hayashida, N; Shinozaki, K; Nishimura, M; Iba, K

    1995-05-01

    A new lambda phage vector system, lambda TI, has been constructed to facilitate genetic complementation of higher plant mutations. The lambda TI vectors are stable, and by using the Cre-lox site-specific recombination, are automatically convertible into Ti-plasmid binary vectors which are capable of expressing genes in higher plants. Two lambda TI vectors were constructed: (i) lambda TI1, which can generate a Ti-plasmid that contains the cauliflower mosaic virus (CaMV) 35S promoter and is suitable for the expression of cDNA in transformed plants and (ii) lambda TI2, which can generate a Ti-plasmid with the multicloning site (MCS). cDNA and genomic libraries, which were constructed from the cruciferous plant Arabidopsis thaliana in these lambda TI vectors, can be probed by large DNA fragments of more than 100 kb, such as yeast artificial chromosomes (YACs), enabling the direct screening of the clones in the chromosome region containing a specified genetic locus. These libraries will certainly become powerful tools for the genetic complementation of Arabidopsis mutant phenotypes by quickly providing transformation-competent clones.

  4. Effect of irrigation systems on temporal distribution of malaria vectors in semi-arid regions

    Science.gov (United States)

    Ohta, Shunji; Kaga, Takumi

    2014-04-01

    Previous research models have used climate data to explain habitat conditions of Anopheles mosquitoes transmitting malaria parasites. Although they can estimate mosquito populations with sufficient accuracy in many areas, observational data show that there is a tendency to underestimate the active growth and reproduction period of mosquitoes in semi-arid agricultural regions. In this study, a new, modified model that includes irrigation as a factor was developed to predict the active growing period of mosquitoes more precisely than the base model for ecophysiological and climatological distribution of mosquito generations (ECD-mg). Five sites with complete sets of observational data were selected in semi-arid regions of India for the comparison. The active growing period of mosquitoes determined from the modified ECD-mg model that incorporated the irrigation factor was in agreement with the observational data, whereas the active growing period was underestimated by the previous ECD-mg model that did not incorporate irrigation. This suggests that anthropogenic changes in the water supply due to extensive irrigation can encourage the growth of Anopheles mosquitoes through the alteration of the natural water balance in their habitat. In addition, it was found that the irrigation systems not only enable the active growth of mosquitoes in dry seasons but also play an important role in stabilizing the growth in rainy seasons. Consequently, the irrigation systems could lengthen the annual growing period of Anopheles mosquitoes and increase the maximum generation number of mosquitoes in semi-arid subtropical regions.

  5. A full Stokes vector ellipsometry measurement system for in situ diagnostics in dynamic experiments.

    Science.gov (United States)

    Bakshi, L; Eliezer, S; Appelbaum, G; Nissim, N; Perelmutter, L; Mond, M

    2012-05-01

    A fast ellipsometry system with a resolution of only a few nanoseconds that can simultaneously measure all four Stokes parameters was developed for use in dynamic experiments. Due to its fine temporal resolution, the system is useful for a wide variety of dynamic setups, two of which are presented, fast foil heating and shock compression. As a test case the optical properties of nickel were measured in a fast foil heating setup. The complex index of refraction and emissivity at 532 nm and in the range of 1000-1900 K are presented. It was found that the emissivity monotonously increases below and above the melting point while an abrupt increase of about 2% was observed at the phase transition. These results are in accordance with the literature. Shock compression experiments included sample-free surface measurements. Samples of 1020 steel were shocked up to 25 GPa on the Hugoniot curve. The measured optical properties under these conditions showed a significant change; the value of the emissivity was doubled.

  6. Optimal design of an in-situ bioremediation system using support vector machine and particle swarm optimization

    Science.gov (United States)

    ch, Sudheer; Kumar, Deepak; Prasad, Ram Kailash; Mathur, Shashi

    2013-08-01

    A methodology based on support vector machine and particle swarm optimization techniques (SVM-PSO) was used in this study to determine an optimal pumping rate and well location to achieve an optimal cost of an in-situ bioremediation system. In the first stage of the two stage methodology suggested for optimal in-situ bioremediation design, the optimal number of wells and their locations was determined from preselected candidate well locations. The pumping rate and well location in the first stage were subsequently optimized in the second stage of the methodology. The highly nonlinear system of equations governing in-situ bioremediation comprises the equations of flow and solute transport coupled with relevant biodegradation kinetics. A finite difference model was developed to simulate the process of in-situ bioremediation using an Alternate-Direction Implicit technique. This developed model (BIOFDM) yields the spatial and temporal distribution of contaminant concentration for predefined initial and boundary conditions. BIOFDM was later validated by comparing the simulated results with those obtained using BIOPLUME III for the case study of Shieh and Peralta (2005). The results were found to be in close agreement. Moreover, since the solution of the highly nonlinear equation otherwise requires significant computational effort, the computational burden in this study was managed within a practical time frame by replacing the BIOFDM model with a trained SVM model. Support Vector Machine which generates fast solutions in real time was considered to be a universal function approximator in the study. Apart from reducing the computational burden, this technique generates a set of near optimal solutions (instead of a single optimal solution) and creates a re-usable data base that could be used to address many other management problems. Besides this, the search for an optimal pumping pattern was directed by a simple PSO technique and a penalty parameter approach was adopted

  7. Multithreading in vector processors

    Energy Technology Data Exchange (ETDEWEB)

    Evangelinos, Constantinos; Kim, Changhoan; Nair, Ravi

    2018-01-16

    In one embodiment, a system includes a processor having a vector processing mode and a multithreading mode. The processor is configured to operate on one thread per cycle in the multithreading mode. The processor includes a program counter register having a plurality of program counters, and the program counter register is vectorized. Each program counter in the program counter register represents a distinct corresponding thread of a plurality of threads. The processor is configured to execute the plurality of threads by activating the plurality of program counters in a round robin cycle.

  8. Construction of a BioBrick™ compatible vector system for Rhodococcus.

    Science.gov (United States)

    Ellinger, James; Schmidt-Dannert, Claudia

    2017-03-01

    Throughout the past decade, the field of synthetic biology has grown rapidly. By using assembly platforms such as BioBricks™, scientists can quickly and easily build gene circuits or multi-step pathways. One limitation, however, is that most of these parts were designed and characterized with Escherichia coli as the target chassis. As a consequence, there exists a lack of standardized and well characterized or BioBrick™ compatible plasmid backbones that replicate in other potential non-model chassis organisms. The Gram-positive bacteria of the genus Rhodococcus represent an interesting chassis for biotechnological applications due to their tremendous metabolic capabilities. In this report we describe our progress toward developing a BioBrick™ compatible plasmid system for Rhodococcus. We demonstrate its utility for heterologous protein expression through flow cytometric analysis of the lac promoter in the oleaginous strain Rhodococcus opacus PD630. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system

    Science.gov (United States)

    Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang; Piensook, Khanrat; Cardineau, Guy; Zeitlin, Larry; Whaley, Kevin J.; Arntzen, Charles J.

    2010-01-01

    Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of heterooligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the “competing” nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al. 2009). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al. 2000) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for heterooligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for

  10. Establishment of a large semi-field system for experimental study of African malaria vector ecology and control in Tanzania

    NARCIS (Netherlands)

    Ferguson, H.M.; Ng'habi, K.R.; Walder, T.; Kadungula, D.; Moore, S.J.; Lyimo, I.; Russell, T.L.; Urassa, H.; Mshinda, H.; Killeen, G.F.; Knols, B.G.J.

    2008-01-01

    Background - Medical entomologists increasingly recognize that the ability to make inferences between laboratory experiments of vector biology and epidemiological trends observed in the field is hindered by a conceptual and methodological gap occurring between these approaches which prevents

  11. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    Science.gov (United States)

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2016-01-01

    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

  12. Designing the input vector to ANN-based models for short-term load forecast in electricity distribution systems

    International Nuclear Information System (INIS)

    Santos, P.J.; Martins, A.G.; Pires, A.J.

    2007-01-01

    The present trend to electricity market restructuring increases the need for reliable short-term load forecast (STLF) algorithms, in order to assist electric utilities in activities such as planning, operating and controlling electric energy systems. Methodologies such as artificial neural networks (ANN) have been widely used in the next hour load forecast horizon with satisfactory results. However, this type of approach has had some shortcomings. Usually, the input vector (IV) is defined in a arbitrary way, mainly based on experience, on engineering judgment criteria and on concern about the ANN dimension, always taking into consideration the apparent correlations within the available endogenous and exogenous data. In this paper, a proposal is made of an approach to define the IV composition, with the main focus on reducing the influence of trial-and-error and common sense judgments, which usually are not based on sufficient evidence of comparative advantages over previous alternatives. The proposal includes the assessment of the strictly necessary instances of the endogenous variable, both from the point of view of the contiguous values prior to the forecast to be made, and of the past values representing the trend of consumption at homologous time intervals of the past. It also assesses the influence of exogenous variables, again limiting their presence at the IV to the indispensable minimum. A comparison is made with two alternative IV structures previously proposed in the literature, also applied to the distribution sector. The paper is supported by a real case study at the distribution sector. (author)

  13. Designing the input vector to ANN-based models for short-term load forecast in electricity distribution systems

    Energy Technology Data Exchange (ETDEWEB)

    Santos, P.J. [LabSEI-ESTSetubal-Department of Electrical Engineering at Escola Superior de Tecnologia, Polytechnic Institute of Setubal Rua Vale de Chaves Estefanilha, 2910-761 Setubal (Portugal); Martins, A.G. [Department of Electrical Engineering, FCTUC/INESC, Polo 2 University of Coimbra, Pinhal de Marrocos, 3030 Coimbra (Portugal); Pires, A.J. [LabSEI-ESTSetubal-Department of Electrical Engineering at Escola Superior de Tecnologia, Polytechnic Institute of Setubal Rua Vale de, Chaves Estefanilha, 2910-761 Setubal (Portugal)

    2007-05-15

    The present trend to electricity market restructuring increases the need for reliable short-term load forecast (STLF) algorithms, in order to assist electric utilities in activities such as planning, operating and controlling electric energy systems. Methodologies such as artificial neural networks (ANN) have been widely used in the next hour load forecast horizon with satisfactory results. However, this type of approach has had some shortcomings. Usually, the input vector (IV) is defined in a arbitrary way, mainly based on experience, on engineering judgment criteria and on concern about the ANN dimension, always taking into consideration the apparent correlations within the available endogenous and exogenous data. In this paper, a proposal is made of an approach to define the IV composition, with the main focus on reducing the influence of trial-and-error and common sense judgments, which usually are not based on sufficient evidence of comparative advantages over previous alternatives. The proposal includes the assessment of the strictly necessary instances of the endogenous variable, both from the point of view of the contiguous values prior to the forecast to be made, and of the past values representing the trend of consumption at homologous time intervals of the past. It also assesses the influence of exogenous variables, again limiting their presence at the IV to the indispensable minimum. A comparison is made with two alternative IV structures previously proposed in the literature, also applied to the distribution sector. The paper is supported by a real case study at the distribution sector. (author)

  14. New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.

    Science.gov (United States)

    Schwendener, Sybille; Perreten, Vincent

    2015-05-01

    Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Rapid Surveillance for Vector Presence (RSVP: Development of a novel system for detecting Aedes aegypti and Aedes albopictus.

    Directory of Open Access Journals (Sweden)

    Brian L Montgomery

    2017-03-01

    Full Text Available The globally important Zika, dengue and chikungunya viruses are primarily transmitted by the invasive mosquitoes, Aedes aegypti and Aedes albopictus. In Australia, there is an increasing risk that these species may invade highly urbanized regions and trigger outbreaks. We describe the development of a Rapid Surveillance for Vector Presence (RSVP system to expedite presence- absence surveys for both species.We developed a methodology that uses molecular assays to efficiently screen pooled ovitrap (egg trap samples for traces of target species ribosomal RNA. Firstly, specific real-time reverse transcription-polymerase chain reaction (RT-PCR assays were developed which detect a single Ae. aegypti or Ae. albopictus first instar larva in samples containing 4,999 and 999 non-target mosquitoes, respectively. ImageJ software was evaluated as an automated egg counting tool using ovitrap collections obtained from Brisbane, Australia. Qualitative assessment of ovistrips was required prior to automation because ImageJ did not differentiate between Aedes eggs and other objects or contaminants on 44.5% of ovistrips assessed, thus compromising the accuracy of egg counts. As a proof of concept, the RSVP was evaluated in Brisbane, Rockhampton and Goomeri, locations where Ae. aegypti is considered absent, present, and at the margin of its range, respectively. In Brisbane, Ae. aegypti was not detected in 25 pools formed from 477 ovitraps, comprising ≈ 54,300 eggs. In Rockhampton, Ae. aegypti was detected in 4/6 pools derived from 45 ovitraps, comprising ≈ 1,700 eggs. In Goomeri, Ae. aegypti was detected in 5/8 pools derived from 62 ovitraps, comprising ≈ 4,200 eggs.RSVP can rapidly detect nucleic acids from low numbers of target species within large samples of endemic species aggregated from multiple ovitraps. This screening capability facilitates deployment of ovitrap configurations of varying spatial scales, from a single residential block to entire

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Yosuke Sakai

    2016-08-01

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

  19. Application of a wide-range yeast vector (CoMed™ system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts

    Directory of Open Access Journals (Sweden)

    Kunze Gotthard

    2006-11-01

    Full Text Available Abstract Background Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™ system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way. Results The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli-derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFα1/IL-6 fusion was performed in a faithful way. Conclusion rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly

  20. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

    Directory of Open Access Journals (Sweden)

    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  1. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance

    Science.gov (United States)

    Xie, Emily; Kotha, Abhiroop; Biaco, Tracy; Sedani, Nikita; Zou, Jonathan; Stashenko, Phillip; Duncan, Margaret J.; Campos-Neto, Antonio; Cayabyab, Mark J.

    2015-01-01

    The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis) that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases. PMID:26618634

  2. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    DEFF Research Database (Denmark)

    Sorensen, Lisette Quaade; Lysøe, Erik; Larsen, Jesper Erup

    2014-01-01

    The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infe......The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize...... single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results: The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated...... fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the Delta...

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

    Directory of Open Access Journals (Sweden)

    X. Joann You

    2011-01-01

    Full Text Available Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs. Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein—before and after differentiation—all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

  4. Design, development and evaluation of an online grading system for peeled pistachios equipped with machine vision technology and support vector machine

    Directory of Open Access Journals (Sweden)

    Hosein Nouri-Ahmadabadi

    2017-12-01

    Full Text Available In this study, an intelligent system based on combined machine vision (MV and Support Vector Machine (SVM was developed for sorting of peeled pistachio kernels and shells. The system was composed of conveyor belt, lighting box, camera, processing unit and sorting unit. A color CCD camera was used to capture images. The images were digitalized by a capture card and transferred to a personal computer for further analysis. Initially, images were converted from RGB color space to HSV color ones. For segmentation of the acquired images, H-component in the HSV color space and Otsu thresholding method were applied. A feature vector containing 30 color features was extracted from the captured images. A feature selection method based on sensitivity analysis was carried out to select superior features. The selected features were presented to SVM classifier. Various SVM models having a different kernel function were developed and tested. The SVM model having cubic polynomial kernel function and 38 support vectors achieved the best accuracy (99.17% and then was selected to use in online decision-making unit of the system. By launching the online system, it was found that limiting factors of the system capacity were related to the hardware parts of the system (conveyor belt and pneumatic valves used in the sorting unit. The limiting factors led to a distance of 8 mm between the samples. The overall accuracy and capacity of the sorter were obtained 94.33% and 22.74 kg/h, respectively. Keywords: Pistachio kernel, Sorting, Machine vision, Sensitivity analysis, Support vector machine

  5. Mechanism of reduction in titers from lentivirus vectors carrying large inserts in the 3'LTR.

    Science.gov (United States)

    Urbinati, Fabrizia; Arumugam, Paritha; Higashimoto, Tomoyasu; Perumbeti, Anil; Mitts, Kyle; Xia, Ping; Malik, Punam

    2009-09-01

    Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.

  6. Generation of X-CGD cells for vector evaluation from healthy donor CD34+ HSCs by shRNA-mediated knock down of gp91phox

    Directory of Open Access Journals (Sweden)

    Christian Brendel

    2014-01-01

    Full Text Available Innovative approaches for the treatment of rare inherited diseases are hampered by limited availability of patient derived samples for preclinical research. This also applies for the evaluation of novel vector systems for the gene therapy of monogenic hematological diseases like X-linked chronic granulomatous disease (X-CGD, a severe primary immunodeficiency caused by mutations in the gp91phox subunit of the phagocytic NADPH oxidase. Since current gene therapy protocols involve ex vivo gene modification of autologous CD34+ hematopoietic stem cells (HSC, the ideal preclinical model should simulate faithfully this procedure. However, the low availability of patient-derived CD34+ cells limits the feasibility of this approach. Here, we describe a straightforward experimental strategy that circumvents this limitation. The knock down of gp91phox expression upon lentiviral delivery of shRNAs into CD34+ cells from healthy donors generates sufficient amounts of X-CGD CD34+ cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91phox transgene. We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.

  7. Introduction to matrices and vectors

    CERN Document Server

    Schwartz, Jacob T

    2001-01-01

    In this concise undergraduate text, the first three chapters present the basics of matrices - in later chapters the author shows how to use vectors and matrices to solve systems of linear equations. 1961 edition.

  8. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Yamaguchi

    Full Text Available The production of cells capable of expressing gene(s of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  9. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    Although retroviral vector systems have been found to efficiently transduce a variety of cell types in vitro, the use of vectors based on murine leukemia virus in preclinical models of somatic gene therapy has led to the identification of transcriptional silencing in vivo as an important problem....... Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t...

  10. Problems of vector Lagrangians in field theories

    International Nuclear Information System (INIS)

    Krivsky, I.Yu.; Simulik, V.M.

    1997-01-01

    A vector Lagrange approach to the Dirac spinor field and the relationship between the vector Lagrangians for the spinor and electromagnetic fields are considered. A vector Lagrange approach for the system of interacting electromagnetic B=(B μ υ)=(E-bar,H-bar) and spinor Ψ fields is constructed. New Lagrangians (scalar and vector) for electromagnetic field in terms of field strengths are found. The foundations of two new QED models are formulated

  11. Solution to the problem of joint evaluation of the nonstationary model of GSP drift and the state vector of a navigation system

    Science.gov (United States)

    Pogorelov, V. A.; Sokolov, S. V.

    2013-05-01

    The solution to the problem of evaluation in real time of nonstationary coefficients of the polynomial model of drift of a gyro-stabilized platform (GSP) is considered. In order to solve this problem, the estimating equations for the state vector of the navigation system (NS) are derived taking into account correlations between the equations of the object and the observer. Evaluation of the coefficients of the model of the GSP drift is realized based on minimization of nonlinear probabilistic criteria.

  12. Diagnosis of osteoporosis from dental panoramic radiographs using the support vector machine method in a computer-aided system

    International Nuclear Information System (INIS)

    Kavitha, M S; Asano, Akira; Taguchi, Akira; Kurita, Takio; Sanada, Mitsuhiro

    2012-01-01

    Early diagnosis of osteoporosis can potentially decrease the risk of fractures and improve the quality of life. Detection of thin inferior cortices of the mandible on dental panoramic radiographs could be useful for identifying postmenopausal women with low bone mineral density (BMD) or osteoporosis. The aim of our study was to assess the diagnostic efficacy of using kernel-based support vector machine (SVM) learning regarding the cortical width of the mandible on dental panoramic radiographs to identify postmenopausal women with low BMD. We employed our newly adopted SVM method for continuous measurement of the cortical width of the mandible on dental panoramic radiographs to identify women with low BMD or osteoporosis. The original X-ray image was enhanced, cortical boundaries were determined, distances among the upper and lower boundaries were evaluated and discrimination was performed by a radial basis function. We evaluated the diagnostic efficacy of this newly developed method for identifying women with low BMD (BMD T-score of -1.0 or less) at the lumbar spine and femoral neck in 100 postmenopausal women (≥50 years old) with no previous diagnosis of osteoporosis. Sixty women were used for system training, and 40 were used in testing. The sensitivity and specificity using RBF kernel-SVM method for identifying women with low BMD were 90.9% [95% confidence interval (CI), 85.3-96.5] and 83.8% (95% CI, 76.6-91.0), respectively at the lumbar spine and 90.0% (95% CI, 84.1-95.9) and 69.1% (95% CI, 60.1-78.6), respectively at the femoral neck. The sensitivity and specificity for identifying women with low BMD at either the lumbar spine or femoral neck were 90.6% (95% CI, 92.0-100) and 80.9% (95% CI, 71.0-86.9), respectively. Our results suggest that the newly developed system with the SVM method would be useful for identifying postmenopausal women with low skeletal BMD

  13. A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

    Directory of Open Access Journals (Sweden)

    Reza Ebrahimzadeh-Vesal

    2014-08-01

    Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

  14. Multi-objective based on parallel vector evaluated particle swarm optimization for optimal steady-state performance of power systems

    DEFF Research Database (Denmark)

    Vlachogiannis, Ioannis (John); Lee, K Y

    2009-01-01

    In this paper the state-of-the-art extended particle swarm optimization (PSO) methods for solving multi-objective optimization problems are represented. We emphasize in those, the co-evolution technique of the parallel vector evaluated PSO (VEPSO), analysed and applied in a multi-objective problem...

  15. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Science.gov (United States)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

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

    Science.gov (United States)

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

    2017-09-14

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

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

    Science.gov (United States)

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

    2018-01-01

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

  18. Flows of non-smooth vector fields and degenerate elliptic equations with applications to the Vlasov-Poisson and semigeostrophic systems

    CERN Document Server

    Colombo, Maria

    2017-01-01

    The first part of the book is devoted to the transport equation for a given vector field, exploiting the lagrangian structure of solutions. It also treats the regularity of solutions of some degenerate elliptic equations, which appear in the eulerian counterpart of some transport models with congestion. The second part of the book deals with the lagrangian structure of solutions of the Vlasov-Poisson system, which describes the evolution of a system of particles under the self-induced gravitational/electrostatic field, and the existence of solutions of the semigeostrophic system, used in meteorology to describe the motion of large-scale oceanic/atmospheric flows.

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

    Science.gov (United States)

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

    2017-10-01

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

  20. Characterization of the RSL1-dependent conditional expression system in LNCaP prostate cancer cells and development of a single vector format.

    Science.gov (United States)

    Lessard, Julie; Aicha, Sonia Ben; Fournier, Andréa; Calvo, Ezequiel; Lavergne, Eliane; Pelletier, Mélissa; Labrie, Claude

    2007-06-01

    Conditional expression systems are useful tools for the study of gene function but the use of these systems in prostate cancer cells is limited by the undesired biological effects of the inducing ligands. The RheoSwitch system employs RheoSwitch Ligand 1 (RSL1), a non-steroidal analog of the insect hormone ecdysone, to activate a modified nuclear receptor heterodimer that controls target gene expression via GAL4 response elements. This system has not been tested in prostate cancer cells. We established LNCaP human prostate cancer cell lines that constitutively express RheoSwitch transcription factors to quantify RSL1-dependent expression and assess the effects of RSL1 on cell proliferation and endogenous gene expression. Potential RSL1-responsive genes were identified using Affymetrix microarrays and validated by Northern blot hybridization. A single-vector format was developed to establish cell lines that conditionally produce a recombinant protein. Stable cell lines displayed tight and potent (over several orders of magnitude) RSL1-dependent regulation of a transiently transfected luciferase reporter gene. RSL1 did not affect basal or androgen-stimulated cell proliferation and exerted minimal effects on the expression of endogenous genes. Cell lines established using the single-vector system also displayed strictly RSL1-dependent production of the recombinant protein encoded by the stably integrated RSL1-responsive expression cassette. The RheoSwitch system is well suited for conditional gene expression in prostate cancer cells. The single-vector format should facilitate the production of stable cell lines. This system should be useful for the study of proteins involved in prostate cancer in both cell and animal models of the disease. Copyright 2007 Wiley-Liss, Inc.

  1. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice

    Directory of Open Access Journals (Sweden)

    Dario Marangoni

    2016-01-01

    Full Text Available X-linked retinoschisis (XLRS is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1 and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124 injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye, and a 6-month study in Rs1-KO mice (n = 162 dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

  2. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice.

    Science.gov (United States)

    Marangoni, Dario; Bush, Ronald A; Zeng, Yong; Wei, Lisa L; Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bartoe, Joshua T; Palyada, Kiran; Santos, Maria; Hiriyanna, Suja; Wu, Zhijian; Colosi, Peter; Sieving, Paul A

    2016-01-01

    X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

  3. Fast Blood Vector Velocity Imaging using ultrasound: In-vivo examples of complex blood flow in the vascular system

    DEFF Research Database (Denmark)

    Hansen, Kristoffer Lindskov; Udesen, Jesper; Gran, Fredrik

    2008-01-01

    Conventional ultrasound methods for acquiring color flow images of the blood motion are restricted by a relatively low frame rate and angle dependent velocity estimates. The Plane Wave Excitation (PWE) method has been proposed to solve these limitations. The frame rate can be increased, and the 2-D...... vector velocity of the blood motion can be estimated. The transmitted pulse is not focused, and a full speckle image of the blood can be acquired for each emission. A 13 bit Barker code is transmitted simultaneously from each transducer element. The 2-D vector velocity of the blood is found using 2-D...... speckle tracking between segments in consecutive speckle images. The flow patterns of six bifurcations and two veins were investigated in-vivo. It was shown: 1) that a stable vortex in the carotid bulb was present opposed to other examined bifurcations, 2) that retrograde flow was present...

  4. Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

    Science.gov (United States)

    Eftekhariyan Ghamsari, Mohammad Reza; Karimi, Farah; Mousavi Gargari, Seyed Latif; Hosseini Tafreshi, Seyed Ali; Salami, Seyed Alireza

    2014-12-01

    Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.

  5. Space Vector Pulse Width Modulation Technique Based Design and Simulation of a Three-Phase Voltage Source Converter Systems

    OpenAIRE

    Farhan Beg

    2014-01-01

    A Space Vector based Pulse Width Modulation control technique for the three-phase PWM converter is proposed in this paper. The proposed control scheme is based on a synchronous reference frame model. High performance and efficiency is obtained with regards to the DC bus voltage and the power factor considerations of the PWM rectifier thus leading to low losses. MATLAB/SIMULINK are used as a platform for the simulations and a SIMULINK model is presented in the paper. The r...

  6. Speed Sensorless vector control of parallel-connected three-phase two-motor single-inverter drive system

    OpenAIRE

    Gunabalan, Ramachandiran; Sanjeevikumar, Padmanaban; Blaabjerg, Frede; Wheeler, Patrick; Ojo, Joseph Olorunfemi; Ertas, Ahmet H.

    2016-01-01

    This paper presents the characteristic behavior of direct vector control of two induction motors with sensorless speed feedback having the same rating parameters, paralleled combination, and supplied from a single current-controlled pulse-width-modulated voltage-source inverter drive. Natural observer design technique is known for its simple construction, which estimates the speed and rotor fluxes. Load torque is estimated by load torque adaptation and the average rotor flux was maintained co...

  7. Vectorization of phase space Monte Carlo code in FACOM vector processor VP-200

    International Nuclear Information System (INIS)

    Miura, Kenichi

    1986-01-01

    This paper describes the vectorization techniques for Monte Carlo codes in Fujitsu's Vector Processor System. The phase space Monte Carlo code FOWL is selected as a benchmark, and scalar and vector performances are compared. The vectorized kernel Monte Carlo routine which contains heavily nested IF tests runs up to 7.9 times faster in vector mode than in scalar mode. The overall performance improvement of the vectorized FOWL code over the original scalar code reaches 3.3. The results of this study strongly indicate that supercomputer can be a powerful tool for Monte Carlo simulations in high energy physics. (Auth.)

  8. Problems and worked solutions in vector analysis

    CERN Document Server

    Shorter, LR

    2014-01-01

    ""A handy book like this,"" noted The Mathematical Gazette, ""will fill a great want."" Devoted to fully worked out examples, this unique text constitutes a self-contained introductory course in vector analysis for undergraduate and graduate students of applied mathematics.Opening chapters define vector addition and subtraction, show how to resolve and determine the direction of two or more vectors, and explain systems of coordinates, vector equations of a plane and straight line, relative velocity and acceleration, and infinitely small vectors. The following chapters deal with scalar and vect

  9. A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

    Science.gov (United States)

    Sinah, Namita; Williams, Charlotte A; Piper, Robert C; Shields, S Brookhart

    2012-08-23

    The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.

  10. Vector bright soliton behaviors of the coupled higher-order nonlinear Schrödinger system in the birefringent or two-mode fiber.

    Science.gov (United States)

    Liu, Lei; Tian, Bo; Xie, Xi-Yang; Guan, Yue-Yang

    2017-01-01

    Studied in this paper are the vector bright solitons of the coupled higher-order nonlinear Schrödinger system, which describes the simultaneous propagation of two ultrashort pulses in the birefringent or two-mode fiber. With the help of auxiliary functions, we obtain the bilinear forms and construct the vector bright one- and two-soliton solutions via the Hirota method and symbolic computation. Two types of vector solitons are derived. Single-hump, double-hump, and flat-top solitons are displayed. Elastic and inelastic interactions between the Type-I solitons, between the Type-II solitons, and between the two combined types of the solitons are revealed, respectively. Especially, from the interaction between a Type-I soliton and a Type-II soliton, we see that the Type-II soliton exhibits the oscillation periodically before such an interaction and becomes the double-hump soliton after the interaction, which is different from the previously reported.

  11. One-step Agrobacterium mediated transformation of eight genes essential for rhizobium symbiotic signaling using the novel binary vector system pHUGE.

    Directory of Open Access Journals (Sweden)

    Andreas Untergasser

    Full Text Available Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.

  12. Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.

    Science.gov (United States)

    Kiatpapan, P; Hashimoto, Y; Nakamura, H; Piao, Y Z; Ono, H; Yamashita, M; Murooka, Y

    2000-11-01

    The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

  13. Propagating Gateway Vectors.

    Science.gov (United States)

    Reece-Hoyes, John S; Walhout, Albertha J M

    2018-01-02

    Generating stocks of Entry and Destination vectors for use in the Gateway recombinatorial cloning system requires transforming them into Escherichia coli strain DB3.1, where they can replicate because this strain is immune to the effects of the ccdB gene carried in the Gateway cassette. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids will consequently allow growth of standard cloning strains of E. coli (e.g., DH5α). Therefore, after making new stocks of Gateway plasmids, their ability to grow in cloning strains of E. coli must be tested. This involves obtaining multiple stocks of vector, each arising from a single plasmid grown in a single DB3.1 bacterial colony, and transforming each stock into both DB3.1 and the preferred cloning strain of E. coli in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (i.e., no or few colonies are obtained after transformation) should be used in Gateway cloning reactions. The sequence can be performed in 3 d. © 2018 Cold Spring Harbor Laboratory Press.

  14. High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries.

    Science.gov (United States)

    Hillgenberg, Moritz; Hofmann, Christian; Stadler, Herbert; Löser, Peter

    2006-06-01

    We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10(6) independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human alpha(1)-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    L. Ding

    2014-06-01

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

  17. 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

  18. Effects of phase vector and history extension on prediction power of adaptive-network based fuzzy inference system (ANFIS) model for a real scale anaerobic wastewater treatment plant operating under unsteady state.

    Science.gov (United States)

    Perendeci, Altinay; Arslan, Sever; Tanyolaç, Abdurrahman; Celebi, Serdar S

    2009-10-01

    A conceptual neural fuzzy model based on adaptive-network based fuzzy inference system, ANFIS, was proposed using available input on-line and off-line operational variables for a sugar factory anaerobic wastewater treatment plant operating under unsteady state to estimate the effluent chemical oxygen demand, COD. The predictive power of the developed model was improved as a new approach by adding the phase vector and the recent values of COD up to 5-10 days, longer than overall retention time of wastewater in the system. History of last 10 days for COD effluent with two-valued phase vector in the input variable matrix including all parameters had more predictive power. History of 7 days with two-valued phase vector in the matrix comprised of only on-line variables yielded fairly well estimations. The developed ANFIS model with phase vector and history extension has been able to adequately represent the behavior of the treatment system.

  19. Vector regression introduced

    Directory of Open Access Journals (Sweden)

    Mok Tik

    2014-06-01

    Full Text Available This study formulates regression of vector data that will enable statistical analysis of various geodetic phenomena such as, polar motion, ocean currents, typhoon/hurricane tracking, crustal deformations, and precursory earthquake signals. The observed vector variable of an event (dependent vector variable is expressed as a function of a number of hypothesized phenomena realized also as vector variables (independent vector variables and/or scalar variables that are likely to impact the dependent vector variable. The proposed representation has the unique property of solving the coefficients of independent vector variables (explanatory variables also as vectors, hence it supersedes multivariate multiple regression models, in which the unknown coefficients are scalar quantities. For the solution, complex numbers are used to rep- resent vector information, and the method of least squares is deployed to estimate the vector model parameters after transforming the complex vector regression model into a real vector regression model through isomorphism. Various operational statistics for testing the predictive significance of the estimated vector parameter coefficients are also derived. A simple numerical example demonstrates the use of the proposed vector regression analysis in modeling typhoon paths.

  20. In vivo visualization of RNA in plants cells using the λN₂₂ system and a GATEWAY-compatible vector series for candidate RNAs.

    Science.gov (United States)

    Schönberger, Johannes; Hammes, Ulrich Z; Dresselhaus, Thomas

    2012-07-01

    The past decade has seen a tremendous increase in RNA research, which has demonstrated that RNAs are involved in many more processes than were previously thought. The dynamics of RNA synthesis towards their regulated activity requires the interplay of RNAs with numerous RNA binding proteins (RBPs). The localization of RNA, a mechanism for controlling translation in a spatial and temporal fashion, requires processing and assembly of RNA into transport granules in the nucleus, transport towards cytoplasmic destinations and regulation of its activity. Compared with animal model systems little is known about RNA dynamics and motility in plants. Commonly used methods to study RNA transport and localization are time-consuming, and require expensive equipment and a high level of experimental skill. Here, we introduce the λN₂₂ RNA stem-loop binding system for the in vivo visualization of RNA in plant cells. The λN₂₂ system consists of two components: the λN₂₂ RNA binding peptide and the corresponding box-B stem loops. We generated fusions of λN₂₂ to different fluorophores and a GATEWAY vector series for the simple fusion of any target RNA 5' or 3' to box-B stem loops. We show that the λN₂₂ system can be used to detect RNAs in transient expression assays, and that it offers advantages compared with the previously described MS2 system. Furthermore, the λN₂₂ system can be used in combination with the MS2 system to visualize different RNAs simultaneously in the same cell. The toolbox of vectors generated for both systems is easy to use and promises significant progress in our understanding of RNA transport and localization in plant cells. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  1. Are Bred Vectors The Same As Lyapunov Vectors?

    Science.gov (United States)

    Kalnay, E.; Corazza, M.; Cai, M.

    Regional loss of predictability is an indication of the instability of the underlying flow, where small errors in the initial conditions (or imperfections in the model) grow to large amplitudes in finite times. The stability properties of evolving flows have been studied using Lyapunov vectors (e.g., Alligood et al, 1996, Ott, 1993, Kalnay, 2002), singular vectors (e.g., Lorenz, 1965, Farrell, 1988, Molteni and Palmer, 1993), and, more recently, with bred vectors (e.g., Szunyogh et al, 1997, Cai et al, 2001). Bred vectors (BVs) are, by construction, closely related to Lyapunov vectors (LVs). In fact, after an infinitely long breeding time, and with the use of infinitesimal ampli- tudes, bred vectors are identical to leading Lyapunov vectors. In practical applications, however, bred vectors are different from Lyapunov vectors in two important ways: a) bred vectors are never globally orthogonalized and are intrinsically local in space and time, and b) they are finite-amplitude, finite-time vectors. These two differences are very significant in a dynamical system whose size is very large. For example, the at- mosphere is large enough to have "room" for several synoptic scale instabilities (e.g., storms) to develop independently in different regions (say, North America and Aus- tralia), and it is complex enough to have several different possible types of instabilities (such as barotropic, baroclinic, convective, and even Brownian motion). Bred vectors share some of their properties with leading LVs (Corazza et al, 2001a, 2001b, Toth and Kalnay, 1993, 1997, Cai et al, 2001). For example, 1) Bred vectors are independent of the norm used to define the size of the perturba- tion. Corazza et al. (2001) showed that bred vectors obtained using a potential enstro- phy norm were indistinguishable from bred vectors obtained using a streamfunction squared norm, in contrast with singular vectors. 2) Bred vectors are independent of the length of the rescaling period as long as the

  2. Light axial vector mesons

    Science.gov (United States)

    Chen, Kan; Pang, Cheng-Qun; Liu, Xiang; Matsuki, Takayuki

    2015-04-01

    Inspired by the abundant experimental observation of axial-vector states, we study whether the observed axial-vector states can be categorized into the conventional axial-vector meson family. In this paper we carry out an analysis based on the mass spectra and two-body Okubo-Zweig-Iizuka-allowed decays. Besides testing the possible axial-vector meson assignments, we also predict abundant information for their decays and the properties of some missing axial-vector mesons, which are valuable for further experimental exploration of the observed and predicted axial-vector mesons.

  3. The consequences of poor vectorization

    CERN Multimedia

    CERN. Geneva

    2016-01-01

    This talk briefly discusses the vectorization problem and how it impacts scientific and engineering systems. A simple cost model of designing such system in context of different phases of software lifetime is considered. Finally a concept for scalable solution is presented.

  4. Vectorization of KENO IV code and an estimate of vector-parallel processing

    International Nuclear Information System (INIS)

    Asai, Kiyoshi; Higuchi, Kenji; Katakura, Jun-ichi; Kurita, Yutaka.

    1986-10-01

    The multi-group criticality safety code KENO IV has been vectorized and tested on FACOM VP-100 vector processor. At first the vectorized KENO IV on a scalar processor became slower than the original one by a factor of 1.4 because of the overhead introduced by the vectorization. Making modifications of algorithms and techniques for vectorization, the vectorized version has become faster than the original one by a factor of 1.4 and 3.0 on the vector processor for sample problems of complex and simple geometries, respectively. For further speedup of the code, some improvements on compiler and hardware, especially on addition of Monte Carlo pipelines to the vector processor, are discussed. Finally a pipelined parallel processor system is proposed and its performance is estimated. (author)

  5. Extreme Contrast Direct Imaging of Planets and Debris disks with the Palomar P3K Adaptive Optics System and the Vector Vortex Coronagraph

    Science.gov (United States)

    Wahl, Matthew; Metchev, S. A.; Patel, R.; Serabyn, G.; PALM-3000 Adaptive Optics Team

    2013-01-01

    We present first results from using the PALM-3000 extreme adaptive optics system and imaging camera on the Hale 5m telescope. Observations using the vector vortex coronagraph have given us direct detections of the planets in the HR8799 system and the dusty debris disk around the star HD141569A. Due to the unprecedented inner working angle of the VVC, the data show a clearing within the inner ring inwards to ~20AU along the projected semi-major axis. Our observations of the disk in the K band (2.2 μm) demonstrate the power of the next generation of adaptive optics systems coupled with phase mask coronagraphy. We also show a comparison of the data reduction techniques currently being implemented in the direct imaging field. Specifically, the Locally Optimized Combination of Images (LOCI) and the Karhunen-Loeve Image Processing (KLIP) algorithms, the latter being a more robust method for resolving debris disks.

  6. A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system.

    Science.gov (United States)

    Tang, Na; Zhang, Yaoyao; Pedrera, Miriam; Chang, Pengxiang; Baigent, Susan; Moffat, Katy; Shen, Zhiqiang; Nair, Venugopal; Yao, Yongxiu

    2018-01-29

    Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines. Copyright © 2018 The Pirbright Institute. Published by Elsevier Ltd.. All rights reserved.

  7. Speed Sensorless vector control of parallel-connected three-phase two-motor single-inverter drive system

    DEFF Research Database (Denmark)

    Gunabalan, Ramachandiran; Sanjeevikumar, Padmanaban; Blaabjerg, Frede

    2016-01-01

    observer design technique is known for its simple construction, which estimates the speed and rotor fluxes. Load torque is estimated by load torque adaptation and the average rotor flux was maintained constant by rotor flux feedback control. The technique’s convergence rate is very fast and is robust......This paper presents the characteristic behavior of direct vector control of two induction motors with sensorless speed feedback having the same rating parameters, paralleled combination, and supplied from a single current-controlled pulse-width-modulated voltage-source inverter drive. Natural...... to noise and parameter uncertainty. The gain matrix is absent in the natural observer. The rotor speed is estimated from the load torque, stator current, and rotor flux. Under symmetrical load conditions, the difference in speed between two induction motors is reduced by considering the motor parameters...

  8. Modified gateway system for double shRNA expression and Cre/lox based gene expression

    Directory of Open Access Journals (Sweden)

    Leung Lisa

    2011-03-01

    Full Text Available Abstract Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2. Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.

  9. Modified gateway system for double shRNA expression and Cre/lox based gene expression.

    Science.gov (United States)

    Radulovich, Nikolina; Leung, Lisa; Tsao, Ming-Sound

    2011-03-22

    The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.

  10. Covariant Lyapunov vectors

    International Nuclear Information System (INIS)

    Ginelli, Francesco; Politi, Antonio; Chaté, Hugues; Livi, Roberto

    2013-01-01

    Recent years have witnessed a growing interest in covariant Lyapunov vectors (CLVs) which span local intrinsic directions in the phase space of chaotic systems. Here, we review the basic results of ergodic theory, with a specific reference to the implications of Oseledets’ theorem for the properties of the CLVs. We then present a detailed description of a ‘dynamical’ algorithm to compute the CLVs and show that it generically converges exponentially in time. We also discuss its numerical performance and compare it with other algorithms presented in the literature. We finally illustrate how CLVs can be used to quantify deviations from hyperbolicity with reference to a dissipative system (a chain of Hénon maps) and a Hamiltonian model (a Fermi–Pasta–Ulam chain). This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Lyapunov analysis: from dynamical systems theory to applications’. (paper)

  11. Covariant Lyapunov vectors

    Science.gov (United States)

    Ginelli, Francesco; Chaté, Hugues; Livi, Roberto; Politi, Antonio

    2013-06-01

    Recent years have witnessed a growing interest in covariant Lyapunov vectors (CLVs) which span local intrinsic directions in the phase space of chaotic systems. Here, we review the basic results of ergodic theory, with a specific reference to the implications of Oseledets’ theorem for the properties of the CLVs. We then present a detailed description of a ‘dynamical’ algorithm to compute the CLVs and show that it generically converges exponentially in time. We also discuss its numerical performance and compare it with other algorithms presented in the literature. We finally illustrate how CLVs can be used to quantify deviations from hyperbolicity with reference to a dissipative system (a chain of Hénon maps) and a Hamiltonian model (a Fermi-Pasta-Ulam chain). This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Lyapunov analysis: from dynamical systems theory to applications’.

  12. VectorBase

    Data.gov (United States)

    U.S. Department of Health & Human Services — VectorBase is a Bioinformatics Resource Center for invertebrate vectors. It is one of four Bioinformatics Resource Centers funded by NIAID to provide web-based...

  13. Interactions between parasites and insects vectors

    OpenAIRE

    Hurd,Hilary

    1994-01-01

    This review stresses the importance of studies that will provide a basic understanding of the pathology of parasite-infected vector insects. This knowledge should be a vital component of the very focussed initiatives currently being funded in the areas of vector control. Vector fecundity reduction is discussed as an example of such pathology. Underlying mechanisms are being investigated in a model system, Hymenolepis diminuta-infected Tenebrio molitor and in Onchocerca-infected blackflies and...

  14. Custodial vector model

    DEFF Research Database (Denmark)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan

    2015-01-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a $SU(2)_L\\times SU(2)_R$ spectral global symmetry. This symmetry partially protects the electroweak S-parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum...

  15. Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

    OpenAIRE

    Urbinati, Fabrizia; Arumugam, Paritha; Higashimoto, Tomoyasu; Perumbeti, Anil; Mitts, Kyle; Xia, Ping; Malik, Punam

    2009-01-01

    Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3′-long ter...

  16. A novel hepatitis B virus-derived cis-acting sequence that enhances expression of transgenes delivered by plasmid vectors in eukaryote cell culture systems.

    Science.gov (United States)

    Lee, Seoung-Ae; Kim, Hong; Lee, So-Young; Kim, Bum-Joon

    2017-08-26

    We tested the effectiveness of a novel 13-bp hepatitis B virus (HBV)-derived cis-acting element (CAE) (ACCTCGACAAGGC), called the DT2 CAE, in augmenting transgene expression delivered by plasmid vectors in eukaryotic cells. The addition of the DT2 CAE just upstream of the start codon of several different target proteins (luciferase, EGFP, LHB, HBsAg, and MIF) in DNA plasmid constructs enhanced their translation in a posttranscriptional manner, irrespective of cell type (cell lines or primary cells) or promoter (CMV or HBV preS1 promoters), suggesting its feasibility for enhanced protein production in eukaryotic cell systems. In conclusion, a novel HBV-derived DT2 CAE could be used effectively for enhanced protein production in eukaryotic cell culture systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Distinguishing Parkinson’s disease from atypical parkinsonian syndromes using PET data and a computer system based on support vector machines and Bayesian networks

    Directory of Open Access Journals (Sweden)

    Fermín eSegovia

    2015-11-01

    Full Text Available Differentiating between Parkinson's disease (PD and atypical parkinsonian syndromes (APS is still a challenge, specially at early stages when the patients show similar symptoms. During last years, several computer systems have been proposed in order to improve the diagnosis of PD, but their accuracy is still limited. In this work we demonstrate a full automatic computer system to assist the diagnosis of PD using 18F-DMFP PET data. First, a few regions of interest are selected by means of a two-sample t-test. The accuracy of the selected regions to separate PD from APS patients is then computed using a support vector machine classifier. The accuracy values are finally used to train a Bayesian network that can be used to predict the class of new unseen data. This methodology was evaluated using a database with 87 neuroimages, achieving accuracy rates over 78%. A fair comparison with other similar approaches is also provided.

  18. Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP.

    Science.gov (United States)

    Xu, Jing; Zhu, Wenhua; Guo, Yuanxu; Jiang, Congshan; Hussain, Nazim; Meng, Liesu; Lu, Shemin

    2017-02-01

    The pLKO.1-TRC plasmid has been a popular and widely used vector due to its simple handling and stability. The huge RNAi database, a TRC library, has been established based on this vector. However, this plasmid only has a puromycin-resisted gene for selecting, which limits its application in microscopy and fluorescence-activated cell sorting (FACS). In the present work, PCR, restriction endonuclease digestion and molecular cloning techniques were used to insert the gene decoding green fluorescent protein (GFP) without changing the structure of original plasmid to extend its application. To demonstrate the function of new plasmid, we constructed shNC and shGAPDH plasmids based on newly constructed pLKO-TurboGFP-TRC (pLKOG) and original pLKO plasmids, and then packaged lentivirus particles by 293T cells. The supernatant containing lentiviral particles was collected and then incubated with RAW264.7 cells for infection. After selection for 7 days by using puromycin, the cells were harvested. RT-qPCR and Western blotting were used to detect the target gene expression. FACS was used to detect the green fluorescent of cells. Our results showed that the newly constructed pLKOG plasmid, as a lentiviral vector carrying shRNA, could knock down the target gene expression efficiently and express TurboGFP protein efficiently in the host cells. We conclude that the new plasmid is a convenient vector for selecting positive cells with shRNA by using fluorescent microscope and FACS.

  19. Phlebotomines (Diptera: Psychodidae) in a Hydroelectric System Affected Area from Northern Amazonian Brazil: Further Insights into the Effects of Environmental Changes on Vector Ecology.

    Science.gov (United States)

    Furtado, Nercy Virginia Rabelo; Galardo, Allan Kardec Ribeiro; Galardo, Clicia Denis; Firmino, Viviane Caetano; Vasconcelos Dos Santos, Thiago

    2016-01-01

    During 2012-2015, an entomological survey was conducted as part of a phlebotomine (Diptera: Psychodidae) monitoring program in an area influenced by the Santo Antônio do Jari hydroelectric system (Amapá State, Brazil). The purpose was to study aspects of Amazon/Guianan American cutaneous leishmaniasis (ACL) vectors subjected to stresses by anthropogenic environmental changes. For sampling, CDC light traps were positioned 0.5, 1, and 20 m above ground at five capture locations along the Jari River Basin. Fluctuations in phlebotomine numbers were analyzed to determine any correlation with rainfall, dam waterlogging, and/or ACL cases, from May 2012 to March 2015. We captured 2,800 individuals, and among 45 species identified, Bichromomyia flaviscutellata , Nyssomyia umbratilis , and Psychodopygus squamiventris s.l. were determined to be the main putative vectors, based on current knowledge of the Amazon/Guianan ACL scenario. Rainfall, but not complete flooding, was relatively correlated with phlebotomine fluctuation, mainly observed for Ps. squamiventris s.l. , as were ACL cases with Ny. umbratilis. Behavioral changes were observed in the unexpected high frequency of Bi. flaviscutellata among CDC captures and the noncanopy dominance of Ny. umbratilis , possibly attributable to environmental stress in the sampled ecotopes. Continuous entomological surveillance is necessary to monitor the outcomes of these findings.

  20. Spatial patterns and eco-epidemiological systems – part I: multi-scale spatial modelling of the occurrence of Chagas disease insect vectors

    Directory of Open Access Journals (Sweden)

    Emmanuel Roux

    2011-11-01

    Full Text Available Studies that explicitly and specifically take into account the spatial dimension within the study of eco-epidemiological systems remain rare. Our approach of modelling the spatial and/or temporal properties of the entomological and/or epidemiological data before their mapping with possible explanatory variables, objectively underline the significant patterns at different scales. The domiciliary and peri-domiciliary presence and abundance of juvenile and adult vectors of the Chagas disease (Triatoma sordida and Panstrongylus geniculatus in Bahia state in northeast Brazil, has been modelled by automatically identifying significant multi-scale spatial patterns of the entomological data by the application and adaptation of the spatial modelling methodology proposed by Dray et al. (2006 and based on principal coordinate analysis of neighbour matrices. We found that entomological data can be modelled by a set of eigenvectors that present a significant Moran’s I index of spatial autocorrelation. The models for juvenile and adult vectors are defined by 28 and 32 eigenvectors that explain 82.3% and 79.9%, respectively, of the total data variances. The results support insect presence as the outcome both of a local scale “near-to-near” dispersal and an infestation from the wild, surrounding environment that produces a higher insect density at the village periphery.

  1. A non-pathogenic live vector as an efficient delivery system in vaccine design for the prevention of HPV16 E7-overexpressing cancers.

    Science.gov (United States)

    Hosseinzadeh, Sahar; Bolhassani, Azam; Rafati, Sima; Taheri, Tahereh; Zahedifard, Farnaz; Daemi, Amin; Taslimi, Yasaman; Hashemi, Mehrdad; Memarnejadian, Arash

    2013-01-01

    The attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT (gp96) and L.tar-E7-CT (gp96) led to enhance the levels of IFN-γ and also IgG2a before and after challenge with TC-1. Furthermore, L.tar-E7-CT (gp96) live vaccine indicated significant protective effects as compared to control groups as well as group vaccinated with L.tar-E7. Indeed, the recombinant live vector is capable of eliciting effective humoral and cellular immune responses in mice, but however, further studies are required to increase their efficacy.

  2. Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Moriyama

    Full Text Available Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1 a modified tetracycline (tet-response element composite promoter, (2 a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3 acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV or the elongation factor 1 α (EF-1α promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.

  3. Multicompartment vectors as novel drug delivery systems: selective activation of Tγδ lymphocytes after zoledronic acid delivery.

    Science.gov (United States)

    Agrati, Chiara; Marianecci, Carlotta; Sennato, Simona; Carafa, Maria; Bordoni, Veronica; Cimini, Eleonora; Tempestilli, Massimo; Pucillo, Leopoldo P; Turchi, Federica; Martini, Federico; Borioni, Giorgio; Bordi, Federico

    2011-04-01

    Multicompartment nanoscopic carriers can be easily assembled by inducing the aggregation of anionic "hybrid" niosomes by means of cationic biocompatible polyelectrolytes. The resulting vesicle clusters, whose size and overall net charge can be easily controlled by varying the polyelectrolyte-to-particle charge ratio, show an interesting potential for multidrug delivery. In this article we provide strong evidence for their effective use in vitro as multicompartment vectors selectively directed toward monocyte/macrophage cells, showing that the monocyte/macrophage-mediated activation of Tγδ lymphocytes induced by zoledronic acid is enhanced by a factor 10(3) when the zoledronic acid is intracellularly delivered through these carriers. Furthermore, the multicompartment ɛ-polylysine niosome clusters, with their intrinsic selectivity toward macrophages, appear particularly suitable for implementing therapeutic strategies against chronically infected macrophages. ɛ-polylysine niosome clusters, with their intrinsic selectivity toward macrophages, offer the potential for multidrug delivery. The effectiveness of aminobisphosphonate zoledronate is demonstrated to enhance the recruitment of Tγδ lymphocytes by macrophages by 2 orders of magnitude, suggesting a new therapeutic strategy for addressing pathologies featuring chronically infected macrophages. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Vertical vector face lift.

    Science.gov (United States)

    Somoano, Brian; Chan, Joanna; Morganroth, Greg

    2011-01-01

    Facial rejuvenation using local anesthesia has evolved in the past decade as a safer option for patients seeking fewer complications and minimal downtime. Mini- and short-scar face lifts using more conservative incision lengths and extent of undermining can be effective in the younger patient with lower face laxity and minimal loose, elastotic neck skin. By incorporating both an anterior and posterior approach and using an incision length between the mini and more traditional face lift, the Vertical Vector Face Lift can achieve longer-lasting and natural results with lesser cost and risk. Submentoplasty and liposuction of the neck and jawline, fundamental components of the vertical vector face lift, act synergistically with superficial musculoaponeurotic system plication to reestablish a more youthful, sculpted cervicomental angle, even in patients with prominent jowls. Dramatic results can be achieved in the right patient by combining with other procedures such as injectable fillers, chin implants, laser resurfacing, or upper and lower blepharoplasties. © 2011 Wiley Periodicals, Inc.

  5. Rotations with Rodrigues' vector

    International Nuclear Information System (INIS)

    Pina, E

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears to be a fundamental matrix that is used to express the components of the angular velocity, the rotation matrix and the angular momentum vector. The Hamiltonian formalism of rotational dynamics in terms of this vector uses the same matrix. The quantization of the rotational dynamics is performed with simple rules if one uses Rodrigues' vector and similar formal expressions for the quantum operators that mimic the Hamiltonian classical dynamics.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Sue VandeWoude

    2011-10-01

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

  8. Vector assembly of colloids on monolayer substrates

    Science.gov (United States)

    Jiang, Lingxiang; Yang, Shenyu; Tsang, Boyce; Tu, Mei; Granick, Steve

    2017-06-01

    The key to spontaneous and directed assembly is to encode the desired assembly information to building blocks in a programmable and efficient way. In computer graphics, raster graphics encodes images on a single-pixel level, conferring fine details at the expense of large file sizes, whereas vector graphics encrypts shape information into vectors that allow small file sizes and operational transformations. Here, we adapt this raster/vector concept to a 2D colloidal system and realize `vector assembly' by manipulating particles on a colloidal monolayer substrate with optical tweezers. In contrast to raster assembly that assigns optical tweezers to each particle, vector assembly requires a minimal number of optical tweezers that allow operations like chain elongation and shortening. This vector approach enables simple uniform particles to form a vast collection of colloidal arenes and colloidenes, the spontaneous dissociation of which is achieved with precision and stage-by-stage complexity by simply removing the optical tweezers.

  9. Synthetic Aperture Vector Flow Imaging

    DEFF Research Database (Denmark)

    Villagómez Hoyos, Carlos Armando

    The main objective of this project was to continue the development of a synthetic aperture vector flow estimator. This type of estimator is capable of overcoming two of the major limitations in conventional ultrasound systems: 1) the inability to scan large region of interest with high temporal......, this thesis showed that novel information can be obtained with vector velocity methods providing quantitative estimates of blood flow and insight into the complexity of the hemodynamics dynamics. This could give the clinician a new tool in assessment and treatment of a broad range of diseases....

  10. Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors

    OpenAIRE

    Capasso, Cristian; Hirvinen, Mari; Cerullo, Vincenzo

    2013-01-01

    Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, a...

  11. Introduction to vector velocity imaging

    DEFF Research Database (Denmark)

    Jensen, Jørgen Arendt; Udesen, Jesper; Hansen, Kristoffer Lindskov

    it virtually impossible to compensate for the factor and obtain correct velocity estimates for either CFM or spectral velocity estimation. This talk will describe methods for finding the correct velocity by estimating both the axial and lateral component of the velocity vector. The transverse oscillation...... method introduces an ultrasound field that oscillation not only along the ultrasound beam both also transverse to it to estimate both the lateral and axial velocity for the full velocity vector. The correct velocity magnitude can be found from this as well as the instantaneous angle. This can be obtained...... over the full region of interest and a real time image at a frame rate of 20 Hz can be displayed. Real time videos have been obtained from both our research systems and from commercial BK Medical scanners. The vector velocity images reveal the full complexity of the human blood flow. It is easy to see...